JAGT 3rd Quarter 2017 by KellenComm

The Journal

of the Association of Genetic Technologists

Volume 43 • Number 3 • Third Quarter 2017

Brain Tickler

Column Editor: Helen Lawce

Brain Tickler

A blood sample on an 11-year-old male referred for Klinefelter Syndrome.

Submitted by: Heather Cole and Suzanne Demczuk Cytogenetics Laboratory Royal University Hospital Saskatoon, SK, Canada

The answer to this Brain Tickler appears on page 135.

The Journal of the Association of Genetic Technologists Third Quarter 2017 Volume 43, Number 3

Table of Contents Brain Tickler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inside Front Cover

The official journal of the AGT

Column Editors and Review Board . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 A Note from the Editor. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 Letter to the Editor The Importance of Teaching Genetics to Non-Science Majors Rasha W. Al-Ali. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 Review The t(12;21)(p13;q22) in Pediatric B-Acute Lymphoblastic Leukemia: An Update Carlos A. Tirado, Maximilian Becker, Kristie Liu. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99

Editorial Information Editor Mark Terry, BSc Associate Editors Turid Knutsen, MT(ASCP), CLSp(CG) Helen Lawce, BSc, CLSp(CG) Heather E. Williams, MS, CG(ASCP) CM Su Yang, BSc, CLSp(CG) Book Review Editor Helen Lawce, BSc, CLSp(CG)

Review The AGT Cytogenetics Laboratory Manual Heather E. Williams.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110 Molecular Diagnostics Molecular Diagnostics: A High School Experience Michelle Mah . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111

Copyright © 2017 by the AGT. All rights reserved. Contents are not to be reproduced or reprinted without permission of the AGT Editor. The Journal of the Association of Genetic Technologists is published four times a year and is available to individuals and libraries at a subscription rate of $115 per year. The subscription rate for members of the AGT is included in the annual membership dues. Back issues can be purchased for members at $25 per issue as long as supplies are available. Material intended for publication or correspondence concerning editorial matters should be sent to the editor.

Research Article Elucidation of Novel Chromosomal Abnormalities in Pancreatic Cancer: Conventional and Molecular Cytogenetic Characterization of 16 Pancreatic Cell Lines David Shabsovich and Carlos A. Tirado. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 Teaching Case Myelodysplastic Syndrome with Isolated del(5q) Juli-Anne Gardner, Katherine Devitt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128 Genetics in the News The Milestone of Non-Invasive Prenatal Identification of Chromosomal Abnormalities in Fetal Trophoblasts Recovered from Maternal Blood Jaime Garcia-Heras . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 Brain Tickler Summary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135 Continuing Education Opportunities Test Yourself #3, 2017. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136 AGT Journal Clubs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140 Association Business Letter from the President. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147

JAGT Editor Mark Terry 1264 Keble Lane Oxford, MI 48371 586-805-9407 (cell) Email: markterry@charter.net

Association of Genetic Technologists 42nd Annual Business Meeting Minutes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149 AGT Annual Meeting Sponsors, Exhibitors and Volunteers. . . . . . . . . . . . . . . . . . . . . 150

Placement service items of less than 150 words and advertisements, requests for back issues, reprint orders, and questions about subscriptions and advertising costs should be sent to the AGT Executive Office at AGT-info@kellencompany.com. Acceptance of advertisements is dependent on approval of the editor-in-chief.

AGT 2016 Sponsors and Exhibitors, Annual Meeting Photos. . . . . . . . . . . . . . . . . . . . 151 AGT and FGT Award Winners . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155 Platform, Poster and Student Poster Presentations from the 2017 Annual Meeting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156 Association of Genetic Technologists BOD Contacts . . . . . . . . . . . . . . . . . . . . . . . . . 185 AGT 2018 Call for Abstracts Student Research Award Entries . . . . . . . . . . . . . . . . . . 186 FGT Letter. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188 2018 FGT Grants and Awards Deadlines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189

ISSN 1523-7834

FGT Board of Trustees . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190 Product Order Form . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191 New Membership Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192 2017-2018 Scientific Meetings Schedule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194 Information for Authors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inside Back Cover

The Journal of the Association of Genetic Technologists is indexed in the life sciences database BIOSIS and in the National Library of Medicine’s PubMed. The Journal of the Association of Genetic Technologists 43 (3) 2017

The Journal of the Association of Genetic Technologists Staff

Column Editors Abstract Reviews/Genetics in the News Genetics, Government & Regulation Helen Bixenman, MBA, CLSup, CLSp(CG) Jaime Garcia-Heras, MD, PhD San Diego Blood Bank Director of Cytogenetics 3636 Gateway Center Avenue, Suite 100 The Center for Medical Genetics San Diego, CA 92102 7400 Fannin, Suite 700 619-400-8254 Houston, TX 77054 hbixenman@sandiegobloodbank.org 713-432-1991 713-432-1661 FAX Jennifer Crawford-Alvares jgarcia@geneticstesting.com Cytogenetic Technologist II Section of Hematology/Oncology Brain Tickler/Book Review Editor The University of Chicago Medicine Helen Lawce, BSc, CLSp(CG) 5841 S. Maryland Ave., Rm. I-304 Clinical Cytogenetics Laboratory Chicago, IL Oregon Health Sciences University jen.crawford34@gmail.com 3181 SW Sam Jackson Parkway Office: 773-702-9153 MP-350 Letters to the Editor Portland, OR 97201 Mark Terry, JAGT Editor 503-494-2790 1264 Keble Lane 503-494-6104 FAX Oxford, MI 48371 lawceh@ohsu.edu 586-805-9407 (cell) markterry@charter.net

Meeting Notices Jun Gu, MD, PhD, CG(ASCP)CM University of Texas MD Anderson Cancer Center School of Health Professions Cytogenetic Technology Program 1515 Holcombe Blvd., Unit 2 Houston, TX 77030 713-563-3094 jungu@mdanderson.org Molecular Diagnostics Michelle Mah, MLT, MB(ASCP)CM Advanced Diagnostics Lab Princess Margaret Cancer Centre University Health Network 610 University Ave., Rm 7-707 Toronto, Ontario Canada M5G 2M9 416-946-4501 ext.5036 michelle.j.mah@gmail.com

Special Interests Turid Knutsen, MT(ASCP), CLSp(CG) 17836 Shotley Bridge Place Olney, MD 20832 301-570-4965 turid.knutsen@verizon.net Test Yourself Sally J. Kochmar, MS, CG(ASCP)CM Magee-Womens Hospital Pittsburgh Cytogenetics Lab 300 Halket St., Room 1233 Pittsburgh, PA 15213 412-641-4882 skochmar@upmc.edu

Profiles & Perspectives Hon Fong Louie Mark, PhD, FACMG President KRAM Corporation 2 Pine Top Road Barrington, RI 02806 401-246-0487 HonFong_Mark@Brown.edu

Review Board Linda Ashworth, BSc, CLSp(CG) (Cytogenetics, Molecular genetics) Helen Bixenman, BSc, CLSp(CG), CLSup (Prenatal diagnosis) Judith Brown, MS, CLSp(CG), CLSp(MB) (Cytogenetics) Kim Bussey, PhD (Cancer genetics, Molecular genetics, Microdissection/PCR/DNA) Mona CantĂş, BSc, CLSp(CG) (Cytogenetics) Anthony Ciminski, CG(ASCP)CM Molecular Genetics, Molecular Cytogenetics Adam Coovadia, CLSpP(CG, MG) (Traditional, Molecular, Regulatory) Philip D. Cotter, PhD, FACMG (Prenatal diagnosis, Chromosome rearrangements, Molecular genetics) Jennifer Costanzo, MS, CLSp(CG) (Cytogenetics, Molecular genetics) Janet Cowan, PhD (Cytogenetics, Cancer genetics, FISH, Solid tumors) Lezlie Densmore, BSc, CLSp(CG) (Cytogenetics, Molecular genetics) Janet Finan, BSc, CLSp(CG) (Hemic neoplasms, Somatic cell hybridization) Lakshan Fonseka, MS (Cytogenetics, Molecular genetics)

Sue Fox, BSc, CLSp(CG) (Bone marrow cytogenetics, Prenatal diagnosis, Supervisory/Management) Jaime Garcia-Heras, MD, PhD (Clinical cytogenetics) Robert Gasparini, MS, CLSp(CG) (Prenatal diagnosis, Cytogenetics) Barbara K. Goodman, PhD, MSc, CLSp(CG) (Molecular cytogenetics)

Hon Fong Louie Mark, PhD, FACMG (Molecular genetics, Somatic cell genetics, Cancer cytogenetics, Breast cancer, Trisomies, Laboratory practices, Regulatory practices, FISH) Jennifer L. McGonigle, BA, CLSp(CG) (Cytogenetics) Karen Dyer Montgomery, PhD, FACMG (Cancer cytogenetics, Cytogenetics, Molecular cytogenetics)

Debra Saxe, PhD (Prenatal diagnosis, Cytogenetics) Jack L. Spurbeck, BSc, CLSp(CG) (Cancer cytogenetics, Molecular genetics) Peggy Stupca, MSc, CLSp(CG) (Cytogenetics, Prenatal diagnosis, Breakage syndromes, FISH, Regulations/ QA)

Nancy Taylor, BSc, CLSp(CG), MT(ASCP) (Cytogenetics, Cancer cytogenetics) Stephen R. Moore, PhD, ABMG (Clinical cytogenetics, radiation biology, Thomas Wan, PhD toxicology; clinical molecular genetics) (Cytogenetics, Molecular genetics, Lynn Hoyt, BSc, CLSp(CG), CLSup Rodman Morgan, MS, CLSp(CG) Cancer genetics) (Classical cytogenetics) (Cancer cytogenetics) James Waurin, MSc, CLSp(CG) Peter C. Hu, PhD, MS, MLS(ASCP), CG, MB (Prenatal diagnosis, Counseling) Susan B. Olson, PhD (Cytogenetics, Molecular cytogenetics, (Cancer cytogenetics, Molecular Sara Wechter, BSc Education) genetics, Prenatal diagnosis, OB/GYN, (Cytogenetics, Cancer) Counseling, Cytogenetics) Denise M. Juroske, MSFS, MB(ASCP)CM (Cytogenetics, Molecular, Education) Heather E. Williams, MS, CG(ASCP)CM Jonathan P. Park, PhD (Cytogenetics, Molecular Genetics) (Cytogenetics, Molecular genetics, Julia Kawecki, BSc, CLSp(CG) Cell biology) (Cytogenetics, Molecular genetics) Su Yang, BSc, CLSP(CG) (Education, Traditional Cytogenetics) David Peakman, AIMLT, CLSp(CG) Turid Knutsen, MT(ASCP), CLSp(CG) (Prenatal diagnosis) (Cancer cytogenetics, CGH, SKY) Jason A. Yuhas, BS, CG(ASCP)CM (Cytogenetics, Molecular cytogenetics) Carol Reifsteck, BA Brandon Kubala, BSc, CLSp(CG) (Breakage syndromes, Fanconiâ&#x20AC;&#x2122;s (Traditional Cytogenetics) James Zabawski, MS, CLSp(CG) anemia, Prenatal diagnosis) (Education, Traditional Cytogenetics) Anita Kulharya, PhD Gavin P. Robertson, PhD (Molecular genetics, Clinical (Cytogenetics, Molecular genetics, cytogenetics) Somatic cell genetics, Tumor suppressor Helen Lawce, BSc, CLSp(CG) genes, Cancer genes) (Prenatal diagnosis, Solid tumors, FISH, Laurel Sakaluk-Moody, MSc, MLT(CG) Chromosome structure, Evolution) (Cytogenetics, Developmental biology, Prenatal cytogenetics) Michelle M. Hess, MS, CLSp(CG) (Cytogenetics, Cancer cytogenetics)

The Journal of the Association of Genetic Technologists 43 (3) 2017

A Note from the Editor

Changes

Publication and the Journal

This issue is jammed with materials. In addition to several research and/or review articles, some short instructional and/ or op-eds, we have a review of the newly published The AGT Cytogenetics Laboratory Manual. It’s been a long time coming, but everything I’m hearing from people who have their hands on it, is that it’s a beautiful and remarkable resource. Much thanks to editors Marilyn Arsham, Helen Lawce and the late Margaret Barch. In addition, there are abstracts, posters and other materials that were presented at this year’s Annual Meeting in St. Louis. Enjoy.

As this changeover began implementation in late-May (to be completed by the end of the year), I had several discussions with Denise and Christie and the board on publication of The Journal of the AGT. The reason for those discussions is that although I am hired to act as editor of the journal, a job I’ve done for almost 17 years now, the graphic design, publication, printing, mailing, and distribution of the journal was handled by the management company, first Applied Measurement Professionals and most recently Kellen. This presented some issues, which are not trivialities. Nor have they been completely resolved—as of this writing we are still looking at proposals from graphic designers. However, one change that will occur is that the journal will no longer be published in paper. Several years ago we began the transition to e-publishing, partly to save costs, and partly because of demand for it. However, a number of people were willing to pay extra for paper … quite a bit more, in the last year or two. But, when evaluating the actual numbers of the membership—around 600 people—and those paying more for paper—about 20—it became clear that print publishing and mailing presented a larger problem than it benefited. So going forward, the journal will be completely e-published.

Management Company As most of you likely know by now, there has been a major change with the Association of Genetic Technologists. When the organization's began (as the Association of Cytogenetic Technologists) over 40 years ago, it was small, but grew. At its peak it had over 2,000 members. As the activities, and business and organization's responsibilities grew, it began to exceed the time, energy and resources of the membership’s volunteers. So they hired a professional management association to handle much of the business-related activities of the organization, including organizing the annual meeting. In recent years, since approximately 2008 or 2009, several things began to happen. One, membership began to shrink. There are likely a number of reasons for that—it’s a national trend among all professional organizations, the core membership’s field (cytogenetics) began to change dramatically, employers stopped paying for membership (assuming they ever did), and some of the main reasons for membership in the first place evolved. In other words, networking, troubleshooting and continuing education opportunities shifted online and are far more available than they were pre-Internet. Two, the economy, notably in 2008 and 2009, went through a dramatic crisis, which affected AGT’s investments and costs—including one of the big revenue generators, the annual meeting. (Shrinking membership also affects revenue). So the bottom line became, in fact, the bottom line. Investments tanked, revenue decreased, while expenses stayed the same or increased. In taking a very hard look—and I’ve been in on these discussions for about eight years now—at the organization’s budget, one very large area of expense was obvious—the fees spent on the management company. It took several years to come to the very hard fact that AGT could no longer afford a professional management company. As the association’s new president, Jason Yuhas, writes in his note, and which you likely already received in an e-newsletter, AGT has ended its relationship with the management company, currently Kellen, and will be going it alone. Denise Juroske Short, who has been the Secretary/Treasurer of the organization for several years, is a former technologist as well as a PhD, has been hired to act as the organization’s Executive Director, and is currently working with our soon-to-be-former Executive Director Christie Ross, on learning many of the nuances of the job.

Consistency Amidst Change For my part, I’m very sad to see us end our relationship with a management company. Over my years with the journal and AGT I have enjoyed working with the staff at AMP and Kellen, and call many of the former Executive Directors and meeting planners friends—Cathy Berra, Stephanie Newman, Joyce Miller, Monica Evans-Lombe and Christie Ross, to name the ones I’ve worked with most directly, as well as Trisha Bergant and Mary Jones. Yet, I do understand the necessity for it from a financial standpoint. So here’s what I expect, going forward. I expect there will be some headaches—mostly Denise’s and the board’s. There may be some on my part as I work with whatever graphic designer we hire and we transition to a slightly different publication and business model. But importantly, I know that Denise and the board and myself will work very hard to make sure that the journal and the other activities of the association continue with the same quality and level of service. I’m confident we will. I also believe that to assure that, the organization can use volunteers in a number of different capacities. If interested, feel free to contact any board member, Denise or myself, to talk about what you can do to help move AGT forward. Cheers, Mark Terry, Editor

The Journal of the Association of Genetic Technologists 43 (3) 2017

Letter to the Editor

The Importance of Teaching Genetics to Non-Science Majors So, who is to blame here, and how do we fix things?

How did an urge to educate the public about a field that has an ever-increasing visibility to the world become one of my life’s greatest passions? Truly great achievements and advancements in the world of medical genetics are splashed across the newspaper daily, readily accessible to the masses. There has never been a more exciting time to learn about genetic diagnosis and prognosis—but topics like gene editing and potential cures for humanity’s most devastating disorders require an understanding of this science that most readers do not currently possess. This article emphasizes how genetics education is essential in opening the door to the public to understand the latest in the field’s research and enabling people to engage in informed debates about the future of genetics applications. No day passes without us coming across an article about the new CRISPR, gene editing, or cancer genetics in major media outlets. Further, the majority of the general public lacks the basic genetic knowledge or terminology to understand the content in the article beyond its (usually) catchy title. My husband and close friends forward me genetics articles all the time with seemingly important headlines—not because they thought these would be a great read but because they simply “sounded” interesting. They typically ask me, the geneticist, to explain, in layman’s terms, the “meat” of the argument and the science behind it—and to judge whether or not a seemingly important headline is matched by a substantively critical revelation. (I admittedly do the same thing with my husband when I encounter unfamiliar articles in political science…) Of course, the vast majority of readers do not have a geneticist on speed dial to brief them on the latest developments. Importantly, I am not talking about prestigious, peer-reviewed, scientific journal articles—or even pieces in more popular scientific magazines or websites. Rather, family and friends typically encounter the bulk of their “public genetics” from Google, Yahoo, and the morning paper on the way to work—light reading that supposes an equal understanding of karyotyping as it does global news, fashion, sports, and weather. Another big concern is how, at the grocery store or dinner with friends, simple questions like, “Why can’t a cat and a human make babies” (or, the best at the neighborhood bar, “Why can’t monkeys and eagles have babies?!”)—and even less ridiculous ones like, “Why haven’t we found a cure for cancer yet?”—get me excitedly talking about genetics. These checkout line questions force me to think on my feet and produce answers that are succinct, clear, and believable. Truthfully, I get so excited talking about genetics—but the lack of basic vocabulary most people have on the subject makes quick conversations and easy answers to these questions difficult. How often, after all, are educators put on the spot to provide simple responses to usually complex science questions in two minutes or less? How can I possibly have a conversation about oncogenes and tumor suppressors if questioners do not know the basics about chromosomes and genes?

I have always been a firm believer that teaching genetics must be a requirement for all college students, no matter their major. Today, this is truer than ever—and urgent. Do I believe that teaching genetics to all students would change that much in our lives? I absolutely do! Only when the general public is able to engage in conversations about the most important topics in genetics—from drug discoveries and genetic testing to gene editing and targeted treatments—will people be prepared to make decisions about, for example, whether to buy an at-home genetics testing kit or engage intelligently with their physician about personalized medicine. Also, genetics ethics these days touches so many fields, including economics, politics, and religion. I am saddened when non-specialists reach conclusions and opine with apparent expert knowledge without actually understanding the science and context of the topic. I came to appreciate shortly after I started teaching Human Genetics for Non-Majors many years ago that our biggest hurdle as educators is all of the new terminology that students encounter. I reassure them early on that they will enjoy this exciting ride learning about our bodies, how we function, diseases, genetic testing, genetic engineering, cancer genetics, and much more. But first, I push them in the first weeks to focus on the foundational concepts and familiarizing themselves with terms that majors see as second nature. I emphasize to my students that only then will they be able to appreciate their magnificent journey of learning—yes, magnificent—and understand the world around them on a much deeper level. Watching them unlock the keys to this very relevant yet mysterious world called genetics is so incredibly satisfying. Nothing compares with the satisfying sparkle in their eyes when once alien topics now seem personal. Knowing that my non-majors, who generally study topics like fashion merchandising, accounting, and education, are the future professionals in my community is such a relief. After all, with their rudimentary understanding of genetics, they leave class prepared to take on science news in the papers and have informed conversations with colleagues over happy hour as they do with their health care professionals. It also means that in the years to come, the odds of getting “Why can’t…?” question at Starbucks will be (thankfully) lower! Rasha W. Al-Ali, M.S. Geneticist and Adjunct Professor Marymount University ralali@marymount.edu

The Journal of the Association of Genetic Technologists 43 (3) 2017

Review

The t(12;21)(p13;q22) in Pediatric B-Acute Lymphoblastic Leukemia: An Update Maximilian Becker1,2,6, Kristie Liu6 and Carlos A. Tirado3,4,5,6 1. 2. 3. 4. 5. 6.

Centro de Investigaciones Tecnológicas, Biomédicas y Medioambientales, Lima, Peru Centro Nacional de Salud Publica, Instituto Nacional de Salud, Lima, Peru Allina Health, Minneapolis, MN 55407 HPS, Minneapolis, MN 55407 The University of Minnesota, School of Medicine. Department of Laboratory Medicine and Pathology, Minneapolis, MN 55407 The International Circle of Genetics Studies, Los Angeles, CA 90024

Abstract Pediatric B-cell acute lymphoblastic leukemia (B-ALL) is the most common hematological malignancy in children, and the t(12;21)(p13;q22) occurs in approximately 25% of these cases, making it is the most prevalent chromosomal abnormality. The t(12;21) translocation results in the ETV6/RUNX1 fusion protein which disrupts hematopoietic differentiation and proliferation, and can be present as a sole abnormality or within the context of a complex karyotype characterized by three or more chromosomal abnormalities. The prognosis of t(12;21) within a complex karyotype is extensively debated. In this review, we discuss the literature regarding t(12;21) and summarize the cytogenetic features found in 363 pediatric cases compiled from the Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer. Cytogenetically, most of the cases had secondary chromosomal abnormalities, about half of which were in the context of a complex karyotype. Trisomy 21 was found to be the most common numerical abnormality in almost one-fifth of the cases, and deletions on chromosome 12 and 6 occurred in 16.9% and 12.5% of cases, respectively. In general, t(12;21) in B-ALL is associated with a favorable prognosis. Herein, we found no significant difference in survival outcome of t(12;21) with a non-complex or complex karyotype.

Introduction

the presence of an extra copy of a normal chromosome 21 and the presence of an extra copy of the derivative chromosome 21. Molecular cytogenetic tools such as fluorescence in situ hybridization (FISH) with whole-chromosome or locus-specific probes, reverse-transcriptase PCR (RT-PCR), or spectral karyotypic analysis (SKY) are used to detect the ETV6-RUNX1 fusion. The prognostic impact of t(12;21)(p13;q22) in B-ALL has been extensively debated, particularly regarding standard treatment regimens and the incidence of late relapses (Forestier et al., 2008).

B-Acute lymphoblastic leukemia (B-ALL), a malignant disorder of the lymphoid progenitor cells, is attributed to approximately 80% of pediatric leukemia, making it the most frequent malignancy in childhood (Pui 2009). The most common chromosomal abnormality in pediatric B-ALL is the non-random translocation t(12;21), occurring in 25% of the cases, with the highest incidence among those aged from 3 to 6 years. (Rubnitz et al., 1997; Yehuda-Gafni et al., 2002; Hübner et al., 2003; Alvarez et al., 2005; Borst et al., 2012). The presence of t(12;21) is associated with a good prognosis, with a reported ten-year Event-Free Survival rate (EFS) of 72% (Olsson et al., 2013). This translocation is associated with a B-lineage immunophenotype, frequently with co-expression of myeloid antigens CD13 and/ or CD33 (Kempski et al., 1999). It leads to the expression of the ETV6-RUNX1 transcription factor, which promotes B-cell progenitor self-renewal and proliferation (Fischer et al., 2005; Mullighan, 2012). However, the ETV6/RUNX1 fusion protein alone does not induce leukemia: transcripts have been detected at birth, in blood spots from children who only years later developed ALL (Mori et al., 2002; Lausten-Thomsen et al., 2011), and neither ETV6/RUNX1 knock-in mouse models nor carriers of ETV6/RUNX1-positive pre-leukemic cells will all develop leukemia (Fischer et al., 2005). Therefore, it has been suggested that it requires additional abnormalities to cause leukemia. Arraybased genome-wide profiling studies have identified recurring copy number alterations (CNA) of transcription factors involved in the regulation of G1 cell cycle progression, B-lymphocyte development and proliferation, apoptosis and hematopoiesis (Mullighan et al., 2007; Enshaei et al., 2013; Öfverholm et al., 2013; Grausenburger et al., 2016). Because of the cytogenetically cryptic nature of the ETV6-RUNX1 fusion due to the similarity of the exchanged bands, G-banding cannot distinguish between

Molecular characterization of ETV6-RUNX1 The gene ETV6 (previously known as TEL) is located at band 12p13 and encodes an ETS family transcription factor containing three functional domains: The N-terminal PNT domain and the central domains act as strong transcriptional repressors, while the C-terminal ETS domain enables DNA binding. The PNT domain enables homo- and heterodimerization with various proteins (Kim et al., 2001; Gunji et al., 2004), including H-L(3)MBT, an HDACindependent tumor suppressor and member of the PcG protein family, which potentiates the repression of ETV6-responsive promoters (Boccuni et al., 2003). The central domain establishes binding of several transcription factors including SMRT, Sin3A, N-CoR, and Tip60, which in turn recruit histone deacetylases (HDACs) that alter the local chromatin structure to influence transcription (Chakrabarti and Nucifora 1999; Guidez et al., 2000; Wang and Hiebert 2001; Nordentoft 2003). It is theorized that several corepressors are required for ETV6-mediated repression, as deletion of the binding-site of Sin3A in the central domain fails to repress ETV6 target promoters (Fenrick et al., 2000). ETV6 is crucial for hematopoiesis and embryonic development since gene knockout in mice causes defects in the developing vascular network and leads to early embryonic lethality (Wang et al., 1997). Rearrangements and numerical abnormalities of

The Journal of the Association of Genetic Technologists 43 (3) 2017

Review The t(12;21)(p13;q22) in Pediatric B-Acute Lymphoblastic Leukemia: An Update ETV6 are associated with infantile fibrosarcoma, and germline The reciprocal translocation t(12;21)(p13;q22) fuses the mutations in ETV6 with red cell macrocytosis and predisposition N-terminal PNT and the central domain of ETV6 to almost to lymphoblastic leukemia (Adem et al., 2001; Noetzli et al., 2015). the entire RUNX1 protein (Golub et al., 1995; Romana et al., The gene RUNX1 (previously known as AML1) is located on 1996). The resulting chimeric transcription factor ETV6-RUNX1 band 21q22 and encodes a RUNT domain transcription factor. converts RUNX1 from an activator to a repressor, as it binds to The N-terminal RUNT homology domain (RHD) mediates RUNX1 target sequences like the TCR-beta enhancer and the binding to DNA, and heterodimerizes with CBF-beta to form IL3 promoter to inhibit their basal activity (Hiebert et al., 1996; to RUNX1 target sequences Uchida like the TCR-beta enhancer and the IL3 the Core Binding Factora repressor, (CBF), a as keyit binds regulator of hematopoietic et al., 1999). Heterodimerization of wild-type ETV6 with promoter to inhibit their basal activity 1996; Uchida etprevents al., 1999). development during embryogenesis (Hiebert et al., 1996,(Hiebert Hart et al., ETV6/RUNX1 its transcriptional repression (McLean of the wild-type ETV6 with prevents its transcriptional repression and Foroni, 2002). TheHeterodimerization central region and C-terminal allowETV6/RUNX1 et al., 1996; Gunji et al., 2004). ETV6-RUNX1 also recruits (McLean et al., 1996. Gunji such et al., as 2004). ETV6-RUNX1 also recruits transcriptional co-the NCOR histone deacetylase recruitment of chromatin-modifying enzymes histone transcriptional co-repressors like repressors the NCOR histone deacetylase and Sin3A to RUNX1 target genes to alter gene to alter gene expression. acetyltransferases (p300, MOZ), like histone deacetylase-containing and Sin3A to RUNX1 target genes expression. Interestingly, the fusion protein binds to Sin3A more stably than either wild-type corepressors (TLE, Sin3A), or the chromatin regulator Polycomb Interestingly, the fusion protein binds to Sin3A more stably than protein (Fenrick et al., 1999; Guidez et al., 2000). shRNA-mediated knockdown and gene Repressive complex PRC1 (via BMI1) (Imai of et al., 1998;positive Kitabayashi either expression profiling t(12;21) and negative caseswild-type of B-ALLprotein showed(Fenrick the effectetofal., 1999; Guidez et al., 2000). et al., 1998; Levanon etETV6/RUNX1 al., 1998; Kitabayashi et al., 2001; Zhao shRNA-mediated knockdown and on genome-wide expression patterns: upregulated genes are involved in cellgene expression profiling of et al., 2007; Guo et al.,activation, 2011; Yu immune et al., 2012). Thereby, RUNX1 t(12;21) while positive and negative cases of B-ALL showed the effect of response, apoptosis and differentiation, downregulated genes include locally alters chromatinkey structure andofregulates the transcription ETV6/RUNX1 oncells genome-wide regulators PI3K/AKT/mTOR signaling and hematopoietic stem (Ross 2003;expression Fuka et patterns: upregulated of target genes involved in pathways like hematopoiesis (i.e.to TGF-β genes are involved in cell activation, immune response, apoptosis al., 2011). ETV6/RUNX1 induces resistance -mediated inhibition of proliferation, and blocks apoptosis by enhancing expression of survivin of the inhibitor apoptosis IL3, GMCSF, TCR-beta) and cell-cycle regulation (i.e. p21) and(a member differentiation, whileofdownregulated genes include key by suppressing and miRNA-320 (Ford et al., Diakos et al., 2010). (De Braekeleer et al., family) 2009; Bakshi et al., miRNA-494 2010). Knockout of regulators of 2009; PI3K/AKT/mTOR signaling and hematopoietic There is evidence that ETV6/RUNX1 increases self-renewal and survival properties preRUNX1 causes impaired fetal liver hematopoiesis and embryonic stem cells (Ross, 2003; Fuka etofal., 2011). ETV6/RUNX1 induces human of cord blood cells, and that it retainsresistance self-renewal of hematopoietic stem of proliferation, and lethality, and inducibleleukemic inactivation RUNX1 in adult mice toabilities TGF-β-mediated inhibition cells cells and maintains them in a quiescent state, allowing pre-leukemic cells to persist in the of survivin (a member leads to impaired lymphoid and megakaryocyte development blocks apoptosisstem by enhancing expression marrow (Hong et al., 2008; Schindler et al., 2009). Together with the finding that the fusion and thrombocytopenia bone (Ichikawa et al., 2004; Growney 2005;). of the inhibitor of apoptosis family) by suppressing miRNA-494 transcript has been found in neonatal blood spots from children who only years later developed Mutations and translocations of RUNX1 frequently occur in and miRNA-320 (Ford et al., 2009; Diakos et al., 2010). There is ALL, but also in children who did not develop ALL at later ages, this may explain that the myeloid and lymphoid disorders (Grossmanncells et al., 2011; Mangan in utero, evidence that ETV6/RUNX1 self-renewal and survival positive pre-leukemic arise predominantly and quietly persist until theyincreases acquire one and Speck, 2011). properties of pre-leukemic human cord or more subsequent genetic “hits” to induce overt leukemia (Mori et al., 2002; Kjeldsen 2016). blood cells, and that it 12p13.1

Chromosome 12

Homo & Heterodimerization

ETV6

NH2

PNT

Regulatory domain

338

ETS

424

452

COOH

453

COOH

21q22

Chromosome 21 DNA binding NH2

331

N-CoR mSin3A SMRT

L(3)MBT

RUNX1

DNA Binding

Transcriptional repression

124 127

RHD

CBFβ TLE Bmi1

Transactivation Inhibition 177

296

mSin3A

TD p300

371

412

TLE

MOZ

t(12;21)(p13;q22)

Der(12)t(12;21) ETV6/RUNX1

NH2

PNT IL-3 repression

TCR-beta repression

Regulatory domain

RHD IL-3 repression

N-CoR

mSin3A

COOH

mSin3A

Figure 1. Overview of theoflocalization, functional domains and main proteinofinteractions of RUNX1 and ETV6/ 1. ofOverview the localization, domains and main protein RUNX1 andETV6, ETV6/RUNX1. Figure 1.Figure Overview the localization, functional domainsfunctional and main protein interactions of ETV6, RUNX1interactions and ETV6/RUNX1.ETV6, Ideograms of chromosomes 12 and 21 indicating the position of the ETV6 and RUNX1 of genes, as well as the12 translocation on the 21 derivative chromosome Der(12)(t(12;21). The functional domains are as follows: Forwell ETV6, as the N-terminal RUNX1. Ideograms ofchromosomes chromosomes 12indicating and indicating the position ofmain the ETV6 andasRUNX1 genes, Ideograms and 21 the position of the ETV6 and RUNX1 genes, as well the indicated translocation onas the pointed domain (PNT) and the C-terminal E26-Transforming Speciﬁc domain (ETS). For RUNX1, the N-terminal Runt homology domain (RHD), the C-terminal transactivation domain (TD) derivative chromosome Der(12)(t(12;21). Theconverts main functional domains are indicated as follows: For ETV6, the N-terminal pointed the translocation derivative chromosome der(12)(t(12;21). The main functional are indicated as of and inhibition domainon (ID). the The translocation t(12;21)(p13;q22) RUNX1 into a constitutive transcriptional repressor, which bindsdomains to DNA through the Runt domain. Recruitment domain (PNT) and the C-terminal E26-Transforming Specific domain (ETS). For RUNX1, the N-terminal Runt homology domain HDACs (via N-CoR) by the ETV6 component and recruitment of the corepressors mSin3A by both the ETV6 and the RUNX1 component changes the patterns of gene expression of RUNX1 follows: For ETV6, theBakshi N-terminal pointed2003; domain (PNT) and the C-terminal E26-Transforming Specific domain (ETS). target genes. Based et al.transactivation 2010; Boccuni et al. Nucifora 1999; De Braekeleer et al.translocation 2009; Fenrick ett(12;21)(p13;q22) al. 1999; Guidez et al.converts 2000; GunjiRUNX1 et al. 2004; Guo et al. (RHD), theon:C-terminal domainChakrabarti (TD) and&inhibition domain (ID). The 2011; Golub et al.N-terminal 1995; Hart & Foroni 2002;homology Hiebert et al. 1996; Imai et al. 1998; Kim etthe al. 2001; Kitabayashi et al. 1998; Kitabayashi et al. domain Levanon(TD) al. 1998; et al. 1996; For RUNX1, the Runt C-terminal transactivation andRomana into a constitutive transcriptional repressor, domain which binds(RHD), to DNA through the Runt domain. Recruitment of2001; HDACs (viaetN-CoR) byinhibition Wang & Hiebert 2001; Yu et al. 2012; Zhao et al. 2007. ! the ETV6 component and recruitment of the corepressors Sin3A by both the ETV6 and the RUNX1transcriptional component changes repressor, the patterns which domain (ID). The translocation t(12;21)(p13;q22) converts RUNX1 into a constitutive of gene expression of RUNX1 target genes. Based on: Golub al., 1995;(via Hiebert et al., 1996; Romana et component al., 1996; Imai etand al., 1998; Recruitment binds to DNA through the Runt domain. ofetHDACs N-CoR) by the ETV6 recruitment of the corepressors Sin3A by both the ETV6 and the RUNX1 component changes the patterns of gene expression of RUNX1 target genes. Based on: Golub et al., 1995; Hiebert et al., 1996; Romana et al., 1996; Imai et al., 1998; Kitabayashi et al., 1998; Levanon et al., 1998; Chakrabarti and Nucifora, 1999; Fenrick et al., 1999; Guidez et al., 2000; Kim et al., 2001; Kitabayashi et al., 2001; Wang and Hiebert, 2001; Hart and Foroni, 2002; Boccuni et al., 2003; Gunji et al., 2004; Zhao et al., 2007; De Braekeleer et al., 2009; Bakshi et al., 2010; Guo et al., 2011; Yu et al., 2012. The Journal of the Association of Genetic Technologists 43 (3) 2017

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Review The t(12;21)(p13;q22) in Pediatric B-Acute Lymphoblastic Leukemia: An Update retains self-renewal abilities of hematopoietic stem cells and maintains them in a quiescent state, allowing pre-leukemic stem cells to persist in the bone marrow (Hong et al., 2008; Schindler et al., 2009). Together with the finding that the fusion transcript has been found in neonatal blood spots from children who only years later developed ALL, but also in children who did not develop ALL at later ages, this may explain that the positive preleukemic cells arise predominantly in utero, and quietly persist until they acquire one or more subsequent genetic “hits” to induce overt leukemia (Mori et al., 2002; Kjeldsen 2016).

ETV6 gene deletions also prevailed among minimal residual disease poor responding cases, ETV6 deletions are as frequent at diagnosis as they are at relapse, and the gene is not expressed in the remaining, non-deleted cases. This suggests that it does not play a major role in the development of drug resistance, while also implicating deletions of NR3C1 as a factor to predict unfavorable prognosis in relapsed ETV6/RUNX1-positive B-ALL cases (Grausenburger et al., 2016). However, regarding the prognostic significance of secondary abnormalities, long-term follow-up of ETV6–RUNX1 ALL in one study indicated that NCI risk, rather than secondary genetic abnormalities, is the key risk factor (Enshaei et al., 2013).

Secondary genetic aberrations

Indeed, more than 80% of initial ETV6/RUNX1-positive ALL patients present secondary genetic alterations. These consist of (partial) deletions of the non-translocated Biological Cytogenetic No. tested Prev % (95% CI) Gene ETV6 gene, an extra copy of RUNX1 or a duplication of process CDKN2A1 location 9p21 208 27.9 (26.3-29.4) the derivative chromosome der(21)t(12;21) in 70%, 23%, CDKN2B2 9p21 161 26.1 (23.7-28.5) G1/S cell cycle and 10% of the cases, respectively (Raynaud et al., 1996; 3 progression CDKN1B 9p21.3 47 31.9 (26.9-36.9) O'Connor et al., 1998). Other frequent abnormalities 4 RB1 13q14.2 94 6.4 (4.5-8.2) include duplication of the normal or derivative 9p13 208 24.5 (21.2-27.8) PAX51 chromosome 21 and gain of chromosome 10. Deletions EBF1 13q14.2 94 10.6 (9.6-11.7) of the long arm of chromosome 6 occur between 10% B-lineage IKZF14 7p13-p11.1 94 1.1 (0.8-1.3) development and 23% (Kempski et al., 1999; Attarbaschi et al., 2004), and proliferation VPREB15 22q11.22 532 38.5 (36.4-40.7) and the short arm of chromosome 9 at around 10% 11p13 94 13.8 (11.8-15.8) RAG24 (Lilljebjörn et al., 2007; Betts et al., 2008; Borst et al., 12q22 208 21.6 (19.8-23.5) BTG11 2012). The majority of secondary aberrations in t(12;21)Glucocorticoid signaling 5q31.3 43 14.0 (12.6-15.3) NR3C16 positive ALLs most likely occur postnatally (Bateman et al., 2010), and abnormalities such as gain of chromosome 15q14 47 8.5 (7.2-9.8) Apoptosis BMF3 12 and/or chromosome 21 are more frequent in first Hematopoiesis ETV61 12p12 341 65.4% (60.0-70.8) relapse than in initial disease (Peter et al., 2009). Genome-wide analysis using SNP arrays is currently Table 1: Prevalence of secondary abnormalities in ETV6-RUNX1-positive ALL cases detected by SNP arrays. 95% CI: 95% Confidence Interval. the most efficient method to identify deletions and Data sources: amplifications of pathogenic genes in areas of copy 1 M ullighan et al., 2007; Enshaei et al., 2013; Öfverholm et al., 2013; Grausenburger number aberrations (CNA), and has become essential in et al., 2016. helping to identify the mechanisms of leukemogenesis. 2 Enshaei et al., 2013; Öfverholm et al., 2013; Grausenburger et al., 2016. One study that utilized SNP arrays in 242 pediatric 3 Öfverholm et al., 2013; Grausenburger et al., 2016. 4 Mullighan et al., 2007; Öfverholm et al., 2013; Grausenburger et al., 2016. ALL patients revealed that deletions, amplifications, 5 Mangum et al., 2013; Öfverholm et al., 2013; Grausenburger et al., 2016. point mutations, and structural rearrangements in 6 Grausenburger et al., 2016. key regulatory genes of B-lymphocyte development and differentiation are present in up to 40% of cases Diagnostic significance of ETV6-RUNX1 fusion (Mullighan et al., 2007). Other studies found multiple (>6) CNAs transcript detection in t(12;21) per case, including recurrent deletions in genes involved in B-cell lineage development and differentiation (PAX5, RAG2, EBF1, Patients are initially classified based on the following metrics: VPREB1), cell-cycle control (CDKN2A, CDKN2B, CDKN1B, age, initial white blood cell count, extramedullary disease status, RB1), but also genes responsible for apoptosis (BMF), hematopoiesis steroid pretreatment, cytogenetic abnormalities and measurement (ETV6) and glucocorticoid signaling (BTG1) (Table 1) (Raynaud of minimal residual disease (MRD) one week into treatment and et al., 1996; Mullighan et al., 2007; Enshaei et al., 2013; Mangum at the end of induction therapy (Baughn et al., 2014). Cytogenetic et al., 2013, Öfverholm et al., 2013; Grausenburger et al., 2016). findings are then used to classify patients into subgroups based Resistance to glucocorticoids (GC), an important component on risk of relapse, currently two subgroups are associated with a of all major childhood ALL treatment protocols, causes ineffective favorable prognosis: the cryptic translocation t(12;21)(p13;q22) blast cell clearance, relapse and treatment failure especially in and the high-hyperdiploid karyotype (51-65 chromosomes) that ETV6/RUNX1-positive patients, and seems to play an important, includes trisomies of chromosomes 4 and 10 (Shurtleff et al., 1995; but less known role in B-ALL. One study found deletions in genes Mullighan, 2012). Recent data suggests a favorable prognosis of associated with GC signaling in more than half of a cohort of ETV6-RUNX1 that exceeds 5-year EFS of more than 90% (Table relapsed ETV6/RUNX1 cases, but only those affecting the gene 2). For ETV6-RUNX1-positive B-ALL, the t(12;21) translocation encoding for the glucocorticoid receptor NR3C1 are associated is one of the best molecular markers to monitor MRD and can with poor response to treatment and disease recurrence. Although be identified by quantitative real-time polymerase chain reaction The Journal of the Association of Genetic Technologists 43 (3) 2017

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Review The t(12;21)(p13;q22) in Pediatric B-Acute Lymphoblastic Leukemia: An Update The t(12;21)(p13;q22) translocation within the context of a complex karyotype

(Q-PCR) or multiplex-nested RT-PCR (Gabert et al., 2003; Taube et al., 2004). Possible prognostic indicators include two copies of the fusion signal (Martineau et al., 2005; Stams et al., 2006), loss of the second ETV6 signal (Attarbaschi et al., 2004; Stams et al., 2006) and loss of the second RUNX1 or extra signal (Martineau et al., 2005), a near-tetraploid modal number (Attarbaschi et al., 2004) and a complex karyotype (Zemanova, 2006).

ETV6/RUNX1 positive ALL is frequently associated with complex karyotypes, which has been suggested to carry a poorer prognosis in other types of leukemia (Raynaud et al., 1999; Jarošová et al., 2003). Jarošová et al. observed in their study that complex karyotypes in ALL patients, in which t(12;21) was a subgroup, was correlated with a lower event free survival (EFS) rate, and that it served as a unfavorable prognostic indicator. However, given

Author, year

Trial/treatment protocol

No. of Patients

Treatment outcome

Median follow-up (yrs)

Kato, 2016

L92-13

10-yr DFS: 93.8 ± 6.1%

16.7

Al-Shehhi, 2013

MRC ALL97

None of the patients relapsed

Bokemeyer, 2013

ALL-REZ BFM

15-yr EFS: 81.0 ± 6.9%

10.1

Olsson, 2013

NOPHO-ALL

230

10-yr EFS: 72.0 ± 10.0%

Enshaei, 2013

MRC ALL97/99

368

8-yr EFS: 85.0 ± 1.8%

9.2

Borst, 2012

NOPHO ALL-92/2000

133

EFS 93.5%

N/A

Bhojwani, 2012

Total XV

5-yr EFS: 96.8 ± 2.4%

N/A

Al-Bahar, 2010

Hospital based

3-yr EFS: 45%

N/A

Moorman, 2010

MRC ALL97/99

368

5-yr EFS: 89.0 ± 1.5%

8.2

Schmiegelow, 2010 Forestier, 2008

NOPHO ALL-92

174

5-yr EFS: 80.0 ± 3.0%

N/A

NOPHO ALL-2000

201

5-yr EFS: 86.0 ± 3.0%

N/A

NOPHO ALL-92

171

5-yr EFS: 80.0 ± 3.0% 10-yr EFS 75.0 ± 4.0% 10-yr OS: 88.0 ± 3.0%

7.75

Rubnitz, 2008

POG ALinC 16

244

5-yr EFS: 86.0 ± 2.0%

Loh, 2006

DFCI 95-01

341

5-yr EFS: 89.0 ± 4.0%

5.2

Seeger, 1998

ALL-REZ BFM 90-96

EFS: 79.0 ± 33%

1.8

McLean, 1996

DFCI ALL Consortium

None of the patients relapsed

8.3

Table 2: Treatment outcome of selected studies of patients with t(12;21) translocation. EFS: Event-free survival; OS: Overall survival; N/A: Not available.

Relapse

their broader range of examined chromosomal abnormalities, it may be possible that this is not the case for t(12;21)-associated leukemia cases (Jarošová et al., 2003). There are conflicting data about the prognostic effect of t(12;21) in the context of a complex karyotype. On the one hand, Jabber Al-Obaidi et al. detected abnormalities of 12p as part of a complex karyotype in a case which relapsed at 10 months, four months after an autologous bone marrow transplant (Jabber Al-Obaidi et al., 2002). Also, in a cohort of 107 t(12;21)-positive children, a complex karyotype indicated a poor prognosis. In the group of children with a noncomplex karyotype (n=69), 10.1% of the cases relapsed while 31.6% of the children with complex karyotype relapsed (n=38), but heterogeneity of treatment protocols may account for these findings (Zemanova et al., 2006). On the other hand, Konn et al. did not support the finding of shorter EFS t(12;21) positive patients with complex karyotype, as many long-term survivors had diagnostic karyotypes as complex as those in relapsed

Although ETV6/RUNX1 positive BCP ALL is generally considered to have an excellent prognosis, reports of frequent late relapses as well as similar incidences of t(12;21) in newly diagnosed and relapsed cases have cast doubts on its favorable prognostic impact (Harbott, 1997; Seeger et al., 1998; Forestier et al., 2008). Forestier et al. found that among 171 cases positive for the ETV6RUNX1 fusion, the EFS decreased after five years, in contrast to other genetic subgroups who maintained similar EFS up to 10 years. They found the most relapses in boys and almost half of the relapses occurred beyond five years from diagnosis. However, they found successful treatment of patients in second remission, as the overall survival after 10 years was 88%. The treatment protocol of this study used a short period of asparaginase therapy, and studies with an extended treatment did not find t(12;21)-positive relapses (Loh et al., 1998; Loh et al., 2006).

The Journal of the Association of Genetic Technologists 43 (3) 2017

102

Review The t(12;21)(p13;q22) in Pediatric B-Acute Lymphoblastic Leukemia: An Update patients. The authors pointed out that the involvement of specific chromosomes, rather than quantity, in complex rearrangements serves as a more accurate prognostic indicator, as they detected no difference in EFS of patients with complex karyotypes compared to those without (Konn et al., 2010). In agreement with these findings, Alvarez et al. reported a patient who, in spite of a complex karyotype, remained in first CR after 81 months (Alvarez et al., 2005). Also, Martinez-Ramirez reported that all six patients with ETV6/RUNX1 fusion achieved complete clinical remission; three cases presented a non-complex karyotype, two cases had single alterations and one had a complex karyotype (Martínez-Ramírez et al., 2001). This could potentially be due to the reduced proliferation capacity that the resultant fusion protein has on B-cell progenitors, which could act against the leukemia itself even in the presence of additional abnormalities (Fischer et al., 2005; Linka et al., 2013). Additionally, because t(12;21) leukemia requires additional secondary abnormalities to progress to an overt form, it is possible that it can exist as a complex karyotype without carrying the poor prognosis. The four-year Disease-free survival (pDFS) of patients treated with the DCOG ALL-7/8/9 protocol that did not present additional genetic changes (pDFS 53±17%) and of those with an extra der(21)t(12;21) (pDFS 60±22%) was poorer than the DFS of patients with other additional genetic changes in ETV6 or RUNX1 (pDFS 79±6%) (Stams et al., 2006).

Methods Data source 363 cases with t(12;21)(p13;q22) were compiled from the Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer (Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer 2015) from a total 57 references.

Statistical Analysis The event-free survival (EFS) probability was calculated using Kaplan–Meier estimates. Relapse times and survival after relapse were calculated only using cases where relapse or mortality occurred, and relapse times were given from the first relapse. Univariate analyses of EFS was performed using the log-rank test. All statistical analyses were performed using IBM SPSS Statistics software version 24.

complex karyotype, while 138 patients (38.3%) had a complex karyotype (n=360). The percentages of male and female cases in both groups constitute around 58% of cases, and females around 42% of cases. More than 58% of the cases with non-complex karyotype were diagnosed between the age 3 to 6, and only 24.2% of cases were older than 6. Among the cases with a complex karyotype, more than 44% were older than 6 at diagnosis (Table 3). The mean count of white blood cells was higher in patients with a non-complex karyotype (26.1*10^9/L, n=109) than in patients with a complex karyotype (17.9*10^9/L, n=61). Non-complex Karyotype (n=222) Nº of cases % of patients

Complex Karyotype (n=138) Nº of cases % of patients

Less than 3

(17.4%)

(8.1%)

Between 3 and 6

(58.3%)

(47.5%)

More than 6

(24.2%)

(44.4%)

Female

(42.7%)

(41.3%)

Male

126

(57.3%)

(58.7%)

Age at diagnosis

Gender

Table 3. Demographics of t(12;21)-positive ALL patients at diagnosis Non-complex Karyotype (n=222)

Complex Karyotype (n=138)

Nº of cases

Mean

Median

Mean

Median

The mean bone marrow blast percentage was 68.95%, and Blood count median was 80.00% for the non-complex karyotype group (n=44), WBC Count (*10^9/L) 109 median26.1 17.9 6.4 and mean 70.24% and 71.50%8.80 for the 61 complex karyotype BM(n=16). Blast (%) Mean peripheral 44 68.9 blast 80.0 percentage 16 70.2 non71.5 blood of the (n -= 4); -no PB complex Blast (%) karyotype4 was 38.50%; 38.5median 21.5 was 21.50% 0 data was available for the complex karyotype (Table 4). t(12;21) Outcome ALL generally has a good prognosis. Event-free survival (EFS) EFS (mo) 107 51.1 38 63 61.5 50 was higher in cases with complex karyotype (mean 61.5, median Relapse (mo) 19 50.2 48 18 37.5 34 50, n=63) than in cases with non-complex karyotype (mean 51.1, Survival after relapse (mo) 11 31.8 25 14 34.5 25 median 38, n=107), but this Karyotype was not (n=222) statistically significant (p=0.28, Non-complex Complex Karyotype (n=138) Nº of cases % of patients Nº of cases of patients log-rank test) (Figure 2). Relapses occurred more %frequently in at diagnosis casesAge with complex karyotype (27%, n=17) than in cases with a Less than 3 28 (17.4%) 8 (8.1%) non-complex karyotype (17.8%, n=19). For the patients with a Between 3 and 6 94 (58.3%) 47 (47.5%) Nº of(24.2%) casesuntil % relapse total patients non-complex the time higher More thanAbnormality 6 karyotype, 39 44 of was (44.4%) (mean 50.21Gender months; median months), than in patients with complex Trisomy 21 4865 17.91% karyotype (mean 37.47, median 34), but the survival time after Female 12p 94 (42.7%) 57 (41.3%) deletions 51 14.05% relapse in both groups Malewas similar 126 (57.3%) (Table 4). 81 (58.7%) 6q deletions

11.29%

Trisomy 10

5.51%

MonosomyNon-complex X 19 Karyotype (n=222) 5.23% del(12)(p13) 19 Nº of cases

Results

Blood count

t(12;21)(p11;q22) translocation cases from the Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer

Complex Karyotype (n=138)

5.23% Nº of cases Median

Mean

Median

6.4

4.13%

WBC Count (*10^9/L) 109 del(12)(p12)

26.1

8.80 3.03% 61

17.9

BM Blast (%)

44 del(12)(p11)

68.9

80.0

70.2

71.5

del(6)(q21)

38.5

21.5

PB Blast (%) Outcome

363 cases were compiled from the Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer (Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer 2015). Not all patients had data available for every examined parameter. 222 cases (61.7%) presented a non-

Monosomy 8

Mean

1.65% 1.65%

EFS (mo)

107

51.1

61.5

Relapse (mo)

50.2

37.5

Survival after relapse (mo)

31.8

34.5

Table 4. Clinical parameters of t(12;21)-positive ALL patients

Abnormality

Nº of cases

% total of patients

Trisomy 21

17.91%

12p deletions 51 The Journal of the Association of Genetic Technologists 43 (3) 2017 6q deletions 41

14.05%

103

11.29%

Trisomy 10

5.51%

Monosomy X

5.23%

del(12)(p13)

5.23%

Blood count WBC Count (*10^9/L) BM Blast (%) PB Blast (%) Outcome EFS (mo) Relapse (mo) Survival after relapse (mo)

Nº of cases

Mean

Median

Nº of cases

Mean

Median

109 44 4

26.1 68.9 38.5

8.80 80.0 21.5

61 16 0

17.9 70.2 -

6.4 71.5 -

19 11

51.1 50.2 31.8

38 48 25

63 18 14

61.5 37.5 34.5

50 34 25

Review107

The t(12;21)(p13;q22) Pediatric ble 4. Clinical parameters of t(12;21)-positivein ALL patients B-Acute Lymphoblastic Leukemia: An Update 100!

Discussion Complex Karyotype (n = 63)!

The cytogenetically cryptic chromosomal abnormality t(12;21) is present in about 25% of all ALL cases of pediatric acute lymphoblastic leukemia. In the present review of the literature, we p = 0.28 ! 80! present a total of 363 cases, 224 with a non-complex karyotype and 139 with a complex karyotype. Based on statistical breakdown of the cases compiled from the Mitelman Database of Chromosome 60! Aberrations and Gene Fusions in Cancer, and in corroboration with the present literature, we conclude that a complex karyotype 40! in t(12;21) ALL does not confer to a poor survival. Although the median relapse time was less in patients with a complex karyotype, the survival after relapse time was similar. Consistent with previous 20! studies (Rubnitz et al., 1997; Hübner et al., 2003), we found that patients with a non-complex karyotype had a peak incidence of Non-complex Karyotype (n=222) Complex Karyotype (n=138) diagnosis at a range from three to six years, but almost half of the Nº of cases % of patients Nº of cases % of patients 0! patients with a complex karyotype were diagnosed at an age older Age at diagnosis than six years. As the fusion protein ETV6/RUNX1 is considered a 0! 28 50! 200! 100! Less than 3 (17.4%) 8 150! (8.1%) weak leukemogenic factor which requires additional abnormalities Between 3 and 6 94 (58.3%) 47 (47.5%) Months from diagnosis! More than 6 39 (24.2%) 44 (44.4%) to produce leukemia, it seems plausible that it takes more time to Figure 2. Event-free survival (EFS) of t(12;21)-positive patients with acquire the high number of secondary abnormalities that define a Gender non-complex or complex karyotypes. A) Kaplan-Meier curve showing complex karyotype at the point of diagnosis. 94 of t(12;21)-positive (42.7%) 57 (41.3%) gure 2. Event-freeFemale survival (EFS) patients with non-complex or complex Event free survival (%)!

Non-complex Karyotype (n = 107)!

no significant difference in EFS in t(12;21)-positive patients with non-

Male complex (57.3%) 81 log-rankintest). (58.7%) ryotypes. A) Kaplan-Meier curve showingkaryotype no significant difference EFS in t(12;21)-positive or126 complex (p=0.28, Frequently Found Secondary Aberrations tients with non-complex or complex karyotype (p=0.28, log-rank test).

Cytogenetically, most cases presented secondary abnormalities, but less than 40% presented a complex karyotype which is usually Nº of cases Mean Median Nº of cases Mean Median correlated with a poor prognosis (Mrózek, 2008). Consistent with Most cases hadabnormalities secondary abnormalities (n=296), about of which Most cases (82.2%) had(82.2%) secondary (n=296), about half (46.6%) Blood count previous observations, we found trisomy 21 in roughly 18%, and half of (46.6%) of karyotypes which were(n=138), in the context of three complex karyotypes ere in the context complex defined as or more structural or trisomy 10 in 6% of the cases, (Paulsson et al., 2013; Lundin et WBC Count (*10^9/L) 109 26.1 8.80 61 17.9 6.4 merical chromosomal abnormalities. Twenty cases (5.51%) had trisomy 10, and sixty-five (n=138), defined as three or more structural or numerical al., 2014). Additional secondary abnormalities include deletions of BM Blast (%) 44 68.9 80.0 16 70.2 71.5 7.91%) had trisomy 21 as a secondary abnormality. Additional recurring abnormalities included chromosomal abnormalities. Twenty cases (5.51%) had trisomy 10, chromosome 12 in almost 17%, and deletions in chromosome 6 in PB Blast (%) 4 38.5 21.5 0 letions of 12pand in fifty-one (14.05%), and deletions 6q in forty-one cases (11.29%). Of sixty-fivecases (17.91%) had trisomy 21 as aofsecondary abnormality. almost 13% of the cases. ose, recurrentAdditional secondary abnormalities were del(12)(p13) in 5.23%, del(12)(p12) in 3.03% and Outcome recurring abnormalities included deletions of 12p in EFS (mo) cases (14.05%), 107 51.1 38 of 6q63in forty-one 61.5 cases 50 fifty-one and deletions Trisomy 21 (11.29%). secondary were Relapse (mo) Of those, recurrent 19 50.2 48abnormalities 18 37.5del(12) 34 Gain of chromosome 21 is a nonrandom chromosomal (p13) 5.23%, del(12)(p12) in 31.8 3.03% 25 and del(6)(q21) in34.5 1.65%25 of Survivalin after relapse (mo) 11 14 abnormality frequently observed in acute lymphoblastic leukemia cases (Table 5). (ALL), occurring in approximately 23% of childhood and adult ALLs with chromosomal abnormalities (Berger, 1997). As RUNX1 is located on chromosome 21, its overexpression in trisomy 21 Abnormality Nº of cases % total of patients might contribute to hematological abnormalities associated with Trisomy 21 65 17.91% these individuals (Speck and Gilliland, 2002). It is particularly 12p deletions 51 14.05% frequent among cases with hyperdiploid and tri- and tetraploid 6q deletions 41 11.29% ALL, where gain of chromosome 21 was most common (Forestier Trisomy 10 20 5.51% et al., 2000). In one study, gain of chromosome 21 was found in all Monosomy X 19 5.23% cases with hyperploid pre-B childhood ALL (Paulsson et al., 2005). del(12)(p13) 19 5.23% +21 is a common secondary change in t(12;21)(p13;q22) occurring Monosomy 8 15 4.13% in approximately 16% of the cases (Loncarevic et al., 1999; del(12)(p12) 11 3.03% Raynaud et al., 1999; Attarbaschi et al., 2004). However, there are no significant differences between ETV6-RUNX1-positive patients del(12)(p11) 6 1.65% with and without a trisomy 21 with respect to the presenting del(6)(q21) 6 1.65% features and treatment outcome (Attarbaschi et al., 2004), and there was no significant difference in EFS between trisomy 21 Table 5. Most frequent chromosome alterations found in t(12;21)patients with and without ETV6/RUNX1-fusion (Karrman et al., positive B-ALL patients (n=363). 2006).

econdary Abnormalities Non-complex Karyotype (n=222) Secondary Abnormalities

Complex Karyotype (n=138)

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Review The t(12;21)(p13;q22) in Pediatric B-Acute Lymphoblastic Leukemia: An Update Trisomy 10

cases (Al-Sheihi et al., 2010). The resultant protein fuses the entire RUNX1 protein to the C-terminal of ETV6, resulting in conservation of the oligomerization domain of ETV6 at the N-terminal that has been shown to actively recruit corepressors and histone modifying proteins to target genes (Fischer et al., 2005). Functionally, ETV6-RUNX1 is hypothesized to deregulate hemapoietic differentiation of the B lymphoid lineage, and additionally acts to sequester the normal copy of ETV6 (Fischer et al., 2005). As a leukemic factor, the resultant fusion protein is noted to have a very weak effect of leukemogenesis (Fischer et al., 2005), and requires additional abnormalities in order to effect leukemia (Stanchescu et al., 2009). Studies support this claim by noting that t(12;21) can arise in utero without progression to overt leukemia, and that knock-in mouse models with the fusion protein did not all progress to leukemia (Borst et al., 2012). Interestingly, Fischer et al. noted that while the fusion protein has been shown to slow differentiation of B-cell progenitors into mature B-cells, it does not completely inhibit differentiation; additionally, it also has a repressive effect on proliferation of those progenitor cells (Fischer et al., 2005). Linka et al. similarly noted that ETV6-RUNX1 downregulated genes involved in proliferation (Linka et al., 2014), and genome-wide SNP array analysis revealed that ETV6-RUNX1positive patients show recurrent deletions of genes involved in B-cell lineage development, cell-cycle control, glucocorticoid signaling, apoptosis and hematopoiesis (Mullighan et al., 2007; Mangum et al., 2013; Öfverholm et al., 2013; Grausenburger et al., 2016). However, Torrano et al. noted that the ETV6-RUNX1 fusion protein also causes overexpression of erythropoietin receptor (EPOR) and promotion of B-cell progenitor survival via the downstream JAK-STAT signaling (Torrano et al., 2011).

Trisomy 10 has been found in up to 63% of high-hyperdiploid B-ALL cases (Heerema et al., 2000), and up to 11% of patients with t(12;21)-positive ALL (Martineau et al., 2005). Trisomy 10 has been identified as a cytogenetic predictor of improved outcome in children with high-hyperpolidy ALL. Patients with a trisomy of chromosome 10 had significantly improved outcome compared with their counterparts who lacked the given trisomy (Heerema et al., 2000).

Deletions on chromosome 12 12p deletions are associated with a loss of the second copy of ETV6, and have been related to a poor prognosis in childhood AML (Hirsch et al., 2013). ETV6 might thus be hypothesized to function as a tumor suppressor, which is supported by the fact that it recruits histone deacetylases and corepressors to target genes in order to repress transcription of target genes (Coniat et al., 1999; Fischer et al., 2005). While some studies correlated the loss of the second ETV6 with a favorable prognosis (Stams et al., 2006; Konn et al., 2010), others found a worse prognosis (Attarbaschi et al., 2004), or no correlation at all (Alvarez et al., 2005; Kawamata et al., 2008).

Deletions on chromosome 6 Chromosome 6 and 9 abnormalities are a common secondary event in t(12;21) ALL, occurring in 10% of cases (Borst et al., 2012). Specifically, Borst et al. noted that deletions of the long arm of chromosome 6 and the short arm of chromosome 9 occurred with 13% and 10% frequency, respectively (Borst et al., 2012). Deletion 6q has also been observed in a broader range of leukemias and neoplasms in both adult and pediatric cases, and has been suggested to contain a tumor suppressor gene in the 6q16-23 region (Betts et al., 2008). In addition to deletions, Betts et al. noted that unbalanced rearrangements involving chromosome 6 occur as secondary events in t(12;21) leukemia, and hypothesize that deletions of 6q material can occur due to either internal deletions proper or unbalanced translocations; however, they did not attribute any prognostic significance to these abnormalities, as their molecular functions remain unknown (Betts et al., 2008).

Prognosis Although the overall prognosis for patients with t(12;21) (p13;q22)-positive childhood acute lymphoblastic leukemia is good, cases with a complex karyotype are defined as unfavorable. Jarošová et al. noted in their study that complex karyotypes in ALL patients, among which t(12;21) was a subgroup, was correlated with a lower event free survival (EFS), and that it served as a poor prognostic indicator (Jarošová et al., 2003). However, given their broader range of examined chromosomal abnormalities, it may be possible that this is not the case for t(12;21) leukemias, as Konn et al. pointed out that the involvement of specific chromosomes, rather than quantity, in complex rearrangements serves as a more accurate prognostic indicator, as they detected no difference in EFS of patients with complex karyotypes compared to those without (Konn et al., 2010). In our report presented here, we found that cases with a complex karyotype had shorter time of relapse but a similar overall survival compared to cases with a non-complex karyotype. This could potentially be due to the reduced proliferative effect of the resultant fusion protein on B-cell progenitors, which could act against the leukemia itself, even in the presence of additional abnormalities (Fischer et al., 2005). Additionally, because t(12;21) leukemia requires additional secondary abnormalities to progress to an overt form, it is possible that it can exist as a complex karyotype without carrying the poor prognosis.

Techniques used to detect the t(12;21)(p13;q22) As t(12;21) is a cytogenetically cryptic translocation, it cannot be detected by traditional karyotyping. Most studies detected the ETV6/RUNX1 fusion genes by RT-PCR and/or commercially available extra signal (ES) dual color translocation probes.

Specific functions of fusion protein in leukemogenesis The t(12;21)(p13;q22) results in fusion of the ETV6 gene on chromosome 12 to RUNX1 on chromosome 21, and is the most common cytogenetic abnormality in pediatric B-ALL (DouetGuilbert et al., 2003). The fusion results in ETV6-RUNX1 on the derivative chromosome 21, and the reciprocal RUNX1ETV6 expressed concomitantly on chromosome 12 in 70% of

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Bateman CM, Colman SM, Chaplin T, Young BD, Eden TO, Bhakta M, Gratias EJ, van Wering ER, Cazzaniga G, Harrison CJ, Hain R, Ancliff P, Ford AM, Kearney L, Greaves M. Acquisition of genome-wide copy number alterations in monozygotic twins with acute lymphoblastic leukemia. Blood. 2010;115(17): 3553–3558. Baughn LB, Biegel JA, South ST, Smolarek TA, Volkert S, Carroll AJ, Heerema NA, Rabin KR, Zweidler-McKay PA, Loh M, Hirsch B. Integration of cytogenomic data for furthering the characterization of pediatric B-ALL: a multi-institution, multi-platform microarray study. Cancer Genet. 2014; 1–40. Berger R. Acute lymphoblastic leukemia and chromosome 21. Cancer Genet Cytogenet. 1997;94(1): 8–12. Betts DR, Stanchescu R, Niggli FK, Cohen N, Rechavi G, Amariglio N, Trakhtenbrot L. SKY reveals a high frequency of unbalanced translocations involving chromosome 6 in t(12;21)-positive acute lymphoblastic leukemia. Leuk Res. 2008;32(1): 39–43. Bhojwani D, Pei D, Sandlund JT, Jeha S, Ribeiro RC, Rubnitz JE, Shurtlieff S et al. ETV6-RUNX1-positive childhood acute lymphoblastic leukemia: improved outcome with contemporary therapy. Leukemia. 2012;26(2): 265–270. Boccuni P, MacGrogan D, Scandura JM, Nimer SD. The Human L(3) MBT Polycomb Group Protein Is a Transcriptional Repressor and Interacts Physically and Functionally with TEL (ETV6). J Biol Chem. 2003;278(17): 15412–15420. Bokemeyer A, Eckert C, Meyr F, Koerner G, voan Stackelberg A, Ullmann R, Turkmen S, Henze G, Seeger K. Copy number genome alterations are associated with treatment response and outcome in relapsed childhood ETV6/RUNX1-positive acute lymphoblastic leukemia. Haematologica. 2014;99(4): 706–714. Borst L, Wesolowska A, Joshi T, Borups R, Nielsen FC, Andersen MK, Jonsson OG, Wehner PS, Wesenberg F, Frost BM, Gupta R, Schmiegelow K. Genome-wide analysis of cytogenetic aberrations in ETV6/RUNX1-positive childhood acute lymphoblastic leukaemia. Br J Haematol. 2012;157(4): 476–482. Chakrabarti SR, Nucifora G. The leukemia-associated gene TEL encodes a transcription repressor which associates with SMRT and mSin3A. Biochem Biophys Res Comm. 1999;264(3): 871–877. Coniat MB, Poirel H, Leblanc T, Bernard OA, Berger R. Loss of the TEL/ ETV6 gene by a second translocation in ALL patients with t(12;21). Leuk Res. 1999;23(10): 895–899. De Braekeleer E, Férec C, De Braekeleer M. RUNX1 translocations in malignant hemopathies. Anticancer Res. 2009;29(4): 1031–1037. Diakos C, Zhong S, Xiao Y, Zhou M, Vasconcelos GM, Krapf G, Yeh RF, Zheng S, Kang M, Wiencke JK, Pombo-de-Oliveeira MS, PanzerGrumayer R, Wiemels JL. TEL-AML1 regulation of survivin and apoptosis via miRNA-494 and miRNA-320a. Blood. 2010;116(23): 4885–4893. Douet-Guilbert N, Morel F, Le Bris MJ, Herry A, Le Calvez G, Marlon V, Abgrall JF, Berthou C, De Braekeleer M. A f luorescence in situ hybridization study of TEL-AML1 fusion gene in B-cell acute lymphoblastic leukemia (1984–2001). Cancer Genet Cytogenet. 2003;144(2): 143–147. Enshaei A, Schwab CJ, Konn ZJ, Mitchell CD, Kinsey SE, Wade R, Vora A, Harrison CJ, Moorman AV. Long-term follow-up of ETV6– RUNX1 ALL reveals that NCI risk, rather than secondary genetic abnormalities, is the key risk factor. Leukemia. 2013;27(11): 2256–2259. Fenrick R, Amann JM, Lutterbach B, Wang L, Westerndorf JJ, Downing JR, Hiebert SW. Both TEL and AML-1 contribute repression domains to the t(12;21) fusion protein. Mol Cell Biol. 1999;19(10): 6566–6574. Fenrick R, Wang L, Nip J, Amann JM, Rooney RJ, Walker-Daniels J, Crawford HC, Hulboy DL, Kinch MS, Matrisian LM, Hiebert SW. TEL, a putative tumor suppressor, modulates cell growth and cell morphology of ras-transformed cells while repressing the transcription of stromelysin-1. Mol Cell Biol. 2000;20(16): 5828–5839.

In summary, we provide here a review of the literature concerning t(12;21)(p13;q22), a nonrandom rearrangement occurring in about 25% of case of B-cell lineage childhood acute lymphoblastic leukemia. t(12;21) results in the expression of the ETV6/RUNX1 fusion transcript and ETV6/RUNX1 fusion protein, which leads to expansion of B-cell precursors with enhanced selfrenewal capacity and impaired differentiation to more mature B-cell stages. The translocation already occurs in utero but for itself is insufficient to generate an overt leukemia. Additional secondary aberrations necessary for disease development have been identified to include deletions of the non-translocated ETV6 gene and other key regulators of pathways that are important for B-cell lineage development and differentiation, cell-cycle control and hematopoiesis. Furthermore, expression of ETV6/RUNX1 causes expression differences for genes involved in differentiation, apoptosis and immune response. About one third of our cases derived from the Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer database carried a complex karyotype, defined as three or more chromosomal abnormalities; about 18% of cases had trisomy 21, and around 5% had trisomy 10 as secondary abnormality. Deletions of chromosome 12 and chromosome 6 were most frequent. We found that t(12;21) in the context of both non-complex and complex karyotype carries a good prognosis; mean survival times in our review were 31.82 and 34.54 months for non-complex and complex karyotypes, respectively. Further study into the role of secondary aberrations in t(12;21)-positive B-ALL could contribute to a more accurate diagnosis and risk stratification which may lead to a more personalized treatment in t(12;21) B-ALL.

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Review The t(12;21)(p13;q22) in Pediatric B-Acute Lymphoblastic Leukemia: An Update Martínez-Ramírez A, Urioste M, Contra T, Cantalejo A, Tavares A, Portero JA, Lopez-Ibor B, Bernacer M, Soto C, Cigudosa JC, Benitez J. et al. Fluorescence in situ hybridization study of TEL/AML1 fusion and other abnormalities involving TEL and AML1 genes. Correlation with cytogenetic findings and prognostic value in children with acute lymphocytic leukemia. Haematologica. 2001;86(12): 1245–1253. McLean TW, Ringold S, Neuberg D, Stegmaier K, Tantravahi R, Ritz J, Koeffler HP, Takeuchi S, Janssen JW, Seriu T, Bartram CR, Sallan SE, Gilliland DG, Golub TR. TEL/AML-1 dimerizes and is associated with a favorable outcome in childhood acute lymphoblastic leukemia. Blood. 1996;88(11): 4252–4258. Moorman AV, Ensor HM, Richards SM, Chilton L, Schwab C, Kinsey SE, Vora A, Mitchell CD, Harrison CJ. Prognostic effect of chromosomal abnormalities in childhood B-cell precursor acute lymphoblastic leukaemia: results from the UK Medical Research Council ALL97/99 randomised trial. Lancet Oncol. 2010;11(5): 429–438. Mori H, Colman SM, Xiao Z, Ford AM, Healy LE, Donaldson C, Hows JM, Navarrete C, Greaves M. Chromosome translocations and covert leukemic clones are generated during normal fetal development. Proc Natl Acad Sci U S A. 2002;99(12): 8242–8247. Mrózek K. Cytogenetic, molecular genetic, and clinical characteristics of acute myeloid leukemia with a complex karyotype. Semin Oncol. 2008;35(4): 365–377. Mullighan CG. Molecular genetics of B-precursor acute lymphoblastic leukemia. J Clin Invest. 2012;122(10): 3407–3415. Mullighan CG, Goorha S, Radtke L, Miller CB, Coustan-Smith E, Dalton JD, Girtman K, Mathew S, Ma J, Pounds SB, Su X, Pui CH, Relling MV, Evans WE, Shurtleff SA, Downing JR. Genome-wide analysis of genetic alterations in acute lymphoblastic leukaemia. Nature. 2007;446(7137): 758–764. Noetzli L, Lo RW, Lee-Sherick AB, Callaghan M, Noris P, Savoia A et al. Germline mutations in ETV6 are associated with thrombocytopenia, red cell macrocytosis and predisposition to lymphoblastic leukemia. Nat Genet. 2015;47(5): 535–538. Nordentoft L, Jorgensen P. The acetyltransferase 60 kDa trans-acting regulatory protein of HIV type 1-interacting protein (Tip60) interacts with the translocation E26 transforming-specific leukaemia gene (TEL) and functions as a transcriptional co-repressor. Biochem J. 2003;374(Pt 1): 165–173. O'Connor HE, Butler TA, Clark R, Swanton S, Harrison CJ, Secker-Walker LM, Foroni L. Abnormalities of the ETV6 gene occur in the majority of patients with aberrations of the short arm of chromosome 12: a combined PCR and Southern blotting analysis. Leukemia. 1998;12(7): 1099–1106. Olsson L, Castor A, Behrendtz M, Giloglav A, Forestier E, Paulsson K, Johansson B. Deletions of IKZF1 and SPRED1 are associated with poor prognosis in a population-based series of pediatric B-cell precursor acute lymphoblastic leukemia diagnosed between 1992 and 2011. Leukemia. 2013;28(2): 302–310. Öfverholm I, Tran AN, Heyman M, Zachariadis V, Nordenskjold M, Nordgren A, Barbany G. Impact of IKZF1 deletions and PAX5 amplifications in pediatric B-cell precursor ALL treated according to NOPHO protocols. Leukemia. 2013;27(9): 1936–1939. Paulsson K, Morse H, Fioretos T, Behrendtz M, Strombeck B, Johansson B. Evidence for a single-step mechanism in the origin of hyperdiploid childhood acute lymphoblastic leukemia. Genes Chromosomes Cancer. 2005;44(2): 113–122. Paulsson K, Forestier E, Andersen MK, Autio K, Barbany G, Borgstrom G, Cavelier L, Golovleva L, Heim S, Heinonen K, Hovland R, Johansson JK, Kjeldsen E et al. High modal number and triple trisomies are highly correlated favorable factors in childhood B-cell precursor high hyperdiploid acute lymphoblastic leukemia treated according to the NOPHO ALL 1992/2000 protocols. Haematologica. 2013;98(9): 1424– 1432.

Kitabayashi I, Aikawa Y, Hguyen LA, Yokoyama A, Ohki M. Activation of AML1-mediated transcription by MOZ and inhibition by the MOZCBP fusion protein. EMBO J. 2001;20(24): 7184–7196. Kitabayashi I, Yokoyama A, Shimizu K, Ohki M. et al. Interaction and functional cooperation of the leukemia-associated factors AML1 and p300 in myeloid cell differentiation. EMBO J. 1998;17(11): 2994–3004. Kjeldsen E. Data on affected cancer-related genes in pediatric t(12;21)positive acute lymphoblastic leukemia patients harboring unbalanced der(6)t(X;6) translocations. Data Brief. 2016;8: 894–903. Konn ZJ, Martineau M, Brown N, RIchards S, Swansbury J, Talley P, Wright SL, Harrison CJ. Cytogenetics of long-term survivors of ETV6-RUNX1 fusion positive acute lymphoblastic leukemia. Genes Chromosomes Cancer. 2010;49(3): 253–259. Lausten-Thomsen U, Madsen HO, Vestergaard TR, Hjalgrim H, Hestering J, Schmiegelow K. Prevalence of t(12;21)[ETV6-RUNX1]-positive cells in healthy neonates. Blood. 2011;117(1): 186–189. Levanon D, Goldstein RE, Bernstein Y, Tang H, Goldenberg D, Stifani S, Paroush Z, Groner Y. Transcriptional repression by AML1 and LEF-1 is mediated by the TLE/Groucho corepressors. Proc Natl Acad Sci U S A. 1998;95(20): 11590–11595. Lilljebjörn H, Heidenblad M, Nilsson B, Lassen C, Horvat A, Heldrup J, Behrendtz M, Johansson B, Andersson A, Fioretos T. Combined high-resolution array-based comparative genomic hybridization and expression profiling of ETV6/RUNX1-positive acute lymphoblastic leukemias reveal a high incidence of cryptic Xq duplications and identify several putative target genes within the commonly gained region. Leukemia. 2007;21(10): 2137–2144. Linka Y, Ginzel S, Borkhardt A, Landgraf P. Identification of TEL-AML1 (ETV6-RUNX1) associated DNA and its impact on mRNA and protein output using ChIP, mRNA expression arrays and SILAC. Genom Data. 2014;2: 85–88. Linka Y, Ginzel S, Kruger M, Novosel A, Gombert M, Kremmer E, Harbott J, Thiele R, Borkhardt A, Landgraf P. The impact of TEL-AML1 (ETV6-RUNX1) expression in precursor B cells and implications for leukaemia using three different genome-wide screening methods. Blood Cancer J. 2013;3(10): e151. Loh ML, Silverman LB, Young ML, Neuberg D, Golub TR, Sallan SE, GIlliland DG. Incidence of TEL/AML1 fusion in children with relapsed acute lymphoblastic leukemia. Blood. 1998;92(12): 4792–4797. Loh ML, Goldwasser MA, Silverman LB, Poon WM, Vattikuti S, Cardoso A, Neuberg DS, Shannon KM, Sallan SE, Gilliland DG. Prospective analysis of TEL/AML1-positive patients treated on Dana-Farber Cancer Institute Consortium Protocol 95-01. Blood. 2006;107(11): 4508–4513. Loncarevic IF, Roitzheim B, Ritterbach J, Viehmann S, Borkhardt A, Lampert F, Harbott J. Trisomy 21 is a recurrent secondary aberration in childhood acute lymphoblastic leukemia with TEL/AML1 gene fusion. Genes Chromosomes Cancer. 1999;24(3): 272–277. Lundin C, Forestier E, Klarskov Andersen M, Autio K, Barbany G, Cavelier L, Golovleva I, Heim S, Heinonen K, Hovland R, Johannsson JH, Kjeldsen E, Nordgren A, Palmqvist L, Johansson B; Nordic Society of Pediatric Hematology Oncology (NOPHO); Swedish Cytogenetic Leukemia Study Group (SCLSG); NOPHO Leukemia Cytogenetic Study Group (NLCSG). Clinical and genetic features of pediatric acute lymphoblastic leukemia in Down syndrome in the Nordic countries. J Hematol Onco. 1999;7: 32. Mangan JK, Speck NA. RUNX1 mutations in clonal myeloid disorders: from conventional cytogenetics to next generation sequencing, a story 40 years in the making. Crit Rev Oncog. 1999;16(1-2): 77–91. Mangum DS, Downir J, Mason CC, Jahromi MS, Joshi D, Rodic V, et al. VPREB1 deletions occur independent of lambda light chain rearrangement in childhood acute lymphoblastic leukemia. Leukemia. 2013;28(1): 216–220. Martineau M, Jalali GR, Barber KE, Broadfield ZJ, Cheung KL, Lilleyman J, Moorman AV, Richards S, Robinson HM, Ross F, Harrison CJ. ETV6/ RUNX1 fusion at diagnosis and relapse: Some prognostic indications. Genes Chromosomes Cancer. 2005;43(1): 54–71.

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Review The t(12;21)(p13;q22) in Pediatric B-Acute Lymphoblastic Leukemia: An Update Taube T, Eckert C, Korner G, Henze G, Seeger K. Real-time quantification of TEL–AML1 fusion transcripts for MRD detection in relapsed childhood acute lymphoblastic leukaemia. Leuk Res. 2004;28(7): 699–706. Torrano V, Procter J, Cardus P, Greaves M, Ford AM. ETV6-RUNX1 promotes survival of early B lineage progenitor cells via a dysregulated erythropoietin receptor. Blood. 2011;118(18): 4910–4918. Uchida H, Downing JR, Miyazaki Y, Frank R, Zhang J, Nimer SD. Three distinct domains in TEL-AML1 are required for transcriptional repression of the IL-3 promoter. Oncogene. 1999;18(4): 1015–1022. Wang L, Hiebert SW. TEL contacts multiple co-repressors and specifically associates with histone deacetylase-3. Oncogene. 2001;20(28): 3716– 3725. Wang LC, Kuo F, Fujiwara Y, Gilliland DG, Golub TR, Orkin SH. Yolk sac angiogenic defect and intra-embryonic apoptosis in mice lacking the Ets-related factor TEL. EMBO J. 1997;16(14): 4374–4383. Yehuda-Gafni O, Cividalli G, Abrahmov A, Weintrob M, Neriah SB, Cohen R, Abeliovich D. Fluorescence in situ hybridization analysis of the cryptic t(12;21) (p13;q22) in childhood B-lineage acute lymphoblastic leukemia. Cancer Genet Cytogenet. 2002;132(1): 61–64. Yu M, Mazor T, Huang HT, Kathrein KL, Woo AJ et al. Direct Recruitment of Polycomb Repressive Complex 1 to Chromatin by Core Binding Transcription Factors. Mol Cell. 2012;45(3): 330–343. Zemanova Z, Michalova K, Babicka L, Pavlistova L, Jarosova M, Holzerova M et al. Clinical relevance of complex chromosomal aberrations in bone marrow cells of 107 children with ETV6/RUNX1 positive acute lymphoblastic leukemia (ALL). Blood. 2006;108(11): 2278. Zhao X, Jankovic V, Gural A, Huang G, Pardanani A, Menendez S et al. Methylation of RUNX1 by PRMT1 abrogates SIN3A binding and potentiates its transcriptional activity. Genes Dev. 2008;22(5): 640–653.

Peter A, Heiden T, Tuabe T, Korner G, Seeger K. Interphase FISH on TEL/ AML1positive acute lymphoblastic leukemia relapses - analysis of clinical relevance of additional TELand AML1copy number changes. Eur J Haematol. 2009;83(5): 420–432. Pui CH. Acute Lymphoblastic Leukemia: Introduction. Semin Hematol. 2009;46(1): 1–2. Raynaud S, Cave H, Baens M, Bastard C, Cacheux V, Grosgeorge J, Guidal-Giroux C, Guo C, Vilmer E, Marynen P, Grandchamp B. The 12;21 translocation involving TEL and deletion of the other TEL allele: two frequently associated alterations found in childhood acute lymphoblastic leukemia. Blood. 1996;87(7): 2891–2899. Raynaud SD, Dastsugue N, Zoccola D, Shurtleff SA, Mathew S, Raimondi SC. Cytogenetic abnormalities associated with the t(12;21): a collaborative study of 169 children with t(12;21)-positive acute lymphoblastic leukemia. Leukemia. 1999;13(9): 1325–1330. Romana SP, Le Coniat M, Poirel H, Marynen P, Bernard O, Berger R. Deletion of the short arm of chromosome 12 is a secondary event in acute lymphoblastic leukemia with t(12;21). Leukemia. 1996;10(1): 167–170. Romana SP, Le Coniat M, Poirel H, Marynen P, Bernard O, Berger R. Deletion of the short arm of chromosome 12 is a secondary event in acute lymphoblastic leukemia with t(12;21). Leukemia. 1996;10(1): 167–170. Ross ME, Zhou X, Son gG, Shurtleff SA, Girtman K, Williams WK, Liu HC, Mahfouz R, Raimondi SC, Lenny N, Patel A, Downing JR. Classification of pediatric acute lymphoblastic leukemia by gene expression profiling. Blood. 2003;102(8): 2951–2959. Rubnitz JE, Downing JR, Pui CH, Shurtleff SA, Raimondi SC, Evans WE, Head DR, Crist WM, Rivera GK, Hancock ML, Boyett JM, Buijs A, Grosveld G, Behm FG. TEL gene rearrangement in acute lymphoblastic leukemia: a new genetic marker with prognostic significance. J Clin Oncol. 1997;15(3): 1150–1157. Schindler JW, Van BUren D, Foudi A, Krejci O, Qin J, Orkin SH, Hock H. TEL-AML1 Corrupts Hematopoietic Stem Cells to Persist in the Bone Marrow and Initiate Leukemia. Cell Stem Cell. 2009;5(1): 43–53. Schmiegelow K, FOrestier E, Hellebostad M, Heyman M, Kristinsson J, Soderhall S, Taskinen M; Nordic Society of Paediatric Haematology and Oncology. Long-term results of NOPHO ALL-92 and ALL2000 studies of childhood acute lymphoblastic leukemia. Leukemia. 2010;24(2): 345–354. Seeger K, Adams HP, Buchwald D, Beyermann B, Kremens B, Niemeyer C, Ritter J, Schwabe D, Harms D, Schrappe M, Henze G. TEL-AML1 fusion transcript in relapsed childhood acute lymphoblastic leukemia. The Berlin-Frankfurt-Münster Study Group. Blood. 1998;91(5): 1716– 1722. Shurtleff SA, Buijs A, Behm FG, Rubnitz JE, Raimondi SC, Hancock ML, Chan GC, Pui CH, Grosveld G, Downing JR. TEL/AML1 fusion resulting from a cryptic t(12;21) is the most common genetic lesion in pediatric ALL and defines a subgroup of patients with an excellent prognosis. Leukemia. 1995;9(12): 1985–1989. Speck NA, Gilliland DG. Core-binding factors in haematopoiesis and leukaemia. Nat Rev Cancer. 2002;2(7): 502–513. Stams WAG, Beverloo HB, den Boer ML, de Menezes RX, Stigter RL, van Drunen E, Ramakers-van-Woerden NL, Loonen AH, van Wering ER, Janka-Schaub GE, Pieters R. Incidence of additional genetic changes in the TEL and AML1 genes in DCOG and COALL-treated t(12;21)positive pediatric ALL, and their relation with drug sensitivity and clinical outcome. Leukemia. 2006;20(3): 410–416. Stanchescu R, Betts DR, Rechavi G, Amariglio N, Trakhtenbrot L. Involvement of der(12)t(12;21)(p13;q22) and as well as additional rearrangements of chromosome 12 homolog in ETV6/RUNX1-positive acute lymphoblastic leukemia. Cancer Genet Cytogenet. 2009;190(1): 26–32.

Corresponding author: Carlos A. Tirado, Ph.D. Carlos.Tirado@allina.com tirad017@umn.edu

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Review

The AGT Cytogenetics Laboratory Manual 4th Edition. Eds. Marilyn S. Arsham, Margaret J. Barch, Helen J. Lawce John Wiley & Sons, Hoboken, NJ, 2017. Hardcover. 1,218 Pages. $187.99 U.S. ISBN: 9781119061175 The AGT Cytogenetics Laboratory Manual is an insightful read essential for all cytogenetic and molecular professionals. As a reference text, the AGT manual covers the history and basics of cytogenetics necessary for the practice of clinical diagnostics and research. The manual addresses cytogenetic practices related to cell culture, human chromosome identification, chromosome abnormalities, computerized and microscopy-based imaging, laboratory safety and compliance, quality, management, laboratory information systems, genetic resources in the context of everyday laboratory practice, and emerging developments in the field of cytogenomics. The manual presents basic cytogenetic concepts starting with cell biology and a historical perspective of the field while building the reader’s knowledge in a logical progression through twenty-five chapters. The manual is easy to read, contains numerous protocols, color photographs and illustrations filled with invaluable references. Chapters on cytogenetic-specific methods for constitutional abnormalities, chromosome instabilit y sy ndromes, hematologic malignancies, solid tumors, Fluorescence In Situ Hybridization (FISH), multicolor FISH (SKY and M-FISH) and CGH, and genomic microarray technologies are must-reads for new learners of cytogenomics. The laboratory management chapter (22) provides a brief introduction including conceptions and functions, personnel management, quality management and control, and budget development. The system approach to quality chapter (21) addresses process m a n a gement , d o c u ment at io n a nd records, and continual improvement and monitoring. The Laboratory information system chapter (23) includes an extensive review of various cytogenetic LIS, software architecture and hardware considerations, and trends for the future. The Animal cytogenetics chapter (24) is expansive and includes many species, including a discussion of investigations in amphibians, the first animal class studied by Walther Flemming in 1879! The AGT manual is a practical reference for everyday laboratory investigations. Laboratories, educators, and students will be well equipped with the information necessary to explore the field. The manual is a fundamental text for molecular and cytogenetic laboratory professionals or as a text for technical training curriculums. This book has and continues to be a foundational text for clinical cytogenetics and genomics fellowship programs.

Many chapters can support cytogenetic technology and molecular biology diagnostic training program curricula including “Chapter 7: Human chromosomes: identification and variations,” “Chapter 8: ISCN: the universal language of cytogenetics,” and “Chapter 18: Genomic microarray technologies for the cytogenetics laboratory.” The chapter on human chromosomes is simply amazing. It is detailed and concise with ISCN information and illustrations to assist the novice in understanding the fundamental concepts and terminology related to banding techniques. “Chapter 9: Constitutional chromosome abnormalities,” includes a table of the more common recognized abnormalities by chromosome, abnormality, and phenotypic characteristics common at presentation; “Figure 9.1 Mapping recurrent constitutional gains and losses” provides ISCN ideograms at the 850-band level for quick reference. “Chapter 11: Cytogenetic analysis of hematologic malignant disease” includes a table of common recurring aberrations with frequencies, prognoses, and variants; “Figure 11.2 Hematological rea r ra ngement s (G-ba ndi ng)” is a wonderful visual resource. “Chapter 12: Cytogenetic methods and findings in human solid tumors” includes a table of recurrent aberrations, loss of heterozygosity and associated genes; numerous figures in this chapter contain representative karyograms as visual resources. Chapters 19 and 20, “Mathematics for the cytogenetic technologist” and “Selected topics on safety, equipment maintenance, and compliance for the cytogenetics laboratory” include overviews of general concepts that help with technical tasks in the laboratory and familiarization with routine equipment. These chapters will benefit new trainees as they gain fundamental knowledge related to their future positons in the clinical laboratory. The AGT Cytogenetics Manual should be on the bookshelves of laboratory directors, operations managers, and genetic specialists as a quick reference for common laboratory issues, knowledge, and as a visual guide for analyses. Without hesitation, and with much excitement I recommend this book to all cytogenetic and molecular professionals. The manual serves as a reference for all laboratory members from the newest learners to the most tenured. Heather E. Williams, MS, CG(ASCP) CM Viapath King’s College Hospital, Haematological Malignancy Diagnostic Centre, Cytogenetics Laboratory, Denmark Hill, Cheyne Wing, London, UK, SE5 9RS

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Molecular Diagnostics

Column Editor: Michelle Mah, MLT, MB(ASCP)CM

Molecular Diagnostics: A High School Experience I volunteered in May at a science symposium hosted by and how biochemical signals are produced and captured for a local university chapter which is part of a larger national data analysis. In Sanger sequencing, in addition to regular nonprofit organization focused on STEM (science, technology, nucleotides, fluorescent nucleotides are added that physically engineering, and mathematics) outreach for children and block the addition of the next nucleotide. This results in youths. It’s a great Canadian educational organization and templates of varying lengths with different fluorescence. I am very proud to be part of their professional network of Commonly, the templates are denatured and run through volunteers. The theme of the symposium revolved around capillary electrophoresis where the fragments are separated cancer. Over 100 grade 11 and 12 students came out to the based on charge and size. The outcome is a coloured community to learn from university students and researchers electropherogram that depicts different fluorescent intensities about molecular biology, different types and causes of cancer, representative of different nucleotides. FIGURE 1 detection and treatment. It was a day packed with talks from industry and healthcare professionals in the morning, and over a dozen engaging activities in the afternoon. I took pa r t i n t he a f ter noon hands-on portion. My activity was titled “Let’s Sequence!” and focused on teaching the basic steps of the polymerase chain reaction (PCR) and sequencing technologies. Afterward, students were given a chance to analyze single nucleotide changes on DNA electropherograms. The expectation was to expose students to the idea of personalized medicine. Now, I had some initial ideas about execution—the most straightforward was to put together a PowerPoint presentation. I could also pull some great videos to showcase advanced technologies and we could work through some examples of examining sequencing electropherograms. I was going to facilitate it like a high school lesson. When I received event details, I was informed each activity was only timed for 10 to 15 minutes for groups of three Unlike Sanger reactions, which take reactions, place in PCR tubes,take the two most NGS Unlike Sanger which place in common PCR tubes, to six students. We would also be in a large auditorium, so in the clinical labthe anchor on ain solid In sequencing two these most amplified commontemplates NGS assays thematrix. clinical lab anchorby acoustics and layout would not be amenable forassays any lesson-type synthesis technology, DNA targets are hybridized to complementary adapterInoligonucleotides these amplified templates on a solid matrix. sequencing presentation. So how does one teach molecular diagnostics in attached on surfaces inside a glass flowtechnology, cell. This flowDNA cell, which ranges from a to by synthesis targets arein size hybridized 10 minutes to secondary students? microscope slide to the size of a cell phone, is then loaded into the sequencer. Sequencing complementary adapter oligonucleotides attached on surfaces Admittedly the situation was daunting, but we worked enzymes and fluorescent nucleotides are then into the cell and the in incorporation inside a glass flow cell.flooded This flow cell,flow which ranges size from of out the possibility of presenting in a poster-style fashion and base calling. Alternatively, sequencing, DNA afor microscope slide to the size in ofsemiconductor a cell phone, is then loaded into everything would be on two poster-sized slides.nucleotides I sat downare onimaged a are attached beads using molecular tags. These DNA-bead complexes are flooded sequencer. Sequencing enzymes and fluorescent nucleotides Saturday afternoon and learned a good lesson targets in making visual to the intosequencing wells contained ion chip.into The ion is loaded into the the sequencer where areelectrical then flooded thechip flow cell and incorporation content. I managed to transition from Sanger to in an nucleotides flow across wells. Nucleotide incorporation changes the of then nucleotides arethe imaged for base calling. Alternatively, inlocal next-generation sequencing (NGS) in a fewenzymes figures Iand would of adetails well andfor this change in pH is measured as a chemical signalare for data processing. semiconductor sequencing, DNA targets attached to beads like to share in this column. I have added pH more using molecular tags. These DNA-bead complexes are flooded this audience, but this marks the most visualFIGURE column2 I have into wells contained in an electrical ion chip. The ion chip contributed to date. It should hopefully be a good introduction is loaded into the sequencer where enzymes and nucleotides to new members as well. then flow across the wells. Nucleotide incorporation changes All sequencing reactions start with PCR amplification of the local pH of a well and this change in pH is measured as a target regions of interest. Most often it is a couple hundred base chemical signal for data processing. FIGURE 2 pairs of a disease associated gene. The difference between one target sequencing and NGS is the greater number of targets

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Molecular Diagnostics

While the technologies may be complex, I think the concept of how molecular diagnostics is used to help patients is not. Students enjoyed looking for sequence changes and asked some really good questions. For example, they asked what the height of the fluorescent intensities mean in an electropherogram? How can more than one patient be sequenced at a time in NGS? And how long do different reactions take? It was also great to bring in some NGS flow cells and ion chips for students to share and compare the difference between a 30ul volume in

a 200ul PCR tube for one patient and the massively parallel sequencing of genomic targets in square microns of surface area for dozens of patients. The look of fascination on students makes me wonder what the next decade will usher in for molecular diagnostics. For my Canadian friends, I encourage you all to visit the organization’s website Let’s Talk Science (http://www.letstalkscience.ca/) for opportunities to promote genetics technology!

AGT Website: www.AGT-info.org Member’s Only area – User Name: First initial, Last initial and AGT ID# (Ex. CR12345) Password: genetics The Journal of the Association of Genetic Technologists 43 (3) 2017

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Research Article

Elucidation of Novel Chromosomal Abnormalities in Pancreatic Cancer: Conventional and Molecular Cytogenetic Characterization of 16 Pancreatic Cell Lines David Shabsovich1,5 and Carlos A. Tirado1,2,3,4 1. 2. 3. 4. 5.

The International Circle of Genetic Studies, Los Angeles, CA 90024 AllinaHealth, Minneapolis, MN 55407 HPA, Minneapolis, MN 55407 The University of Minnesota School of Medicine, Department of Laboratory Medicine and Pathology, Minneapolis, MN 55401 University of California - Los Angeles, Los Angeles, CA 90024

Abstract Pancreatic carcinoma is a major cause of cancer-related death in the United States, with a five-year survival rate of approximately 5%. Cytogenetic analysis has identified clinically significant chromosomal abnormalities in numerous malignancies, but it is not utilized in the clinical management of pancreatic carcinoma. We performed conventional and molecular cytogenetic analysis of 16 pancreatic carcinoma cell lines using Giemsa banding and DNA-based fluorescence in situ hybridization (FISH). Conventional cytogenetic analysis revealed a diversity of recurrent and clonal numerical and structural abnormalities in all cell lines analyzed, many of which occurred at loci of genes implicated in pancreatic or related cancers. FISH analysis revealed significant decreases in copy number of numerous tumor-suppressor genes including TP53, CDKN2A, and SMAD4. In some cell lines, amplification of oncogenes HER2 and MYC was also observed. Finally, novel rearrangements involving ARID1A and TGFBR2 were identified in a small subset of cell lines by means of molecular cytogenetic analysis. All in all, these data provide additional insight into recurrent chromosomal abnormalities in pancreatic carcinoma that can potentially be utilized as biomarkers in the clinical management of the disease. Investigation of other aberrations as well as correlation of recurrent ones with clinicopathologic features is warranted in order to assess the utility of cytogenetic analysis of pancreatic carcinoma.

Introduction

of 16 pancreatic carcinoma cell lines using Giemsa banding and DNA-based fluorescence in situ hybridization (FISH), with the goal of identifying recurrent chromosomal abnormalities in pancreatic cancer that may be useful in the clinical management of the malignancy.

Pancreatic carcinoma is a major cause of cancer-related death in the United States, with a five-year survival rate of approximately 5% (Raimondi et al., 2009; Yadav et al., 2016). Clinically, the malignancy is treated using a combination of surgical and chemotherapeutic approaches, albeit with limited success. Diagnostically, standard histopathological approaches are utilized to characterize the particular type of pancreatic carcinoma, which consequently guides treatment decisions and determines prognoses. Cytogenetic analysis has identified clinically significant chromosomal abnormalities in numerous malignancies, but it is not utilized in the clinical management of pancreatic carcinoma. This is due in part to the immense cytogenetic heterogeneity previously identified in pancreatic carcinoma specimens, both between tumors (intertumor) and even within tumors (intratumor) (Griffin et al., 1995). This heterogeneity is likely due to a complex cytogenetic evolution coupled with the frequent late detection of the malignancy, allowing for additional abnormalities to accrue. It has been established that pancreatic carcinoma is likely driven by stepwise mutations involving four potent tumor suppressor genes: KRAS, TP53, CDKN2A, and SMAD4 (Iacobuzio-Donahue et al., 2012). Mutations or other gene-disrupting events have been shown to occur in a stepwise fashion as the neoplasm proceeds through stages of pancreatic intraepithelial neoplasia (PanIN) and becomes a fully developed tumor (Iacobuzio-Donahue et al., 2012). Throughout this process, numerous additional abnormalities likely occur, both at cytogenetically detectable and submicroscopic levels, but the recurrence and functional/clinical relevance of a number of these aberrations remains uncertain. We performed conventional and molecular cytogenetic analysis

Materials and Methods 16 pancreatic carcinoma cell lines were obtained from the American Tissue Culture Collection (ATCC). Source information for these cell lines is listed in Table 1. Cell were Name AsPC-1 MIA PaCa-2 BxPC-3 Capan-2 CFPAC-1 HPAF-II Panc 02.13 Panc 03.27 Panc 10.05 Panc 02.03 Panc 04.03 Panc 05.04 Capan-1 HPAC PANC-1

Specimen adenocarcinoma carcinoma adenocarcinoma adenocarcinoma adenocarcinoma adenocarcinoma adenocarcinoma adenocarcinoma adenocarcinoma adenocarcinoma adenocarcinoma adenocarcinoma adenocarcinoma adenocarcinoma epithelioid carcinoma

Sex/Age F/62 M/56 F/61 M/56 M/26 M/44 F/64 F/65 M/? F/70 F/70 F/77 M/40 F/64 M/56

Panc 08.13

adenocarcinoma

M/85

Table 1. Source information for 16 pancreatic carcinoma cell lines utilized in this study, all of which were obtained from ATCC.

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Research Article Elucidation of Novel Chromosomal Abnormalities in Pancreatic Cancer: Conventional and Molecular Cytogenetic Characterization of 16 Pancreatic Cell Lines Results

cultured in standard conditions using appropriate growth media, supplements, and antibiotics. Conventional cytogenetic analysis was performed using standard cytogenetic techniques and 20 metaphases were analyzed per cell line. A composite karyotype was constructed for each cell line and conveyed via nomenclature outlined in ISCN 2016 guidelines. DNA-based fluorescence in situ hybridization was conducted using the probes listed in Table 2, all acquired from either Empire Genomics or Abbott Molecular. 200 nuclei were analyzed per probe set per cell line.

Chromosome

Conventional cytogenetic analysis revealed a variety of numerical and structural abnormalities present in the 16 cell lines analyzed. Table 3 depicts composite karyotypes for each cell line determined by analysis of 20 metaphases per cell line. Figure 1 depicts the percentage of cell lines with a numerical abnormality involving a particular chromosome, and Figure 2 depicts the percentage of cell lines with a structural abnormalities involving a particular chromosome, including marker chromosomes. Figure 3 depicts representative karyotypes from four of the sixteen pancreatic carcinoma cell lines analyzed.

Probe/Probe Set

Locus/Loci

ARID1A Break Apart FISH Probe*

1p36.1

TGFBR2 Break Apart FISH Probe*

3p24.1

MYC (8q24) Break Apart Rearrangement Probe**

8q24

CDKN2A (9p21) Spectrum Orange CEP 9 Spectrum Green**

9p21 Centromere 9

TP53 (17p13) Spectrum Orange CEP17 Spectrum Green**

17p13 Centromere 17

Vysis PathVysion HER2 DNA Probe Kit**

17q12 Centromere 17

Custom SMAD4 (18q21.2) Spectrum Orange* CEP18 Spectrum Green*

18q21.2 Centromere 18

Table 2. Information regarding DNA-based FISH probes, including name and localization, utilized in this study. *Probe acquired from Empire Genomics (700 Michigan Ave, # 200, Buffalo, NY 14203, USA) **Probe acquired from Abbott Molecular (100 Abbott Park Road, Abbott Park, IL 60064, USA)

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Composite Karyotype 50-56,XY,-Y,add(1)(p13),+2,+3,+5,+7,add(7)(q22),+8,+9,add(9)(p22),+11,+12,-13,-14,-15,-17,+20,+1-4mar[cp20]

MIA PaCa-2

57-66,<3n>,X,-X,-Y,add(X)(p11.2),add(X)(q26),add(1)(p13),-2,-3,-4,+5,add(6)(q13),-8,-8,add(8)(q24),-9,-11,12,add(12)(p12),-13,-14,-14,add(14)(q32),-15,-16,add(16)(p11.2),add(20)(q11.2)x2,-21,-22,+5-7mar[cp20]

BxPC-3

52-60,<3n>,XX,-X,add(1)(q11.2),-2,-2,add(3)(p11.2),add(3)(q21),-4,add(4)(q21),-5,-6,+7,add(7)(q36),+8,-9,add(9) (p13),-10,add(10)(p13),+11,-12,+14,+15,-17,-18,+20,-21,-22,+2-4mar[cp20]

Capan-2

63-75,<3n>,XX,-Y,+add(1)(p13),-3,+4,+5,-6,+7,-8,add(9)(p21),+10,+11,+11,add(11)(p11.2),i(11)(p10),-12,-14,i(14) (q10)x2,-18,-19,+20,-22,+2-5mar[cp20]

CFPAC-1

64-77,<3n>,XX,-Y,+1,add(1)(p13)x2,+2,add(7)(p11.2),-8,-9,-11,-12,add(13)(q11.2)x3,-14,add(14)(p13)x2,add(14) (p11.2)x2,-17,-18,-18,add(19)(p13),add(20)(q13.3),+21,-22,+7-9mar[cp20]

HPAF-II

54-56,X,-Y,+X,+add(1)(p13),+2,-3,+5,+7,+add(8)(p21),add(9)(q34),+add(11)(p11.2),+add(12)(p11.2),+del(12) (p12),der(13;14)(p10;q10)x2,+14,+add(14)(p12),-16,+18,+20,+20,+22,+1-3mar[cp20]

Panc-02.13

70-77,<3n>,XXX,+1,add(1)(p13),add(1)(p31),i(1)(p11.2),+2,add(3)(p13),-6,+7,add(7)(q22),add(8)(p11.2),+12,+12,13,add(14)(p12),+16,-17,-18,-19,+20,-21,-21,-22,+9-10mar[cp20]

Panc-03.27

57-73,<3n>,add(1)(p31),add(3)(p13),-4,+5,add(6)(p11.2),+add(6)(q13),add(8)(p21),add(10)(p11.2),-12,add(12) (p11.2),add(13)(p11.2),+add(13)(q12),+14,add(15)(p11.2),-16,add(16)(p11.2),+17,-18,-19,add(19)(q13.3),-21,-22,+23mar[cp20]

Panc-10.05

35-40,X,-Y,add(1)(p13),add(3)(p13),-4,add(6)(q21),i(8)(q10),add(10)(p13),-12,-13,-14,-15,-17,-18,-21,-22,22,+2mar[cp20]

Panc-02.03

66-77,<3n>,X,-X,-X,add(1)(p22),add(1)(q11.2),+der(1;3)(q10;p10)x2,-3,add(3)(p13),+5,+5,-6,-7,-8,add(8) (p11.2),add(9)(p13),add(10)(q26),+11,+11,add(11)(p11.2),add(11)(q23),-14,add(14)(p11.2),+20,-21,-22,+29mar[cp20]

Panc-04.03

54-62,<3n>,X,-X,-Y,+1,der(1)add(p13)dup(q21q23),-3,-5,add(6)(q17),-8,add(9)(p13),-12,+13,+14,-15,add(17) (p11.2),-18,-21,-22,+1-3mar[cp20]

Panc-05.04

83-93,<4n>,XXXX,add(X)(p22.1),add(X)(q22),-1,-2,-3,-3,-7,add(10)(p11.2)x2,add(11)(p11.2)x2,add(14)(p11.2),18,-18,+2-8mar[cp20]

Panc-08.13

50-53,X,-Y,add(1)(p13),+3,psudic(9)(p13),+11,add(11)(p11.2),-13,i(13)(q10),-14,+15,add(15)(p13),+17,-18,-19,-20,22,+2-9mar[cp20]

Panc-1

46-57,X,+X,-Y,+1,add(1)(p13),+2,+3,add(6)(q13),+7,-8,i(9)(q10),del(9)(q12q13),-10,+11,add(11)(p11.2),+12,13,add(13)(p13),-14,add(15)(p13)x2,-16,+19,add(19)(q13.3),+20,-21,-22,+1-4mar[cp20]

HPAC

57-61,<3n>,X,-X,-X,-6,+8,+8,del(9)(q12q13),-10,del(11)(p11.2),add(12)(q24.1),add(13)(p13),-15,-16,-17,add(17) (p11.2),-18,-19,+20,-21,+2-8mar[cp20]

Capan-1

55-62,X,-Y,+1,+1,add(1)(p13),i(1)(p10),-3,add(3)(q21),+6,+6,add(6)(q13)x2,+7,add(7)(q32),add(7)(q11.2),8,+9,+9,add(9)(p13),add(9)(p12),add(9)(q34),+11,+add(11)(p11.2),+12,dup(12)(q21;q24.1),+13,add(13)(p13),15,+17,+17,add(17)(p11.2),+20,+8-10mar[cp20]

Table 3. Composite karyotypes of 16 pancreatic carcinoma cell lines included in this study from analysis of 20 metaphases per cell line, depicted using ISCN 2016 guidelines.

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Figure 1. Percentage of cell lines with numerical abnormality, a change in chromosome number/ploidy that does not affect chromosome structure, depicted by chromosome.

Figure 2. Percentage of cell lines with structural abnormality, a change in chromosome structure that does not affect the number of chromosomes, depicted by chromosome. â&#x20AC;&#x153;MARâ&#x20AC;? indicates marker chromosome, or chromosomes of unknown origin.

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A. HPAF-II: 54-56,X,-Y,+X,+add(1)(p13),+2,-3,+5,+7,+add(8) (p21),add(9)(q34),+add(11)(p11.2), +add(12)(p11.2),+del(12) (p12),der(13;14)(p10;q10)x2,+14,+add(14)(p12),16,+18,+20,+20,+22,+1-3mar[cp20]

B. Panc-04.03: 54-62,<3n>,X,-X,-Y,+1,der(1)add(p13) dup(q21q23),-3,-5,add(6)(q13),-8,add(9)(p13),-12,+13,+14,15,add(17)(p11.2),-18,-21,-22,+1-3mar[cp20]

C. BXPC-3: 52-60,<3n>,XX,-X,add(1)(q11.2),-2,-2,add(3) (p11.2),add(3)(q21),-4,add(4)(q21),-5,-6,+7,add(7)(q36),+8,9,add(9)(p13),-10,add(10)(p13),+11,-12,+14,+15,-17,-18,+20,-21,22,+2-4mar[cp20]

D. Capan-2: 63-75,<3n>,XX,-Y,+add(1)(p13),-3,+4,+5,-6,+7,8,add(9)(p21),+10,+11,+11,add(11)(p11.2),i(11)(p10),-12,-14,i(14) (q10)x2,-18,-19,+20,-22,+2-5mar[cp20]

Figure 3. Representative karyotypes from pancreatic carcinoma cell lines HPAF-II (A), Panc-04.03 (B), BxPC-3 (C), and Capan-2 (D).

FISH analysis likewise revealed a spectrum of abnormal signal patterns using the probes listed in Table 2. Probes targeting the tumor suppressor genes (TSGs) CDKN2A, TP53, and SMAD4 revealed deletions of these TSGs in numerous contexts, depicted in Table 4 and Figures 4, 5, and 6, respectively. Furthermore, the average copy number per signal was computed for each of

these probe sets, indicating statistically significant differences (p<0.05) between the copy number of each TSG and that of the centromeric control of the chromosome on which it is located (Figure 6). The copy numbers were computed as follows for each of these probes: CEP9 (2.94), CDKN2A (1.75), CEP17 (2.93), TP53 (2.08), CEP18 (2.14), and SMAD4 (1.42).

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TP53 (R) centromere 17 (G)

SMAD4 (R) centromere 18 (G)

Signal Pattern

Number of Nuclei (n=200)

Signal Pattern

Number of Nuclei (n=200)

Signal Pattern

Number of Nuclei (n=200)

AsPC-1

2R2G 1R2G

49 151

2R2G

200

2R2G 1R2G

116 84

MIA PaCa-2

2R2G 1R2G 0R2G

11 74 115

3R3G 2R3G 2R2G

139 19 42

3R3G

200

BxPC-3

2R2G 1R2G 0R2G

14 57 129

2R2G

200

2R2G 1R1G

172 28

Capan-2

3R4G 2R4G 2R3G

41 122 37

2R3G 2R2G

89 111

2R2G

200

CFPAC-1

3R3G 2R3G

37 163

2R4G 2R3G

169 31

0R4G 0R3G 0R2G

HPAF-II

2R4G 2R3G

149 51

2R2G

200

2R2G

Panc-02.13

1R6G 0R6G 1R5G 0R5G

91 72 30 7

3R4G 2R4G 2R3G 2R2G

13 143 37 7

3R3G 2R2G

164 36

Panc-03.27

3R3G

200

3R4G 2R4G 2R3G

11 120 69

2R3G 2R2G

79 121

15 3 27 155

1R2G 1R1G

173 27

11 160 29 200

Panc-10.05

2R2G

200

2R3G 1R3G 2R2G 1R2G

Panc-02.03

2R3G

200

2R4G 2R3G

173 27

2R3G

200

Panc-04.03

2R3G 2R2G

166 34

2R3G 1R3G

187 13

1R2G 1R1G

134 66

Panc-05.04

2R3G 2R2G

178 22

5R5G 4R4G

83 117

0R3G 0R2G

137 23

Panc-08.13

3R3G 2R3G

66 134

2R3G 1R3G 1R2G

123 48 29

1R2G 0R2G

192 8

Panc-1

1R2G 0R2G

154 46

3R3G 2R2G

147 53

1R1G

200

HPAC

3R3G

200

2R3G 2R2G

170 30

1R2G 0R2G

152 48

Capan-1

0R4G 0R3G

21 179

2R4G 2R3G

173 67

0R4G 0R3G

19 181

Table 4. Interphase FISH analysis of tumor suppressor genes (TSGs) implicated in pancreatic cancer, including CDKN2A/p16 (9p21), TP53 (17p13.1), and SMAD4 (18q21.2). For each FISH experiment, the gene of interest (TSG) was labeled with a red probe (R), and the centromere on which the gene is located was labeled with a green probe (G), serving as a positive control. For each TSG/ centromere combination, 2R2G was considered a cytogenetically normal signal pattern. 200 interphase nuclei were scored per TSG/centromere combination per cell line, and all normal and abnormal signal patterns as well as their corresponding frequencies were noted. The Journal of the Association of Genetic Technologists 43 (3) 2017

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Figure 4. Examples of abnormal interphase nuclei depicting deletions of CDKN2A/p16 (9p21) using the probe set listed in Table 2. The green signal localizes to the centromere of chromosome 9 and the red signal localizes to the CDKN2A/p16 (9p21) TSG.

Figure 6. Average copy number of TSGs and positive controls (centromeres of chromosomes on which particular TSG is located), averaged among all cell lines. p<0.05 for all comparisons.

Probes targeting the oncogenes MYC (8q24) and HER2 (17q11.2), conversely, revealed amplification, examples of which are depicted in Figures 7 and 8, respectively. Cell line-specific data showing results of hybridization with probes targeting MYC and HER2 is provided in Table 5.

Figure 5. Examples of abnormal interphase nuclei depicting deletions of TP53 (17p13.1) using the probe set listed in Table 2. The green signal localized to the centromere of chromosome 17 and the red signal localized to the TP53 (17p13.1) TSG.

Figure 6. Examples of abnormal interphase nuclei bearing deletions of SMAD4 (18q21.2) using the probe set listed in Table 2. The green signal localizes to the centromere of chromosome 18 and the red signal localized to the SMAD4 (18q21.2) TSG.

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HER2 (R) centromere 17 (G)

Signal Pattern

Number of Nuclei (n=200)

Signal Pattern

Number of Nuclei (n=200)

AsPC-1

200

3R2G 2R2G

44 156

MIA PaCa-2

3Y 2Y

154 46

4R3G 3R3G 3R2G

64 119 17

BxPC-3

3Y 2Y

174 26

2R2G

200

Capan-2

4Y 3Y

154 46

4-7R3G 3R3G 2R2G

34 51 115

CFPAC-1

2R3Y 2R2Y

153 47

5-13R3-4G 4R4G 3R3G

171 21 8

HPAF-II

4Y 3Y 2Y

43 130 27

3R2G 2R2G

69 131

Panc-02.13

4Y 3Y

162 38

5-8R2-4G

200

Panc-03.27

4Y 3Y

73 127

4R4G

200

Panc-10.05

200

3R3G 2R2G

13 187

Panc-02.03

3-10Y

200

4R4G 4R3G

165 35

Panc-04.03

200

3R3G

Panc-05.04

200

5R5G 4R4G

91 109

Panc-08.13

200

3R3G 2R2G

157 43

Panc-1

200

4-7R3G 3R3G 4-7R2G

120 24 56

HPAC

200

3R3G 2R2G

93 107

Capan-1

200

4R4G 3R3G

162 38

Table 5. Interphase FISH analysis of oncogenes implicated in pancreatic cancer, including MYC (8q24) and HER2 (17q11.2). For the experiments involving MYC, the 5’/centromeric region of the gene was labeled with a red probe (R), and the 3’/telomeric region of the gene was labeled with a green probe (G). A cytogenetically normal single pattern for this break-apart probe set was two yellow (red/green colocalization) signals (2Y), indicating two intact (non-rearranged) MYC alleles. For the experiments involving HER2, the HER2 locus was labeled with a red probe (R) and centromere 17 was labeled with a green probe (G), serving as a positive control. For the experiments involving HER2, 2R2G was considered a cytogenetically normal signal pattern. 200 interphase nuclei were scored per gene of interest per cell line, and all normal and abnormal signal patterns as well as their corresponding frequencies were noted.

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Figure 8. Interphase nucleus and metaphase spread depicting amplification of HER2 (17q11.2) oncogene in CFPAC-1 pancreatic adenocarcinoma cell line, detected using the probe set listed in Table 2. The green signal represents the centromere of chromosome 17, and the red signal represents the HER2 (17q11.2) oncogene.

Figure 7. Interphase nucleus and metaphase spread depicting amplification of MYC (8q24) oncogene in Panc-02.03 pancreatic adenocarcinoma cell line, detected using the probe set listed in Table 2. The yellow signals represent intact (non-rearranged) MYC alleles.

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Research Article Elucidation of Novel Chromosomal Abnormalities in Pancreatic Cancer: Conventional and Molecular Cytogenetic Characterization of 16 Pancreatic Cell Lines A break-apart probe set targeting ARID1A (1p36.11) revealed a single cell line—HPAF-II—that showed evidence of a deletion or unbalanced rearrangement involving loss of the 5’/telomeric portion of ARID1A, evidenced by a 1G1Y signal pattern (Figure 9). Additionally, a break-apart probe set targeting TGFBR2

(3p24.1) showed evidence of a biallelic rearrangement, evidenced by a 2R2G signal pattern, in a single cell line—Panc-02.13 (Figure 10). The results of all cell line-specific hybridizations using these probes are provided in Table 6.

5’ ARID1A (R) 3’ ARID1A (G)

5’ TGFBR2 (R) 3’ TGFBR2 (G)

Signal Pattern

Number of Nuclei (n=200)

Signal Pattern

Number of Nuclei (n=200)

AsPC-1

200

MIA PaCa-2

2Y 1Y

183 17

200

BxPC-3

3Y 2Y

121 79

200

Capan-2

200

CFPAC-1

200

HPAF-II

1G1Y

200

Panc-02.13

200

2R2G

200

Panc-03.27

200

3Y 2Y

81 119

Panc-10.05

2Y 1Y

32 178

2Y 1Y

187 23

Panc-02.03

200

Panc-04.03

200

Panc-05.04

200

Panc-08.13

200

Panc-1

2Y 1Y

147 53

200

HPAC

200

Capan-1

200

4Y 3Y

157 43

Table 6. Interphase FISH analysis of target genes ARID1A (1p36.11) and TGFBR2 (3p24.1). For each FISH experiment, the 5’/telomeric region of the gene of interest was labeled with a red probe (R), and the 3’/centromeric region of the gene of interest was labeled with the green probe (G). A cytogenetically normal single pattern for these break-apart probe sets was two yellow (red/green colocalization) signals (2Y), indicating two intact (non-rearranged) alleles of the gene of interest. 200 interphase nuclei were scored per gene of interest per cell line, and all normal and abnormal signal patterns as well as their corresponding frequencies were noted.

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Figure 9. Interphase nucleus and metaphase spread depicting 1G1Y abnormal signal pattern of ARID1A (1p36.11), detected in pancreatic adenocarcinoma cell line HPAF-II using the breakapart probe kit listed in Table 2. The green signal represents the 3’/centromeric portion of the ARID1A gene and the yellow (fusion) signal represents an intact (5’ and 3’ colocalized) ARID1A allele.

Figure 10. Interphase nucleus and metaphase spread depicting 2R2G abnormal signal pattern of TGFBR2 (3p24.1), detected in pancreatic adenocarcinoma cell line Panc-02.13 using the break-apart probe kit listed in Table 2. The green signal represents the 3’/centromeric portion of the TGFBR2 gene and the red signal represents the 5’/telomeric portion of the gene.

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Research Article Elucidation of Novel Chromosomal Abnormalities in Pancreatic Cancer: Conventional and Molecular Cytogenetic Characterization of 16 Pancreatic Cell Lines Discussion

was identified among interphase nuclei analyzed, ranging from cytogenetically normal signal patterns to loss of all the alleles of a particular tumor suppressor gene. Due to the heterogeneity of abnormalities involving these tumor suppressor genes, it is uncertain to what extent cytogenetically detectable deletions of these genes contribute to pancreatic carcinogenesis versus aberrations at the genomic level (e.g. loss-of-function mutations). For example, deletion of a particular tumor suppressor gene may be the primary loss-of-function event, or may occur subsequent to a loss-of-function point mutation as a passenger abnormality. Aberrations involving these tumor suppressor genes have also been implicated in pancreatic cancer risk and progression. Campa et al. identified a single nucleotide polymorphism within the CDKN2A/B locus that is associated with increased risk of developing pancreatic cancer, in contrast to the typically somatic (acquired) mutations of the gene observed in pancreatic cancer specimens (Campa et al., 2016). Additionally, Hosoda et al. (2017) assessed high-grade Pan-IN samples and found inactivating mutations of TP53 and SMAD4 to be relatively infrequent in these lesions, suggesting that abnormalities involving these genes occur late in the progression of PanIN and would predominate in invasive pancreatic cancer. All in all, assessment of recurrent abnormalities involving tumor suppressor genes in pancreatic cancer by FISH may be useful in elucidating the interplay between cytogenetic and genomic-level changes in the genesis of the disease and can serve as a useful tool to monitor progression of the disease, as is done in the case of many other malignancies. Although the stepwise accumulation of inactivating aberrations to particular tumor suppressor genes in pancreatic cancer development has been established, the role of oncogenes in this process is more widespread and less consistent. We assessed abnormalities involving two potent oncogenes—MYC (8q24) and HER2 (17q11.2)—in our sample of pancreatic carcinoma cell lines using a break-apart probe for the former gene and locusspecific probe with a centromeric positive control for the latter. In the analysis of MYC, we identified two cell lines that showed evidence of MYC amplification, albeit via different mechanisms. CFPAC-1 showed populations of cells with two to three yellow signals (intact MYC alleles) in addition to two red signals (5’ MYC locus), indicating amplification of only the 5’ signal. Conversely, Panc-02.03 showed cells with three to 10 yellow signals (intact MYC alleles), indicating amplification of the intact MYC locus. The functional implications of these distinct cytogenetic mechanisms of amplification remain to be studied; it is possible that amplification of only the 5’ portion of the MYC gene is sufficient to produce an oncogenic effect. Alternatively, the structure of the break-apart probe may have resulted in the pattern observed in CFPAC-1; since the green probe hybridizes farther away from the MYC gene than the red probe, it could have not been included in the portion that was amplified because it did not contain the oncogenic genetic material. MYC activation has been implicated in pancreatic cancer in numerous capacities, although generally in contexts independent of cytogenetic analysis. Armengol et al. and Mahlamäki et al. utilized comparative genomic hybridization and FISH to identify MYC amplification in seven out of eight and 16 out of 31 pancreatic cancer samples, respectively (Armengol et al., 2000;

Pancreatic cancer is a genetically complex malignancy that most often carries a poor prognosis for patients. In this study, we sought to cytogenetically characterize 16 pancreatic carcinoma cell lines with the goal of identifying recurrent chromosomal abnormalities, as well as novel targets worthy of future investigation. Conventional cytogenetic analysis revealed a spectrum of abnormalities involving all chromosomes. Among all the cell lines we analyzed, the most common numerical abnormalities involved chromosomes X, 8, 11, 12, 14, 15, 18, 20, and 22 and the most common structural abnormalities involved chromosomes 1, 8, 9, and marker chromosomes (chromosomes of unknown structural origin). Previous studies assessing pancreatic carcinoma samples (both cell lines and clinical samples) by conventional cytogenetic means revealed complex karyotypes and cytogenetic heterogeneity both within and between samples (Gorunova et al., 1995; Griffin et al., 1995; Brat et al., 1997; Gorunova et al., 1998; Kowalski et al., 2007). Importantly, Gorunova et al. specifically assessed cytogenetic heterogeneity among pancreatic carcinoma samples, finding extensive heterogeneity—from two to 58 clones—per sample (Gorunova et al., 1998). Despite this, Griffin et al. identified recurrent abnormalities in an analysis of 62 pancreatic adenocarcinoma samples, establishing their nonrandom nature (Griffin et al., 1995). Additionally, Kowalski et al. analyzed potential patterns of cytogenetic evolution within pancreatic adenocarcinoma clinical samples, and identified both consistently early and late karyotypic changes in the evolution of the malignancy, establishing potential pathways through which this process occurs (Kowalski et al., 2007). All in all, our data agreed with these findings—all 16 cell lines showed distinct cytogenetic changes ranging in complexity. When pooled, abnormalities involving each chromosome were identified, albeit at varied frequencies. It is important to note that numerous chromosomes were designated as marker chromosomes due to their complex structural nature, which served as a limitation to precise assessment of the frequency of numerical and structural abnormalities of specific chromosomes. Assessment of tumor suppressor genes is imperative to the cytogenetic analysis of pancreatic cancer as they can readily be assessed and may play multiple integral roles in the pathway of pancreatic carcinogenesis. Iacobuzio-Donahue described the stepwise genetic evolution of pancreatic adenocarcinoma as involving specific genetic changes occurring at particular stages of pancreatic intraepithelial neoplasia (PanIN) based on data gathered from the pancreatic cancer genome sequencing project (Iacobuzio-Donahue, 2012). We analyzed three key genetic events discussed by Iacobuzio-Donahue and other investigators— abnormalities involving CDKN2A (occurring during PanIN2), TP53 (occurring during PanIN-3), and SMAD4 (occurring during PanIN-3)—albeit at the molecular cytogenetic level using interphase and metaphase fluorescence in situ hybridization analysis rather than at the genomic level (Iacobuzio-Donahue, 2012). After pooling the data among all 16 cell lines, we identified statistically significant copy number decreases of each of these tumor suppressor genes relative to the centromeres on which these genes are found. Importantly, profound heterogeneity

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Research Article Elucidation of Novel Chromosomal Abnormalities in Pancreatic Cancer: Conventional and Molecular Cytogenetic Characterization of 16 Pancreatic Cell Lines Mahlamäki et al., 2002). Additionally, He et al. identified that expression levels of c-Myc in human pancreatic cancer correlated with perineural invasion, which in turn is associated with a poor prognosis in the disease (He et al., 2012). Furthermore, Kumari et al. identified that gene silencing of c-MYC in pancreatic cancerderived cell lines, among other targets and treatments, resulted in decreased telomerase activity, which in turn is required for cellular immortalization, also suggesting the importance of c-MYC in pancreatic carcinogenesis (Kumari et al., 2009). Due to such studies elucidating the varied effects of MYC in pancreatic cancer, it is considered a significant candidate for therapeutic targeting (in conjunction with other therapies) as well as diagnosis/ prognosis determination in pancreatic carcinoma (Skoudy et al., 2011; Hessman et al., 2016). To this end, cytogenetic analysis can be useful in providing a more holistic picture of MYC abnormalities—particularly amplification and heterogeneity—in pancreatic carcinogenesis. In addition to amplification of MYC, interphase and metaphase FISH analysis of HER2 (17q11.2) revealed amplification of the oncogene in four out of the 16 cell lines analyzed, with interphase nuclei bearing up to 13 HER2 signals. Because a centromeric positive control was utilized in this case, it was clear that the frequency of HER2 signals in these cases was significantly greater than that of the centromere of chromosome 17. Although HER2 involvement and therapeutic targeting is widely established in particular malignancies, its role in pancreatic cancer is not well studied or understood. Liang et al. identified a correlation between amplification of the TOP2A gene, encoding topoisomerase II alpha, and amplification of HER2 (both identified by FISH) in clinical pancreatic ductal adenocarcinoma samples, suggesting a potential synergistic effect between the two genes (Liang et al., 2007). Chantrill et al. discovered five cases of HER2amplified pancreatic adenocarcinoma in an effort to identify patients that could benefit from established targeted therapies (e.g. HER2-kinase inhibitors) (Chantrill et al., 2015). Although current anti-HER2 therapy is approved for a limited number of malignancies, pancreatic cancer and other malignancies in which HER2 aberrations (amplification, overexpression, etc.) occur, even in a minor subset of patients, may benefit from such targeted therapeutic intervention, particularly in conjunction with other therapies (Yan et al., 2014). Whether HER2 drives pancreatic carcinogenesis or largely acts synergistically with other aberrant genes has yet to be understood and would significantly affect the utility of such a therapeutic approach, but the continued evaluation of HER2 using cytogenetic and other means is warranted in order to better understand the role of the gene in pancreatic cancer and its interplay with other key genes involved. Upon surveying our conventional cytogenetic data and published literature, we utilized break-apart FISH probes to analyze two genes previously not analyzed by molecular cytogenetic analysis, to our knowledge—ARID1A (1p36.11) and TGFBR2 (3p24.1). ARID1A is primarily involved in epigenetic modification by means of chromatin remodeling, and was chosen for further FISH analysis primarily due to the predominance of structural abnormalities involving the short arm of chromosome 1 identified in nearly all 16 cell lines analyzed as well as a growing body of evidence suggesting the role of the gene in pancreatic cancer.

Among the 16 cell lines analyzed, only HPAF-II showed an unbalanced aberration involving specifically ARID1A by evidence of a population of cells showing one green signal, representing the 3’/centromeric portion of one of the ARID1A alleles, and a yellow/fusion signal, representing an intact ARID1A allele. The isolated green signal can be due either to a deletion (interstitial or terminal) with a breakpoint within the ARID1A locus, or an unbalanced rearrangement that ultimately resulted in loss of the 5’/telomeric portion of that ARID1A allele. Abnormalities involving ARID1A have been implicated, albeit at the genomic level, in pancreatic carcinoma in numerous studies (Birnbaum et al., 2011; Jones et al., 2012; Heestand and Kuzrock, 2015; Waddell et al., 2015; Zhu et al., 2015). Zhu et al., for example, identified single nucleotide polymorphism (SNP) variants in genes encoding the SWItch/Sucrose Non Fermentable (SWI/SNF) complex, which is centrally involved in chromatin modeling and is in part encoded by ARID1A (Zhu et al., 2015). Furthermore, Jones et al. identified somatic mutations involving ARID1A in 10 out of 119 (8%) pancreatic cancer samples analyzed, many of which were truncating mutations that lead to inactivation of the gene (Jones et al., 2012). Finally, Birnbaum et al. utilized array comparative genomic hybridization to analyze 39 pancreatic adenocarcinoma samples (both clinical samples and cell lines), and identified heterozygous deletions involving the 1p35-36 band, containing ARID1A, in nearly one-third of the samples (Birnbaum et al., 2011). Although this abnormality occurred in only one out of 16 cell lines (approximately 6%) we analyzed, this proportion is similar to the frequency of somatic mutations reported by Jones et al., further suggesting that abnormalities involving ARID1A are recurrent in pancreatic cancer and can occur at both the cytogenetic and genomic levels (Jones et al., 2012). As is the case with other genes, the relative contribution and interplay of large-scale (cytogenetic) and small-scale (genomic) changes is important to understanding the mechanisms leading to ARID1A inactivation and the utility of molecular cytogenetics in the monitoring of aberrations to this gene. We also pursued molecular cytogenetic analysis of TGFBR2 (3p24.1), both due to the recurrence of structural abnormalities involving the short arm of chromosome 3 in the cell lines we analyzed, as well as the role of disruption of the transforming growth factor-beta (TGF-beta) signal transduction pathway (involved in cell proliferation, cell differentiation, and apoptosis, among other processes) in pancreatic carcinogenesis. Among the 16 cell lines we analyzed by FISH using a break-apart probe set flanking the TGFBR2 gene, a single cell line—Panc-02.13— showed evidence of a gene-specific aberration involving TGFBR2, evidenced by two red signals (representing the 5’/telomeric portion of the gene) and two green signals (representing the 3’/ centromeric portion of the gene), none of which colocalized with one another. This ‘split’ signal pattern suggests the presence of biallelic rearrangements involving both TGFBR2 alleles, which to our knowledge, has not been previously reported. Aberrations involving TGFBR2 have been implicated in pancreatic carcinoma, albeit in a relatively low frequency of studies (Goggins et al., 1998; Venkatasubbarao et al., 1998; Ijichi et al., 2006). Venkatasubbarao et al. reported two cases of pancreatic cancer that showed mutations in the region of TGFBR2 encoding

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Research Article Elucidation of Novel Chromosomal Abnormalities in Pancreatic Cancer: Conventional and Molecular Cytogenetic Characterization of 16 Pancreatic Cell Lines the polyadenine tract (Venkatasubbarao et al., 1998). Goggins et al. reported four additional cases of pancreatic cancer that harbored somatic mutations involving TGFBR2, including homozygous deletions and frameshift mutations (Goggins et al., 1998). Furthermore, Ijichi et al. compared phenotypes of mice with pancreas-specific knockout of TGFBR2, pancreasspecific activation of KRAS via the G12D mutation, and both, and found that only the combination of abnormalities resulted well-differentiated pancreatic carcinoma (Ijichi et al., 2006). However, a pancreas-specific heterozygous deletion of TGFBR2 in conjunction with KRAS activation also resulted in welldifferentiated disease, suggesting the integral, yet synergistic role of TGFBR2 in pancreatic carcinogenesis (Ijichi et al., 2006). Our identification of a biallelic rearrangement involving TGFBR2 provides yet another window into potential mechanisms of inactivation of this gene in pancreatic carcinogenesis, and is worthy of investigation in the context of additional genomic changes in the disease. In this study, we utilized conventional and molecular cytogenetic analysis to characterize 16 pancreatic carcinoma cell lines. Conventional cytogenetic analysis confirmed previously reported findings of cytogenetic heterogeneity, complex structural aberrations, and frequent changes to ploidy. Likewise, molecular cytogenetic analysis of tumor suppressor genes heavily implicated in pancreatic cancer—CDKN2A, TP53, and SMAD4—revealed statistically significant copy number changes to each gene of interest relative to the centromere on which the gene is located. Furthermore, analysis of oncogenes previously implicated in pancreatic cancer—MYC and HER2—revealed amplification in particular subsets of cell lines. Finally, assessment of novel cytogenetic targets—ARID1A and TGFBR2—by FISH revealed gene-specific changes involving each target in a single cell line that have not previously been reported, to our knowledge. Our data provide further insight into recurrent large-scale cytogenetic changes that are observed in pancreatic cancer, and suggest the utility of cytogenetic analysis in the analysis of pancreatic cancer. Further investigation into the interplay between such large-scale (cytogenetic) and small-scale (genomic) changes, functional implications of cytogenetic/genomic aberrations, and correlation of abnormalities with clinicopathologic data is warranted in order to better understand the role and utility of chromosomal abnormalities in pancreatic carcinogenesis.

Campa D1,2, Pastore M1,2, Gentiluomo M1,2, Talar-Wojnarowska R3, Kupcinskas J4, Malecka-Panas E3, Neoptolemos JP5, Niesen W6, Vodicka P7,8, Delle Fave G9, Bueno-de-Mesquita HB10,11,12, Gazouli M13, Pacetti P14, Di Leo M15, Ito H16, Klüter H17, Soucek P18,19, Corbo V20, Yamao K21, Hosono S16, Kaaks R22, Vashist Y23, Gioffreda D24, Strobel O6, Shimizu Y25, Dijk F26, Andriulli A24, Ivanauskas A4, Bugert P17, Tavano F24, Vodickova L8,27, Zambon CF28, Lovecek M29, Landi S1, Key TJ30, Boggi U31, Pezzilli R32, Jamroziak K33, Mohelnikova-Duchonova B18,34, Mambrini A14, Bambi F35, Busch O36, Pazienza V24, Valente R9, Theodoropoulos GE37, Hackert T6, Capurso G9, Cavestro GM15, Pasquali C38, Basso D39, Sperti C38, Matsuo K40, Büchler M6, Khaw KT41, Izbicki J23, Costello E5, Katzke V22, Michalski C6, Stepien A42, Rizzato C43, Canzian F2. Functional single nucleotide polymorphisms within the cyclin-dependent kinase inhibitor 2A/2B region affect pancreatic cancer risk. Oncotarget. 2016 Aug 30;7(35): 57011-57020. Chantrill LA1, Nagrial AM2, Watson C3, Johns AL3, Martyn-Smith M3, Simpson S3, Mead S4, Jones MD5, Samra JS6, Gill AJ7, Watson N7, Chin VT8, Humphris JL3, Chou A9, Brown B10, Morey A10, Pajic M3, Grimmond SM5, Chang DK11, Thomas D8, Sebastian L12, Sjoquist K13, Yip S13, Pavlakis N14, Asghari R15, Harvey S15, Grimison P16, Simes J17, Biankin AV18; Australian Pancreatic Cancer Genome Initiative (APGI); Individualized Molecular Pancreatic Cancer Therapy (IMPaCT) Trial Management Committee of the Australasian Gastrointestinal Trials Group (AGITG). Precision Medicine for Advanced Pancreas Cancer: The Individualized Molecular Pancreatic Cancer Therapy (IMPaCT) Trial. Clin Cancer Res. 2015 May 1;21(9): 2029-37. Goggins M1, Shekher M, Turnacioglu K, Yeo CJ, Hruban RH, Kern SE. Genetic alterations of the transforming growth factor beta receptor genes in pancreatic and biliary adenocarcinomas. Cancer Res. 1998 Dec 1;58(23): 5329-32. Gorunova L, Johansson B, Dawiskiba S, Andrén-Sandberg A, Jin Y, Mandahl N, Heim S, Mitelman F. Massive cytogenetic heterogeneity in a pancreatic carcinoma: fifty-four karyotypically unrelated clones. Genes Chromosomes Cancer. 1995 Dec;14(4): 259-66. Gorunova L, Höglund M, Andrén-Sandberg A, Dawiskiba S, Jin Y, Mitelman F, Johansson B. Cytogenetic analysis of pancreatic carcinomas: intratumor heterogeneity and nonrandom pattern of chromosome aberrations. Genes Chromosomes Cancer. 1998 Oct;23(2): 81-99. Griffin CA, Hruban RH, Long PP, Morsberger LA, Douna-Issa F, Yeo CJ. Chromosome abnormalities in pancreatic adenocarcinoma. Genes Chromosomes Cancer. 1994 Feb;9(2): 93-100. Griffin CA, Hruban RH, Morsberger LA, Ellingham T, Long PP, Jaffee EM, Hauda KM, Bohlander SK, Yeo CJ. Consistent chromosome abnormalities in adenocarcinoma of the pancreas. Cancer Res. 1995 Jun 1;55(11): 2394-9. He C1, Jiang H, Geng S, Sheng H, Shen X, Zhang X, Zhu S, Chen X, Yang C, Gao H. Expression of c-Myc and Fas correlates with perineural invasion of pancreatic cancer. Int J Clin Exp Pathol. 2012;5(4): 339-46. Heestand GM1, Kurzrock R1. Molecular landscape of pancreatic cancer: implications for current clinical trials. Oncotarget. 2015 Mar 10;6(7): 4553-61. Hessmann E1, Schneider G2, Ellenrieder V1, Siveke JT2,3. MYC in pancreatic cancer: novel mechanistic insights and their translation into therapeutic strategies. Oncogene. 2016 Mar 31;35(13): 1609-18. Hosoda W1, Chianchiano P1, Griffin JF2, Pittman ME3, Brosens LA4, Noë M1, Yu J1, Shindo K1, Suenaga M1, Rezaee N2, Yonescu R5, Ning Y5, Albores-Saavedra J6, Yoshizawa N7, Harada K8, Yoshizawa A9, Hanada K10, Yonehara S11, Shimizu M12, Uehara T13, Samra JS14, Gill AJ15, Wolfgang CL2,16, Goggins MG1,17,16, Hruban RH1,16, Wood LD1,16. Genetic analyses of isolated high-grade pancreatic intraepithelial neoplasia (HG-PanIN) reveal paucity of alterations in TP53 and SMAD4. J Pathol. 2017 Feb 11.

References Armengol G1, Knuutila S, Lluís F, Capellà G, Miró R, Caballín MR. DNA copy number changes and evaluation of MYC, IGF1R, and FES amplification in xenografts of pancreatic adenocarcinoma. Cancer Genet Cytogenet. 2000 Jan 15;116(2): 133-41. Birnbaum DJ1, Adélaïde J, Mamessier E, Finetti P, Lagarde A, Monges G, Viret F, Gonçalvès A, Turrini O, Delpero JR, Iovanna J, Giovannini M, Birnbaum D, Chaffanet M. Genome profiling of pancreatic adenocarcinoma. Genes Chromosomes Cancer. 2011 Jun;50(6): 456-65. Brat DJ, Hahn SA, Griffin CA, Yeo CJ, Kern SE, Hruban RH. The structural basis of molecular genetic deletions. An integration of cla ssical cytogenetic a nd molecular a nalyses in pa ncreatic adenocarcinoma. Am J Pathol. 1997 Feb;150(2): 383-91.

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Research Article Elucidation of Novel Chromosomal Abnormalities in Pancreatic Cancer: Conventional and Molecular Cytogenetic Characterization of 16 Pancreatic Cell Lines Iacobuzio-Donahue CA. Genetic evolution of pancreatic cancer: lessons learnt from the pancreatic cancer genome sequencing project. Gut. 2012 Jul;61(7): 1085-94. Ijichi H1, Chytil A, Gorska AE, Aakre ME, Fujitani Y, Fujitani S, Wright CV, Moses HL. Aggressive pancreatic ductal adenocarcinoma in mice caused by pancreas-specific blockade of transforming growth factor-beta signaling in cooperation with active Kras expression. Genes Dev. 2006 Nov 15;20(22): 3147-60. Johansson B, Bardi G, Pandis N, Gorunova L, Bäckman PL, Mandahl N, Dawiskiba S, Andrén-Sandberg A, Heim S, Mitelman F. Karyotypic pattern of pancreatic adenocarcinomas correlates with survival and tumour grade. Int J Cancer. 1994 Jul 1;58(1): 8-13. Jones S1, Li M, Parsons DW, Zhang X, Wesseling J, Kristel P, Schmidt MK, Markowitz S, Yan H, Bigner D, Hruban RH, Eshleman JR, IacobuzioDonahue CA, Goggins M, Maitra A, Malek SN, Powell S, Vogelstein B, Kinzler KW, Velculescu VE, Papadopoulos N. Somatic mutations in the chromatin remodeling gene ARID1A occur in several tumor types. Hum Mutat. 2012 Jan;33(1): 100-3. Kowalski J, Morsberger LA, Blackford A, Hawkins A, Yeo CJ, Hruban RH, Griffin CA. Chromosomal abnormalities of adenocarcinoma of the pancreas: identifying early and late changes. Cancer Genet Cytogenet. 2007 Oct 1;178(1): 26-35. Kumari A1, Srinivasan R, Wig JD. Effect of c-MYC and E2F1 gene silencing and of 5-azacytidine treatment on telomerase activity in pancreatic cancer-derived cell lines. Pancreatology. 2009;9(4): 360-8. Liang ZY1, Wang WZ, Gao J, Wu SF, Zeng X, Liu TH. [Topoisomerase IIalpha and HER2/neu gene alterations and their correlation in pancreatic ductal adenocarcinomas]. Zhonghua Bing Li Xue Za Zhi. Mahlamäki EH1, Bärlund M, Tanner M, Gorunova L, Höglund M, Karhu R, Kallioniemi A. Frequent amplification of 8q24, 11q, 17q, and 20q-specific genes in pancreatic cancer. Genes Chromosomes Cancer. 2002 Dec;35(4): 353-8. Raimondi S1, Maisonneuve P, Lowenfels AB. Epidemiology of pancreatic cancer: an overview. Nat Rev Gastroenterol Hepatol. 2009 Dec;6(12): 699-708. Skoudy A1, Her ná ndez-Mu ñoz I, Navar ro P. Pa ncreatic ductal adenocarcinoma and transcription factors: role of c-Myc. J Gastrointest Cancer. 2011 Jun;42(2): 76-84. Venkatasubbarao K1, Ahmed MM, Swiderski C, Harp C, Lee EY, McGrath P, Mohiuddin M, Strodel W, Freeman JW. Novel mutations in the polyadenine tract of the transforming growth factor beta type II receptor gene are found in a subpopulation of human pancreatic adenocarcinomas. Genes Chromosomes Cancer. 1998 Jun;22(2): 138-44. Waddell N1, Pajic M2, Patch AM3, Chang DK4, Kassahn KS3, Bailey P5, Johns AL6, Miller D3, Nones K3, Quek K3, Quinn MC3, Robertson AJ3, Fadlullah MZ3, Bruxner TJ3, Christ AN3, Harliwong I3, Idrisoglu S3, Manning S3, Nourse C5, Nourbakhsh E3, Wani S3, Wilson PJ3, Markham E3, Cloonan N1, Anderson MJ3, Fink JL3, Holmes O3, Kazakoff SH3, Leonard C3, Newell F3, Poudel B3, Song S3, Taylor D3, Waddell N3, Wood S3, Xu Q3, Wu J6, Pinese M6, Cowley MJ6, Lee HC6, Jones MD7, Nagrial AM6, Humphris J6, Chantrill LA6, Chin V6, Steinmann AM6, Mawson A6, Humphrey ES6, Colvin EK6, Chou A8, Scarlett CJ9, Pinho AV6, Giry-Laterriere M6, Rooman I6, Samra JS10, Kench JG11, Pettitt JA6, Merrett ND12, Toon C6, Epari K13, Nguyen NQ14, Barbour A15, Zeps N16, Jamieson NB17, Graham JS18, Niclou SP19, Bjerkvig R20, Grützmann R21, Aust D21, Hruban RH22, Maitra A23, Iacobuzio-Donahue CA24, Wolfgang CL25, Morgan RA22, Lawlor RT26, Corbo V27, Bassi C28, Falconi M29, Zamboni G30, Tortora G31, Tempero MA32; Australian Pancreatic Cancer Genome Initiative, Gill AJ33, Eshleman JR22, Pilarsky C21, Scarpa A26, Musgrove EA34, Pearson JV1, Biankin AV4, Grimmond SM5. Whole genomes redefine the mutational landscape of pancreatic cancer. Nature. 2015 Feb 26;518(7540): 495-501.

Yadav S1, Sharma P, Zakalik D. Comparison of Demographics, Tumor Characteristics, and Survival Between Pancreatic Adenocarcinomas and Pancreatic Neuroendocrine Tumors: A Population-based Study. Am J Clin Oncol. 2016 Jun 17. Yan M1, Parker BA2, Schwab R2, Kurzrock R2. HER2 aberrations in cancer: implications for therapy. Cancer Treat Rev. 2014 Jul;40(6): 770-80. Zhu B1, Tian J2, Zhong R1, Tian Y2, Chen W1, Qian J3, Zou L1, Xiao M2, Shen N1, Yang H3, Lou J1, Qiu Q2, Ke J1, Lu X3, Song W2, Li H2, Liu L4, Wang L2, Miao X1. Genetic variants in the SWI/SNF complex and smoking collaborate to modify the risk of pancreatic cancer in a Chinese population. Mol Carcinog. 2015 Sep;54(9): 761-8.

Acknowledgement: To all the members of “The International Circle of Genetic Studies” Chapter Los Angeles, who helped with the bench work. Corresponding Author: Carlos A. Tirado, Ph.D. Carlos.tirado@allina.com tirad017@umn.edu

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Teaching Case

Myelodysplastic Syndrome with Isolated del(5q) Juli-Anne Gardner1 and Katherine Devitt1 1. Department of Pathology and laboratory Medicine, University of Vermont Medical Center, Burlington, VT Brief Title: MDS with del(5q) A 75-year-old woman presented to her primary care physician with fatigue and general malaise. A complete blood count (CBC) was notable for macrocytic anemia (hemoglobin 8.5g/dL, MCV 103 fL) and thrombocytosis (platelets 465,000/ÂľL). A bone marrow biopsy revealed a hypercellular marrow with marked megakaryocytic dysplasia. Karyotype analysis demonstrated an interstitial deletion in the long arm of chromosome 5 (A). Myelodysplastic syndrome with isolated del(5q) is a discrete type of myelodysplastic syndrome (MDS) and has a female predominance. Patients typically present with macrocytic anemia and normal or increased platelet counts. Bone marrow studies are most notable for characteristic dysplastic megakaryocytes with monolobated or hypolobated nuclei (B-D). Erythropoiesis may also show dysplastic changes but granulocytic dysplasia is usually minimal or absent. Patients have an excellent prognosis with a low rate of progression to a higher grade MDS or acute myeloid leukemia (AML) and respond well to treatment with the thalidomide analogue lenalidomide (Revlimid). Recent studies have shown

that MDS with del(5q) still has a favorable prognosis even when associated with an additional cytogenetic abnormality unless the abnormality is monosomy 7 or del(7q). Thus, per the 2016 revision of the World Health Organization (WHO) classification, the entity MDS with isolated del(5q) may still be diagnosed if there is one additional chromosomal abnormality not including monosomy 7 or del(7q). While the prognosis is favorable overall, approximately 20% of patients harbor a mutation in TP53 which is associated with an increased risk of progression to AML and a poor response to lenalidomide. Corresponding author: Juli-Anne Gardner, MD, Department of Pathology and Laboratory Medicine, University of Vermont Medical Center, Burlington, VT 05401 (T): 802-487-2700 (F): 802-847-3987 (E): Juli-Anne.Gardner@uvmhealth.org

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Genetics in the News

The Milestone of Non-Invasive Prenatal Identification of Chromosomal Abnormalities in Fetal Trophoblasts Recovered from Maternal Blood Jaime Garcia-Heras Abstract Two recent studies demonstrated that array CGH and NGS allow identification of chromosomal abnormalities in fetal trophoblasts circulating in maternal blood. This remarkable breakthrough paves the way for an improved assay that supersedes the performance of non-invasive prenatal testing (NIPT) in cell-free fetal DNA. Furthermore, it is foreseeable to expand the use of this new genomic analysis in trophoblasts to uncover single gene mutations of clinical significance prenatally.

Introductory background

scanning microscope. Fluorescent images taken from each cell were subsequently studied with image analysis software to identify the fetal trophoblasts. The candidate trophoblast cells with a typical labelling pattern were re-examined by an operator to confirm the trophoblastic origin. Finally, the fetal trophoblasts were picked individually to perform a whole-genome amplification, genotyping, and analysis of copy number by array CGH and NGS. Genotyping of trophoblasts was required to establish fetal gender by Y-specific PCR, or a truly fetal origin by biparental inheritance of polymorphic STR markers in cases of female fetuses or fetuses with an inconclusive Y-PCR analysis. Breman et al. studied pregnancies from women who were scheduled to undergo CVS or amniocentesis for prenatal diagnosis via chromosome analysis or array-CGH. They performed a genome analysis with array-CGH and/or NGS in at least 40 single trophoblasts from 16 pregnancies; 17 cells were normal male cells while the other 23 cells showed abnormal results (one 47,XXY cell, 14 cells from two cases with trisomy 21, one trisomy 18, one trisomy 13, two cells with a 2.7Mb deletion of 15q, and four cells from a case with a 1.2 Mb deletion of 1q). The 47,XXY and the trisomy 18 had previous abnormal results by cffDNA and were confirmed by chorionic villus sampling (CVS). One trisomy 21 was confirmed by cytogenetic follow-up with CVS. The identification of a 2.7Mb deletion of 15q highlighted the potential to detect relatively small deletions. The only false negative result was a 1.2Mb deletion of 1q that was missed in the analysis of four trophoblasts but was detected by array-CGH in a CVS sample. There was a very instructive case of a confined placental mosaicism (CPM) in which a prior NIPT study in cffDNA in maternal blood was positive for trisomy 13. NGS demonstrated that three trophoblasts were normal while one trophoblast showed trisomy 13. A follow-up study by CVS showed a normal karyotype. These results go along with the current consensus that CPM is the origin of false positive NIPT results with cffDNA when follow-up cytogenetic studies in CVS or amniotic fluid are normal (Mardy and Wapner, 2016). The other study described an improved high-throughput method for enrichment and isolation of pure fetal trophoblasts, a subsequent whole genome amplification, and an array-CGH and/or NGS analysis (KĂ¸lvraa et al., 2016). Blood samples were collected from 111 pregnant women (71 low-risk pregnancies and 40 high-risk pregnancies). The samples were fixed and processed for a selective red blood cell lysis and obtained an unenriched pellet of white cells. This pellet was incubated with a cocktail of

The discovery of cell-free fetal DNA (cffDNA) that circulates in maternal blood suggested the possibility of using this cffDNA for non-invasive prenatal diagnosis (Lo et al., 1997). Years later once next-generation sequencing (NGS) was available, NGS analysis of cffDNA isolated from the blood of pregnant women was implemented for non-invasive prenatal screening of the most common fetal aneuploidies, especially trisomy 21 (Palomaki et al., 2012). This novel technology became widely known as NIPT (non-invasive prenatal testing). The worldwide experience with NIPT has been largely successful (Taylor-Phillips et al., 2016), but with growing knowledge of this test, some limitations and pitfalls became apparent. As a result, and while the protocols were being refined, research and development in NIPT started to focus on the search for and studies on how to use cells of unambiguous fetal origin that circulate in maternal blood. After many failures to isolate various types of fetal cells from maternal blood for prenatal diagnosis (Beaudet, 2016), the presence of trophoblasts of fetal origin in peripheral blood from pregnant women suggested it might be possible to use them for non-invasive prenatal testing (Emad et al., 2014; Hatt et al., 2014). Two recent landmark studies accomplished for the first time a detection of chromosome abnormalities by genome-wide microarray and NGS in pure fetal trophoblasts isolated from peripheral blood of pregnant women (Breman et al., 2016; KĂ¸lvraa et al., 2016). Both research studies presented reliable methods for enriching fetal cells from maternal blood, perform whole genome amplification to obtain fetal DNA of suitable quality and quantity, and finally detecting abnormalities that were corroborated by studies in CVS or amniotic fluid.

The results of a novel NIPT approach in fetal trophoblasts isolated from maternal blood The first study by Breman et al. reported the isolation of fetal trophoblasts through a series of labor-intensive steps. First, nucleated cells were separated by density gradients from 30 ml of whole blood collected from pregnant women at 10-16 weeks of gestation. These pools of cells were then stained with an antibody cocktail of two anti-cytokeratins and an antiCD-45 to identify the fetal trophoblasts. A suspension of stained fetal cells (anticytokeratin positive, antiCD-45 negative) was recovered and pipetted into custom well slides that were placed onto a digital

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Genetics in the News The Milestone of Non-invasive Prenatal Identification of Chromosomal Abnormalities in Fetal Trophoblasts Recovered from Maternal Blood selection antibodies and subject to magnetic activated cell sorting enrichment. The enriched pellet of fetal cells was then stained with a cocktail of cytokeratin antibodies, washed, and spread on slides. These slides were used to identify and pick individual fetal trophoblasts using first an automatic scanner and classifier software followed by a visual confirmation of fetal origin. After a whole genome amplification (WGA) of DNA of the trophoblasts that were picked, array-CGH and NGS analysis were performed. Kølvraa et al. focused on reporting results on two high-risk pregnancies affected by chromosomal abnormalities that were seen in CVS/amniocentesis. One was a pregnancy of a female fetus with trisomy 21 identified by the pooled analysis of two trophoblast cells. The other one carried a mosaicism of a cell line with monosomy X and a cell line with 1-2 copies of a ring of chromosome X shown by cytogenetics and array-CGH in amniotic fluid: 45,X[10]/46,X,r(X)(p21.3;q26.3)[9]/47,X,r(X)(p21.3;q26.3) x2[1]. Two trophoblasts were collected from maternal blood and studied by NGS. One cell showed the loss of one copy of chromosome X while the other showed deletions of the distal Xp and Xq compatible with a ring of chromosome X. Breman et al. pointed out several advantages of a cell-based NIPT in fetal trophoblasts compared to the use of cffDNAbased NIPT. A) The findings would be representative of the fetal genome instead of the maternal genome. B) A capability to study multiple cells, look for consistency in the results, and diagnose mosaicism (including confined placental mosaicism). The analysis of 3-5 cells was recommended. C) The possibility to perform very deep sequencing to reliably detect smaller CNVs in single cells compared to what is now achievable with cffDNA. D) The potential to uncover de novo point mutations testing multiple trophoblast cells independently, a method predicted to be more reliable than studying a pool of cff-DNA. Considering the previously published evidence of 1-6 trophoblasts per milliliter of blood in pregnant women (Emad et al., 2014), Breman et al. speculated that improvements in the techniques of cell enrichment and isolation might allow retrieval of more of these cells. Indeed, this seemed to be the case, because on average 12.8 fetal trophoblasts were found in 30ml of maternal blood collected from 111 pregnant women (Kølvraa et al., 2016). There was variability between pregnancies in the number of fetal cells retrieved, however. In addition, Kølvraa et al. confirmed, by FISH with probes for chromosomes X and Y, the specificity of the markers used to identify fetal cells in 11 pregnancies known to carry a male fetus. The fetal gender was accurately established in 116 cells and none of them had two signals with the X probe. A clear-cut case of CPM that involved trisomy 13 was identified in one study (Breman et al., 2016). This finding was not considered a surprise because CPM has been observed in 1-2% of cytogenetic studies in CVS performed at 9-12 weeks of gestation (Ledbetter et al., 1992; Association of Clinical Cytogeneticists Working Party on Chorionic Villi in Prenatal Diagnosis, 1994). Breman et al. anticipated other cases of CPM as they expand the testing to more pregnancies, but stated they had the appropriate procedures in place to manage them. These include the individual analysis of multiple trophoblasts to compare results (probably at least 3-5), drawing another maternal blood sample to recover more fetal trophoblasts for an additional analysis, and cytogenetics

in amniotic fluid according to the guidelines for following up a suspected CPM in CVS. Therefore, there were no concerns about the occurrence of CPM in trophoblasts. Still, Breman et al. acknowledged the need to established the baseline frequency of CPM in trophoblasts because it is currently unknown. Breman et al. discussed whether trophoblast-based NIP would become a routine prenatal test offered to low-risk and highrisk pregnant women, particularly with the goal of detecting subchromosomal abnormalities that are not reliably diagnosed by the current NIPT methods with cffDNA (Yin et al., 2015; Lo et al., 2016). Breman et al. were confident that this goal will be possible in the future considering the breakthroughs in single-cell sequencing to uncover CNVs of 54kb (Navin et al., 2012; Leung et al. 2016) and the efficacy with pre-implantation genetic screening (Kung et al., 2015; Lee et al., 2015). Neither of these studies, which were performed on a small series of patients, validated the procedures, nor established the rates of false positive and false negative results (Breman et al., 2016; Kølvraa et al., 2016). With the exception of only one false negative result of a deletion of 1q previously diagnosed by a microarray in CVS (Breman et al., 2016), all the findings in fetal trophoblasts were confirmed in CVS or amniotic fluid. There are plans to continue this project and validate trophoblast-based NIPT comparing the results with cell-free NIPT and with CVS or amniotic fluids tested with array-CGH. The ultimate goals are to launch a trophoblast-based NIPT assay that gives results equally reliable to array-CGH in CVS or amniocentesis (Breman et al., 2016) and potentially replace cffDNA-based NIPT (Kølvraa et al., 2016). Overall, it was estimated that results could be delivered on a routine basis in less than a week, but given the complexity of the procedures it was deemed to be centralized (Kølvraa et al., 2016), probably in a laboratory with state-of-the-art diagnostic technology.

Comments and discussion This new technology for fetal genetic analysis in trophoblasts brings important improvements that were needed to solve several intermittent problems with NIPT performed on fetal cell-free DNA. The power to exclusively study the fetal genome in trophoblasts without interference from DNA of maternal origin is a significant development. Excess maternal cell-free DNA compared to the smaller fetal fraction in peripheral blood of pregnant women can cause difficulties for accurate interpretation of NIPT results. Therefore, restricting the analysis to fetal trophoblasts would eliminate results that do not reflect the genetic constitution of the fetus and instead are false positives indicative of incidental maternal findings. The latter include occult malignancies (Amant et al., 2015), copy number variations (CNVs) that translated into high risk for autosomal trisomies (Snyder et al., 2015; Chudova et al., 2016; Zhou et al., 2017), and maternal mosaicisms that frequently involve chromosome X and inaccurately suggest risk for a sex chromosome aneuploidy (Wang et al., 2014). The single-cell analysis of multiple trophoblasts is a meaningful advance for a prompt, early and accurate identification of confined placental mosaicism (CPM), a well-recognized cause of false positive results with cffDNA-based NIPT (Mardy and Wapner, 2016). In

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Genetics in the News The Milestone of Non-invasive Prenatal Identification of Chromosomal Abnormalities in Fetal Trophoblasts Recovered from Maternal Blood this regard, there are well documented NIPT results of trisomy 13 (Hall et al., 2013; Zhang et al., 2015), trisomy 18 (Dugo et al., 2014) and trisomy 21 (Lebo et al., 2015) due to CPM that were not confirmed by follow-up studies. These types of cases would benefit from the non-invasive analysis of fetal trophoblasts instead of invasive follow-up procedures with CVS or amniocentesis. It is important to recognize that even when cffDNA results turn out to be false positives related to CPM, they always generate anxiety for the prospective parents and create difficulties for the genetic counseling and management of the pregnancy. They also raise the risk of irreversible parental decisions to terminate pregnancies that probably are chromosomally normal when confirmatory follow-up studies by CVS or amniocentesis are declined. CPM has also been the cause of false negative NIPT results for autosomal trisomies, but these events are rarer (Hartwig et al., 2017). False negative NIPT results for aneuploidies have been suspected when abnormal ultrasound findings are found later in the pregnancy and invasive procedures that showed a chromosomal abnormality confirmed this presumption (Hochstenbach et al., 2015; Lebo et al., 2015). Informative studies in fetal trophoblasts may be possible in other cases of inconsistencies between normal NIPT results and fetal anomalies detected by ultrasound before proceeding with invasive diagnostic procedures that were recommended (Van Opstal et al., 2016). Rare autosomal trisomies for chromosomes 7, 16, 15, 22 and 8 which became increasingly common with the widespread use of NIPT (reviewed by Bianchi, 2017) could also be evaluated for confirmation and assessment of the clinical outcome of pregnancies through a comprehensive analysis in fetal trophoblasts. It is not surprising that the types and frequency of these abnormalities are similar to what has been reported by cytogenetics in CVS, mostly trisomies 2, 3, 7, 8, 9, 16, and 22 associated with CPM (Wolstenholme, 1996). The clinical implications of rare trisomies currently ascertained by NIPT are not fully understood yet and studies in fetal trophoblasts may contribute essential information. Confirmation of all positive results generated by the current NIPT technology with cell-free DNA is another improvement achievable with the genomic analysis of trophoblasts. The few abnormal cases confirmed so far (Breman et al. 2016; Kølvraa et al., 2016) demonstrated that it is possible to meet the standing recommendation for confirmatory studies of all positive NIPT results that today calls for invasive testing (CVS or amniocentesis) (Mardy and Wapner, 2016). The basis of this recommendation is that for trisomies 13, 18 and 21, the rarity of these disorders in the general obstetric population translates into relatively low positive predictive values by NIPT despite low false positive rates (Wang et al, 2015; Taylor-Phillips et al., 2016). Sex chromosome aneuplodies which are often associated with false positive NIPT results (Dugo et al., 2014) could also be confirmed or ruled out through the analysis of trophoblasts. There has been a controversy about the accuracy of detection of microdeletions and microduplications by cffDNA-based NIPT and the wisdom of offering this test in clinical settings that hasn’t been settled yet (Vora and O’Brien, 2014; Yaron et al., 2015). Based on evaluations of few patients (Srinivasan et al., 2013), single case reports (Peters et al., 2011; Jensen et al., 2012) and a

very new targeted capture enrichment assay (Neofytou et al., 2017) some argued it is feasible and clinically useful while others dispute these assertions (Lo et al, 2016). Furthermore, high false positive rates with a SNIP-based NIPT assay for microdeletions of 22q11.2 suggest the need of reflex deep DNA-sequencing for a confirmation (Gross et al., 2016). Other studies reported quite considerable numbers of false positive NIPT results for microdeletions (Sahoo et al., 2016; Schwartz et al., 2017). Given uncertainties about the diagnostic accuracy and the predictive values of the NIPT results, back in 2015 several professional organizations did not recommend NIPT screening for microdeletions (American College of Obstetricians and Gynecologists, Committee Opinion No 640, 2015; Dondorp et al., 2015). There were also recent calls for large validation studies to determine the sensitivity and specificity of current NIPT technologies to detect microdeletions before a clinical use (Saho et al., 2016). All things considered, subsequent recommendations were issued to expand NIPT only targeting clinically significant microdeletions and microduplications with a severe phenotype (Benn, 2015,) or limited to well-characterized genomic disorders based on experimental validation (Pescia et al., 2017). Molecular analysis of fetal trophoblasts with the aid of the latest developments with NGS in single cells was proposed as a suitable alternative for detecting microdeletions and microduplications in an accurate non-invasive way (Breman et al. 2016; Kølvraa et al., 2016). Observations by microarrays of microduplications and microdeletions in 1-1.7% of pregnancies with a normal ultrasound and normal karyotype (Wapner et al., 2012) support the rationale to have a dependable NIPT testing for these abnormalities in the near future. It may be possible to diagnose inherited genetic diseases due to parental gonadal mosaicism at a very early gestational age through the genomic analysis of trophoblasts. Gonadal mosaicism is a condition suspected in healthy couples who had several children affected with a genetic disease of well-established etiology, neither parent is a constitutional carrier of a predisposing abnormality but one of them is suspected to carry gonadal cells with a pathogenic mutation that was transmitted to the offspring. Gonadal mosaicism is rather rare, but is a well-known cause of recurrent dominant, recessive, and X-linked gene disorders (Zlotogora, 1998) and less frequently chromosomal abnormalities (Tosca et al., 2010). Genetic counseling and estimates of recurrence risk in gonadal mosaicism are difficult. Today, the only alternatives for couples seeking a healthy liveborn offspring are invasive prenatal diagnosis with CVS or amniocentesis, and preimplantation genetic diagnosis in embryos generated “in vitro” (assisted reproductive technology or ART). Diagnostic studies in trophoblasts now seem to be an option for the couples who don’t pursue the aforementioned courses of action. Finally, looking into the future and turning attention to gene-related disorders, the detection of deleterious mutations through the analysis of trophoblasts was considered realistic and advantageous compared to the use of cff-DNA of fetal origin (Breman et al. 2016; Kølvraa et al., 2016). This view was justified by data that mutations in ~500 genes related to intellectual disabilities may occur in 3-5 per thousand pregnancies (de Ligt et al., 2013). Although prenatal screening and diagnosis of point

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References

mutations associated with clinically distinct genetic disorders is already feasible with NIPT in cell-free fetal DNA (Drury et al., 2016), there are concerns about false positive results linked to artifacts appearing during DNA amplification and sequencing. In spite of these caveats, recent improvements in DNA sequencing at the single-cell level and the independent analysis of several trophoblasts were deemed adequate steps to minimize such risks and move forward (Breman et al., 2016). Given the modern trend of preconception carrier screening prior to conception to identify mutations associated with many heritable genetic diseases, also called “expanded carrier screening” (Langlois et al., 2015), couples who carry gene mutations may choose to study their pregnancies at risk of being affected through the early testing of trophoblasts. “De novo” gene mutations which are not so rare could be ascertained as well once the technology is tuned up for clinical use. Under such scenarios, over time we can expect a reduction in the population frequency of deleterious gene mutations, of certain genetic diseases, and in the burden of the medical care related to these conditions.

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Conclusion The decisive molecular evaluation accomplished in fetal trophoblasts represents a turning point in non-invasive prenatal testing that most likely will revolutionize the fields of prenatal care, diagnosis and screening, as well as the prevention of genetic diseases. These studies were the first to accomplish a goal that has been elusive for many years: The isolation of pure fetal cells (in this case trophoblasts) that in turn allowed a reliable study of the fetal genome by array-CGH and NGS, and the identification of chromosome abnormalities. This groundbreaking research in cytogenetic diagnosis was credited to persistance in the face of many drawbacks, very meticulous work, and advances in fetal cell isolation technology (Breman et al., 2016). Overall fetal trophoblasts, which have been on the radar for some time, turned out to be the “golden key” that opened doors to unprecedented prospects in non-invasive prenatal genetic studies. Previous investigations focused on the detection of the gene disorders cystic fibrosis (CF) and spinal muscular atrophy (SMA), first in circulating trophoblasts (Mouawia et al., 2012) and then in trophoblasts from the endocervical canal (Pfeifer et al., 2016). Most recently, comprehensive genomic profiling was achieved by a multiplexed targeted sequencing approach with NGS in fetal DNA from trophoblasts that were isolated from the endocervical canal (Jain et al., 2016). Time will tell if the confident prediction of an “era of prenatal diagnosis with trophoblasts” (Beaudet, 2016) settles in the clinical practice after so many years of failed attempts with other fetal cells and eventually fulfills the expectations to supersede the existing tests with cell-free fetal DNA.

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Genetics in the News The Milestone of Non-invasive Prenatal Identification of Chromosomal Abnormalities in Fetal Trophoblasts Recovered from Maternal Blood Palomaki GE, Deciu C, Kloza EM, Lambert-Messerlian GM, Haddow JE, Neveux LM, Ehrich M, van den Boom D, Bombard AT, Grody WW, Nelson SF, Canick JA. DNA sequencing of maternal plasma reliably identifies trisomy 18 and trisomy 13 as well as Down syndrome: An international collaborative study. Genet Med. 2012;14: 296-305. Pescia G, Guex N, Iseli C, Brennan L, Osteras M, Xenarios I, Farinelli L, Conrad B. Cell-free DNA testing of an extended range of chromosomal anomalies: clinical experience with 6,388 consecutive cases. Genet Med. 2017;19: 169-175. Peters D, Chu T, Yatsenko SA, Hendrix N, Hogge WA, Surti U, Bunce K, Dunkel M, Shaw P, Rajkovic A. Noninvasive prenatal diagnosis of a fetal microdeletion syndrome. N Engl J Med. 2011;365: 1847-1848. Pfeifer I, Benachi A, Saker A, Bonnefont JP, Mouawia H, Broncy L, Frydman R, Brival ML, Lacour B, Dachez R, Paterlini-Bréchot P. Cervical trophoblasts for noninvasive single-cell genotyping and prenatal diagnosis. Placenta. 2016;37: 56-60. Sahoo T, Hovanes K, Strecker MN, Dzidic N, Commander S, Travis MK. Expanding noninvasive prenatal testing to include microdeletions and segmental aneuploidy: case for concern? Genet Med. 2016;18: 275-276. Schwartz S, Pasion R, Gadi I, Penton A, Phillips K, Schleede J, Burnside R, Tepperberg J, Papenhausen P, Kohan M, Platt L. Cell free DNA detection of microdeletions: Microarray and FISH follow-up with unexpected and complex findings. Abstract No 756. ACMG Annual Clinical Genetics Meeting. March 21-25, 2017. Snyder MW, Simmons LE, Kitzman JO, Coe BP, Henson JM, Daza RM, Eichler EE, Shendure J, Gammill HS. Copy-number variation and false positive prenatal aneuploidy screening results. N Engl J Med. 2015;372: 1639-1645. Srinivasan A, Bianchi DW, Huang H, Sehnert AJ, Rava RP. Noninvasive detection of fetal subchromosome abnormalities via deep sequencing of maternal plasma. Am J Hum Genet. 2013; 92: 167-176. Taylor-Phillips S, Freeman K, Geppert J, Agbebiyi A, Uthman OA, Madan J, Clarke A, Quenby S, Clarke A. Accuracy of non-invasive prenatal testing using cell-free DNA for detection of Down, Edwards and Patau syndromes: A systematic review and meta-analysis. BMJ Open. 2016;6: e010002. Tosca L, Brisset S, Petit FM, Lecerf L, Rousseau G, Bas C, Laroudie M, Maurin M-L, Tapia S, Picone O, Prevot S, Goosens M, Labrune P, Tachdjian G. Recurrent 70.8 Mb 4q22.2q32.3 duplication due to ovarian germinal mosaicism. Eur J Hum Genet. 2010;18: 882-888. Van Opstal D, Srebniak MI, Polak J, de Vries F, Govaerts LC, Joosten M, Go AT, Knapen MF, van den Berg C, Diderich KE, Galjaard RJ. False negative NIPT results: Risk figures for chromosomes 13, 18, and 21 based on chorionic villi results in 5967 cases and literature review. PLoS One. 2016;11: e0146794. Vora NL, O’Brien BM. Noninvasive prenatal testing for microdeletion syndromes and expanded trisomies. Proceed with caution. Obstet Gynecol. 2014;123: 1097-1099. Wang J-C, Sahoo T, Schonberg S, Kopita KA, Ross L, Patek K, Strom CM. Discordant noninvasive prenatal testing and cytogenetic results: A study of 109 consecutive cases. Genet Med. 2015;17: 234-236. Wang Y, Chen Y, Tian F, Zhang J, Song Z, Wu Y, Han X, Hu W, Ma D, Cram D, Cheng W. Maternal mosaicism is a significant contributor to discordant sex chromosomal aneuploidies associated with noninvasive prenatal testing. Clin Chem. 2014;60: 251-259. Wapner RJ, Martin CL, Levy B, Ballif BC, Eng CM, Zachary JM, Savage M, Platt LD, Saltzman D, Grobman WA, Klugman S, Scholl T, Simpson JL, Mc Call K, Aggarwal VS, Bunke B, Nahum O, Patel A, Lamb AN, Thom EA, Beaudet AL, Ledbetter DH, Schaffer LG, Jackson L. Chromosomal microarray versus karyotyping for prenatal diagnosis. N Engl J Med. 2012; 367: 2175-2184. Wolstenholme J. Confined placental mosaicism for trisomies 2, 3, 7, 8, 9, 16, and 22. Their incidence, likely origins, and mechanisms for cell lineage compartmentalization. Prenat Diagn. 1996;16: 511-524.

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Genetics in the News The Milestone of Non-invasive Prenatal Identification of Chromosomal Abnormalities in Fetal Trophoblasts Recovered from Maternal Blood Zhang H, Gao Y, Jiang F, Fu M, Yuan Y, Guo Y, Zhu Z, Lin M, Liu Q, Tian Z, Zhang H, Chen F, Lau TK, Zhao L, Yi X, Yin Y, Wang W. Non-invasive prenatal testing for trisomies 21, 18 and 13: clinical experience from 146958 pregnancies. Ultrasound Obstet Gynecol. 2015;45: 530-538. Zhou X, Sui L, Xu Y, Song Y, Qi Q, Zhang J, Zhu H, Sun H, Tian F, Wu M, Cram DS, Liu J . Contribution of maternal copy number variations to false-positive fetal trisomies detected by noninvasive prenatal testing. Prenat Diagn. 2017;37: 318-322. Zlotogora J. Germ line mosaicism. Hum Genet. 1998;102: 381-386.

Yaron Y, Jani J, Schmid M, Oepkes D. Current status of testing for microdeletion syndromes and rare autosomal trisomies using cell-free DNA technology. Obstet Gynecol. 2015;126: 1095-1099. Yin A-H, Peng C-F, Zhao X, Caughey BA, Yang J-X, Liu J, Huang W-W, Liu C, Luo D-H, Liu H-L, Chen Y-Y Wu J, Hou R, Zhang M, Ai M, Zheng L, Xue RQ, Mai M-Q, Guo F-F, Qi Y-M, Wang D-M, Krawczyk M, Zhang D, Wang Y-N, Huang Q-F, Karin M, Zhang K . Noninvasive detection of fetal subchromosomal abnormalities by semiconductor sequencing of maternal plasma DNA. Proc Natl Acad Sci USA. 2015;112: 14670-14675.

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Brain Tickler

Brain Tickler Summary (see inside front cover)

Solution: 46,XY,inv(12)(q13.3q23)

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Continuing Education Opportunities

Column Editor: Sally J. Kochmar, MS, CG(ASCP)CM

Test Yourself #3, 2017 Readers of The Journal of the Association of Genetic Technologists are invited to participate in this “open book test” as an opportunity to earn Contact Hours. AGT offers 3 Contact Hours for this Test Yourself based on articles in Volume 43, Number 2, Second Quarter 2017 of the Journal. Test Yourself is free to AGT members and $30 for non-members. To take this exam, send a copy of your completed Answer Sheet along with the completed Contact Hours Reporting Form to the AGT Education Committee Representative in your region. The list of representatives can be found on the AGT website. Non-members should submit a check payable to AGT for $30 with their answer sheet. Entry material must be post-marked on or before December 1, 2017. Passing score is 85% or 17 out of 20 questions answered correctly. Compiled by Doina Ciobanu and Sally Kochmar.

The following questions are from Fonseka L et al. Cytogenetics and Molecular Genetics of Prostate Cancer: A Comprehensive Update. J Assoc Genet Technol. 2015;41(3): 100-111.

5. The FDA approved Exondys 51 on: a. b. c. d.

1. Prostate cancer (PCa) is: I. one of the most commonly diagnosed cancers in males in the U.S. II. linked to mutations in specific genes. III. a leading cause of cancer death in the U.S. IV. a cancer whose expression varies from asymptomatic to aggressive presentation. a. b. c. d.

The following questions are from Brar R et al. Mosaic Trisomy 9p in a Patient with Mild Dystrophic Features and Normal Intelligence. J Assoc Genet Technol. 2017;43(2): 56-58. 6. Which of the following is not a clinical feature of 9p duplications?

I and II II, III and IV I, III and IV All of the above

a. Microbrachycephaly b. Low-set ears c. Heart-related problems d. Delayed bone age

The following questions are from A Note from the Editor. Duchenne Muscular Dystrophy, Genetics, the FDA and Drug Pricing. J Assoc Genet Technol. 2017;43(2): 53-55.

7. Unique facial features of the patient included prominent nasal bridge, low-set ears and a sloping forehead. a. True b. False

1. Duchenne Muscular Dystrophy (DMD):

8. Microarray analysis of this patient revealed a………... duplication of the 9p region.

I. Is a progressive disease II. Has an X-linked dominant inheritance pattern III. Affects mostly boys IV. Is a muscle-wasting disease a. b. c. d.

a. b. c. d.

I, II and IV I, II and III I, III and IV All of the above

I. Was first described in 1970. II. Has been reported in more than 150 patients. III. Is the fourth most common life-compatible trisomy. IV. Has been commonly reported in the literature with characteristic phenotypic features and intellectual disabilities.

The FDA approved Exondys 51 to treat DMD Exondys 51 is an antisense oligonucleotide Exondys 51 is manufactured by Sarepta Therapeutics The most common mutation in DMD is in exon 79

a. b. c. d.

3. Sarepta’s trial included 121 patients. a. True b. False 4. Who was the director of the FDA’s Center for Drug Evaluation and Research? a. b. c. d.

68 Kb 608 Mb 68 bp 68 Mb

9. Whole arm or partial trisomy 9p:

2. All of the following are correct, except: a. b. c. d.

April 25, 2016 May 25, 2016 February 10, 2017 September 9, 2016

I, II and III II, III and IV I, III and IV All of the above

10. Conventional cytogenetic analysis indicated a normal female in…out of … metaphases:

Robert Califf Ronald Farkas Janet Woodcock Luciana Borio

a. b. c. d.

16, 20 4, 20 4, 5 5, 20

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Continuing Education Opportunities

Column Editor: Sally J. Kochmar, MS, CG(ASCP)CM

The following questions are from Mah M. Best Education Practices, Teaching the Next Generation of Technologists NextGeneration Sequencing. J Assoc Genet Technol. 2017;43(2): 61-62.

The following questions are from Dowiak A et al. Cytogenetic Characterization of Myeloid Neoplasms with t(2;3)(p1325;q25-29): An Analysis of 60 Cases. J Assoc Genet Technol. 2017;43(2): 64-68.

11. The goal of the NGS workshop was to demonstrate the importance of supporting the analytical workflow from data generation to variant reporting.

15. Choose the incorrect statement: a. Chromosomal translocations involving 2p and 3q are rare. b. This article reports a study done on 60 patients with myeloid malignancies. c. Translocations involving 2p and 3q are associated with a good prognosis. d. Myeloid malignancies are clonal diseases of hematopoietic stem cells.

a. True b. False 12. All of the following statements are true except: a. The NGS workshop was presented to students at the Michener Institute. b. There are four post-secondary institutions offering the Canadian Medical Association. accredited Clinical Genetics Technology program in Canada. c. The workshop was delivered in Toronto. d. This year, the workshop incorporated feedback for areas to improve from last year.

16. Chromosomal translocations involving 2p and 3q are observed in about…….% of myeloid malignancies. a. 5 b. 50 c. 0.5 d. 0.05

13. According to the author, there is still a learning gap: a. b. c. d.

17. The locus of the EVI1 gene is on 2p21. a. True b. False

in working with HGVS nomenclature. in understanding fundamentals of DNA. in the presentation of software variants. between manual sequence analysis and understanding the use of advanced analytical software.

18. The most common additional abnormality reported in this study was: a. t(9;22) b. monosomy 5 c. deletion 5q d. monosomy 7

14. Individuals who work away from the bench are: I. Genetic counselors II. Variant analysts III. Annotation specialists IV. Genomic specialists a. b. c. d.

19. EVI1 rearrangement was confirmed in…….cases.

I, II and IV I, II and III II, III and IV All of the above

a. 19 b. 14 c. 38 d. 16 20. All of the following are true, except: a. b. c. d.

Answer Sheet 1.____ 2.____ 3.____ 4.____ 5.____

6.____ 7.____ 8.____ 9.____ 10.____

t(2;3) existed as sole abnormality in 22 cases Deletion 5q was observed in 26% of cases t(2;3) was a primary abnormality in 38% of cases t(9;22) occurred in 13% of cases

Please Print Clearly

Answers to Test Yourself #2, 2017

11.____ 12.____ 13.____ 14.____ 15.____

Passing Score: (passing score is 19/22 or 86.3%)

16.____ 17.____ 18.____ 19.____ 20.____

1.c 2.b 3.a 4.b 5.d 6.d

7.d 8.b 9.c 10.d 11.a

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12.c 13.d 14.c 15.d 16.b 17.d

18.d 19.b 20.d 21.c 22.d

Continuing Education Opportunities

Column Editor: Sally J. Kochmar, MS, CG(ASCP)CM

The Association of Genetic Technologists Name:

CEU REPORTING FORM

First Name

Last Name

(check appropriate box) Address

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Address:

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Country: Email Address: Program Title/Activity:

Date(s) of Publications:

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T ES T YOURSE L F , 3 r d QUAR T ER 2015 7 J P544 Qtr

CEU Area (1, 2, 3, 4): →

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ASSOC

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Institution, Association, Journal, etc.

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Send your Test Yourself to your AGT Education Committee Regional Representative. A list of representatives can be found here

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• Explore our degree program in Cytogenetic Technology including on campus part-time enrollment, oncampus full-time enrollment, and hybrid online enrollment • Explore our Internet-Based Review Course in Clinical Cytogenetics with ongoing enrollment • Explore our Annual Comprehensive Review Course in Clinical Cytogenetics to prepare for ASCP-BOC (CG) exam For more information contact Jun Gu, M.D., Ph.D., CG (ASCP) Program Director/Associate Professor jungu@mdanderson.org 1-800-551-9503

Continuing Education Opportunities

The AGT Education Committee’s Journal Club Journal Clubs are a great way to earn Contact Hours without leaving your home or lab! Journal Clubs can be completed as a group or individually. Each Journal Club includes a reading list, several discussion questions and a post-test. The discussion questions provide a starting point for group discussion and give individuals taking a Journal Club questions to consider while reading the articles. The post-test is taken after reading the articles and is returned to the regional representatives of the Education Committee to be graded. Each successfully completed Journal Club is worth 4.0 Contact Hours. Journal Clubs can be ordered through the AGT Executive Office. READING LIST 54 – General Content Area: Chromosome Breakage Syndromes–2006

READING LIST 58 – General Content Area: Solid Tumor and FISH–2007

1. Chromosome Breakage Syndromes and Cancer 2. DEB Test for Fanconi Anemia Detection in Patients with Atypical Phenotype 3. Nijmegen Breakage Syndrome: Clinical Manifestation of Defective Response to DNA Doublestrand Breaks

1. Methylthioadenosine Phosphorylase Gene Deletions are Frequently Detected by Fluorescence in situ Hybridization in Conventional Chondrosarcoma 2. Solid Pseudopapillary Neoplasms of the Pancreas are Associated with FLI-1 Expression, but Not with EWS/FLI-1 Translocation 3. High Incidence of Chromosome 1 Abnormalities in a Series of 27 Renal Oncocytomas: Cytogenetic and Fluorescent In Situ Hybridization Studies

READING LIST 55 – General Content Area: Array Based Prenatal Genetics–2006 1. Array-based Comparative Genomic Hybridization Facilitates Identification of Breakpoints of a Novel der(1)t(1;18) (p36.3;q23)dn in a Child Presenting with Mental Retardation 2. Detection of Cryptic Chromosome Aberrations in a Patient with a Balanced t(1;9)(p34.2;p24) by Array-based Comparative Genomic Hybridization 3. Jumping Translocations in Multiple Myeloma

READING LIST 56 – General Content Area: Leukemia–2007 1. Fluorescence in situ Hybridization Analysis of Minimal Residual Disease and the Relevance of the der(9) Deletion in Imatinib-treated Patients with Chronic Myeloid Leukemia 2. Characterization of the t(17;19) Translocation by Gene-specific Fluorescent in situ Hybridizationbased Cytogenetics and Detection of the E2A-HLF Fusion Transcript and Protein in Patient’s Cells 3. Combination of Broad Molecular Screening and Cytogenetic Analysis for Genetic Risk Assignment and Diagnosis in Patients with Acute Leukemia

READING LIST 57 – General Content Area: Premature Chromosome Condensation–2007 1. Premature Chromosome Condensation in Humans Associated with Microcephaly and Mental Retardation: A Novel Autosomal Recessive Condition 2. Chromosome Condensation: DNA Compaction in Real Time 3. Phosphatase Inhibitors and Premature Chromosome Condensation in Human Peripheral Lymphocytes at Different CellCycle Phases

READING LIST 59 – General Content Area: Treatment of Prader-Willi Syndrome with Growth Hormone–2008 1. Two Years of Growth Hormone Therapy in Young Children with Prader-Willi Syndrome: Physical and Neurodevelopmental Benefits - American Journal of Medical Genetics Part A, Volume 143A, Issue 5, pages 443-448, 1 March 2007 2. Growth Hormone Therapy and Scoliosis in Patients with Prader-Willi Syndrome 3. Cause of Sudden, Unexpected Death of Prader-Willi Syndrome Patients with or without Growth Hormone Treatment

READING LIST 60 – General Content Area: Generics of Autism–2008 1. 15q11-13 GABAa Receptor Genes are Normally Biallelically Expressed in Brain yet are Subject to Epigenetic Dysregulation in Autism-Spectrum Disorders 2. Characterization of an Autism-Associated Segmental Maternal Heterodisomy of the Chromosome 15q11-13 Region 3. 15q Duplication Associated with Autism in a Multiplex Family with a Familial Cryptic Translocation t(14;15)(q11.2;q13.3) Detected Using Array-CGH

READING LIST 61 – General Content Area: Genetics of Nicotine Addiction–2008 1. Fine Mapping of a Linkage Region on Chromosome 17p13 Reveals that GABARAP and DLG4 are Associated with Vulnerability to Nicotine Dependence in European-Americans

2. Genomewide Linkage Scan for Nicotine Dependence: Identification of a Chromosome 5 Risk Locus 3. Genetic Linkage to Chromosome 22q12 for a Heavy-Smoking Quantitative Trait in Two Independent Samples

READING LIST 62 – General Content Area: Somatic Mutation Detection–2007 1. Inferring Somatic Mutation Rates Using the Stop-Enhanced Green Fluorescent Protein Mouse 2. Paternal Age at Birth is an Important Determinant of Offspring Telomere Length 3. Genome-Wide SNP Assay Reveals Structural Genomic Variation, Extended Homozygosity and Cellline Induced Alterations in Normal Individuals

READING LIST 63 – General Content Area: Polyglutamine Neurodegenerative Disorders–2007 1. CAG- Encoded Polyglutamine Length Polymorphism in the Human Genome 2. Polyglutamine Neurodegenerative Diseases and Regulation of Transcription: Assembling the Puzzle 3. Pathogenesis and Molecular Targeted Therapy of Spinal and Bulbar Muscular Atrophy

READING LIST 64 – General Content Area: Clinical Applications of Noninvasive Diagnostic Testing–2008 1. Digital PCR for the Molecular Detection of Fetal Chromosomal Aneuploidy 2. Noninvasive Testing for Colorectal Cancer: A Review 3. Novel Blood Biomarkers of Human Urinary Bladder Cancer

READING LIST 65 – General Content Area: Diabetes–2010 1. The Development of c-MET Mutation Detection Assay 2. Molecular Mechanisms of Insulin Resistance in Chronic Hepatitis C 3. A Genetic Diagnosis of HNF1A Diabetes Alters Treatment and Improves Glycaemic Control in the Majority of Insulin-Treated Patients

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Continuing Education Opportunities READING LIST 66 – General Content Area: Diabetes–2010 1. Distribution of Human Papillomavirus Genotypes in Invasive Squamous Carcinoma of the Vulva 2. Distribution of HPV Genotypes in 282 Women with Cervical Lesions: Evidence for Three Categories of Intraepithelial Lesions Based on Morphology and HPV Type 3. Evaluation of Linear Array Human Papillomavirus Genotyping Using Automatic Optical Imaging Software

READING LIST 67 – General Content Area: Pancreatic Cancer and its Biomarkers–2010 1. Molecular Profiling of Pancreatic Adenocarcinoma and Chronic Pancreatitis Identifies Multiple Genes Differentially Regulated in Pancreatic Cancer 2. Effect of Recombinant Adenovirus Vector Mediated Human Interleukin-24 Gene Transfection on Pancreatic Carcinoma Growth 3. Highly Expressed Genes in Pancreatic Ductal Adenocarcinomas: A Comprehensive Characterization and Comparison of the Transcription Profiles Obtained from Three Major Technologies

READING LIST 68 – General Content Area: Influenza A(H1N1) Virus–2010 1. Detection of Influenza A(H1N1)v Virus by Real-Time RT-PCR 2. Economic Consequences to Society of Pandemic H1N1 Influenza 2009 – Preliminary Results for Sweden 3. Response after One Dose of a Monovalent Influenza A (H1N1) 2009 Vaccine — Preliminary Report

READING LIST 69 – General Content Area: The Development of c-MET Mutation Detection Assay–2010 1. Somatic Mutations in the Tyrosine Kinase Domain of the MET Proto-Oncogene in Papillary Renal Carcinomas 2. Expression and Mutational Analysis of MET in Human Solid Cancers 3. Role of cMET Expression in Non-Small-Cell Lung Cancer Patients Treated with EGFR Tyrosine Kinase Inhibitors

READING LIST 70 – General Content Area: Molecular Cardiology–2010 1. Identification of a Pleiotropic Locus on Chromosome 7q for a Composite Left Ventricular Wall Thickness Factor and Body Mass Index: The HyperGEN Study 2. Novel Quantitative Trait Locus is Mapped to Chromosome 12p11 for Left Ventricular Mass in Dominican Families: The Family Study of Stroke Risk and Carotid Atherosclerosis

3. Genome-Wide Association Study Identifies Single-Nucleotide Polymorphism in KCNB1 Associated with Left Ventricular Mass in Humans: The HyperGEN Study

READING LIST 71 – General Content Area: Detection of Clarithromycin Resistance in H. Pylori–2010 1. Rapid Detection of Clarithromycin Resistance in Helicobacter Pylori Using a PCR-based Denaturing HPLC Assay 2. Rapid Screening of Clarithromycin Resistance in Helicobacter Pylori by Pyrosequencing 3. Quadruplex Real-Time PCR Assay Using Allele-Specific Scorpion Primers for Detection of Mutations Conferring Clarithromycin Resistance to Helicobacter pylori

READING LIST 72 – General Content Area: Werner Syndrome Gene–2010 1. Telomeric protein TRF2 protects Holliday junctions with telomeric arms from displacement by the Werner syndrome helicase 2. WRN controls formation of extrachromosomal telomeric circles and is required for TRF2DeltaBmediated telomere shortening 3. Sequence-specific processing of telomeric 3' overhangs by the Werner syndrome protein exonuclease activity

READING LIST 73 – General Content Area: Diagnosis of Melanoma Using Fluorescence in Situ Hybridization–2011 1. Using Fluorescence in situ Hybridization (FISH) as an Ancillary Diagnostic Tool in the Diagnosis of Melanocytic Neoplasms 2. Transcriptomic versus Chromosomal Prognostic Markers and Clinical Outcome in Uveal Melanoma 3. Detection of Copy Number Alterations in Metastatic Melanoma by a DNA Fluorescence In situ Hybridization Probe Panel and Array Comparative Genomic Hybridization: A Southwest Oncology Group Study (S9431)

READING LIST 74 – General Content Area: Role of Short Interfering RNA in Gene Silencing–2011 1. Highly Specific Gene Silencing by Artificial miRNAs in Rice. 2. Gene silencing by RNAi in mouse Sertoli cells. 3. Retrovirus-delivered siRNA.

READING LIST 75 – General Content Area: Multiple Myeloma: Molecular Markers and Tests–2010 1. Multiple Myeloma: Lusting for NF-B 2. Functional Interaction of Plasmacytoid Dendritic Cells with Multiple Myeloma Cells: A Therapeutic Target 3. High-resolution genomic profiles define distinct clinico-pathogenetic subgroups of multiple myeloma patients

READING LIST 76 – General Content Area: Colorectal Cancer and Loss of Imprinting of IGF2–2010 1. Loss of imprinting of IGF2 as an epigenetic marker for the risk of human cancer 2. Temporal stability and age-related prevalence of loss of imprinting of the insulin-like growth factor-2 gene. 3. Epigenetics at the Epicenter of Modern Medicine

READING LIST 77 – General Content Area: Health Effects Associated with Disruption of Circadian Rhythms–2011 1. Circadian Polymorphisms associated with Affective Disorders 2. A new approach to understanding the impact of Circadian Disruption on Human Health 3. Circadian Rhythm and its Role in Malignancy

READING LIST 78 – General Content Area: Role of Hedgehog Signaling Pathway in Diffuse Large BCell Lymphoma–2010 1. Sonic hedgehog signaling proteins and ATP-bindig cassette G2 are aberrantly expressed in diffuse large B-cell lymphoma 2. Sonic Hedgehog Signaling Pathway is Activated in ALK-Positive Anaplastic Large Cell Lymphoma 3. Sonic Hedgehog is Produced by Follicular Dendritic Cells and Protects Germinal Center B Cells from Apoptosis

READING LIST 79 – General Content Area: Whole Genome Amplification & 1986 Chernobyl, Ukraine Nuclear Power Plant Accident–2010 1. BAC-FISH assays delineate complex chromosomal rearrangements in a case of post-Chernobyl childhood thyroid cancer. 2. Whole Genome Amplification Technologies - Eliminating the Concern Over Running Out of DNA Samples Mid Experiment. 3. A break-apart fluorescence in situ hybridization assay for detecting RET translocation in papillary thyroid carcinoma.

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Continuing Education Opportunities READING LIST 80 – General Content Area: Expression of miRNA in Diffuse Large B-Cell Lymphoma–2010 1. Differentiation stage specific expression of microRNAs in B lymphocytes and diffuse large B-cell lymphomas. 2. Coordinated Expression of MicroRNA-155 and Predicted Target Genes in Diffuse Large B-cell Lymphoma. 3. Specific expression of miR-17-5p and miR127 in testicular and central nervous system diffuse large B-cell lymphoma.

READING LIST 81 – General Content Area: The Genetics of Bipolar Disorder–2010 1. Gene-wide analyses of genomewide association data sets: evidence for multiple common risk alleles for schizophrenia and bipolar disorder and for overlap in genetic risk 2. Subcortical Gray Matter Volume Abnormalities in Healthy Bipolar Offspring: Potential Neuroanatomical Risk Marker for Bipolar Disorder? 3. Genetic and Environmental Influences on Pro-Inflammatory Monocytes in Bipolar Disorder

READING LIST 82 – General Content Area: Role and Detection of Human Endogenous Retroviruses in Rheumatoid Arthritis–2011 1. Increase in Human Endogenous Retrovirus HERV-K(HML-2) Viral Load in Active Rheumatoid Arthritis. 2. A role for human endogenous retrovirus-K (HML-2) in rheumatoid arthritis: investigating mechanisms of pathogenesis 3. Lack of Detection of Human Retrovirus-5 Proviral DNA in Synovial Tissue and Blood Specimens From Individuals With Rheumatoid Arthritis or Osteoarthritis.

READING LIST 83 – General Content Area: Roles of Oncogenes in Breast Cancer–2010 1. The Nuclear Receptor Coactivator Amplified in Breast Cancer-1 Is Required for Neu (ErbB2/HER2) Activation, Signaling, and Mammary Tumorigenesis in Mice. 2. Dysregulated miR-183 inhibits migration in breast cancer cells. 3. Current and emerging biomarkers in breast cancer: prognosis and prediction

READING LIST 84 – General Content Area: Elevated Levels of Human Endogenous Retrovirus-W in Patients With First Episode of Schizophrenia–2010 1. Elevated Levels of Human Endogenous Retrovirus-W Transcripts in Blood Cells From Patients With First Episode Schizophrenia. 2. Endogenous Retrovirus Type W GAG and

Envelope Protein Antigenemia in Serum of Schizophrenic Patients. 3. Reduced Expression of Human Endogenous Retrovirus (HERV)– W GAG Protein in the Cingulate Gyrus and Hippocampus in Schizophrenia, Bipolar Disorder, and Depression.

READING LIST 85 – General Content Area: Esophageal Cancer–2010 1. The Changing Face of Esophageal Cancer 2. Epidermal Growth FactorInduced Esophageal Cancer Cell Proliferation Requires Transactivation of-Adrenoceptors 3. Esophageal cancer risk by type of alcohol drinking and smoking: a casecontrol study in Spain

READING LIST 86 – General Content Area: p53 Family and Its Role In Cancer–2010 1. Telomere dysfunction suppresses spontaneous tumorigenesis in vivo by initiating p53-dependent cellular senescence. 2. Shaping genetic alterations in human cancer: the p53 mutation paradigm. 3. p53 polymorphisms: cancer implications.

READING LIST 87 – General Content Area: Proteins Involved with Chronic Myleloid Leukemia and Other Myleoprolifertive Disorders–2011 1. Gain-of-Function Mutation of JAK2 in Myeloproliferative Disorders. 2. Kinase domain mutants of Bcr enhance Bcr-Abl oncogenic effects. 3. Destabilization of Bcr-Abl/Jak2 Network by a Jak2/Abl Kinase Inhibitor ON044580 Overcomes Drug Resistance in Blast Crisis Chronic Myelogenous Leukemia (CML).

READING LIST 88 – General Content Area: DNA Topology–2010 1. The why and how of DNA unlinking. 2. Bacterial DNA topology and infectious disease. 3. DNA topoisomerase II and its growing repertoire of biological functions.

READING LIST 89 – General Content Area: LPL Waldenstrom Macroglobulinemia–2010 1. Spontaneous splenic rupture in Waldenstrom's macroglobulinemia. 2. How I Treat Waldenstrom's Macroglobulinemia. 3. International prognostic scoring system for Waldenström Macroglobulinemia.

READING LIST 90 – General Content Area: Next Generation Sequencing Platforms–2010

sequencing technologies. 2. Combining Next-Generation Sequencing Strategies for Rapid Molecular Resource Development from an Invasive Aphid Species. 3. Evaluation of next generation sequencing platforms for population targeted sequencing studies.

READING LIST 91 – General Content Area: Hutchinson-Gilford Progeria Syndrome–2011 1. Epidermal expression of the truncated prelamin a causing Hutchinson– Gilford progeria syndrome: effects on keratinocytes, hair and skin 2. Defective Lamin A-Rb Signaling in Hutchinson-Gilford Progeria Syndrome and Reversal by Farnesyltransferase Inhibition 3. Increased expression of the Hutchinson– Gilford progeria syndrome truncated lamin a transcript during cell aging.

READING LIST 92 – General Content Area: Severe Combined Immunodeficiency Screening and Patient Studies–2011 1. Long-term Outcome after Hematopoietic Stem Cell Transplantation of a Singlecenter Cohort of 90 Patients with Severe Combined Immunodeficiency. 2. Why Newborn Screening for Severe Combined Immunodeficiency Is Essential: A Case Report. 3. Development of a Routine Newborn Screening Protocol for Severe Combined Immunodeficiency.

READING LIST 93 – General Content Area: Biological and Physical Hazards Encountered in the Laboratory–2011 1. Lab Safety Matters. 2. Virus Transfer from Personal Protective Equipment to Healthcare Employees’ Skin and Clothing. Emerging Infectious Diseases. 3. Prevalence of Hepatitis C Virus Infection Among Health-Care Workers: A 10-Year Survey.

READING LIST 94 – General Content Area: Rapid whole-genome mutational profiling using nextgeneration sequencing technologies–2011 1. Comparison of next generation sequencing technologies for transcriptome characterization. 2. ShortRead: a bioconductor package for input, quality assessment and exploration of highthroughput sequence data. 3. Next-Generation Sequencing: From Basic Research to Diagnostics.

1. Rapid whole-genome mutational profiling using next-generation

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Continuing Education Opportunities READING LIST 95 – General Content Area: Cell Death–2011 1. Hypoxia induces autophagic cell death in apoptosis-competent cells through a mechanism involving BNIP3. 2. Truncated forms of BNIP3 act as dominant negatives inhibiting hypoxiainduced cell death. 3. Hypoxia-Induced Autophagy Is Mediated through Hypoxia-Inducible Factor Induction of BNIP3 and BNIP3L via Their BH3 Domains.

READING LIST 96 – General Content Area: Genetic Associations of Cerebral Palsy–2011 1. Mannose-binding lectin haplotypes may be associated with cerebral palsy only after perinatal viral exposure. 2. Methylenetetrahydrofolate Reductase Gene Polymorphisms and Cerebral Palsy in Chinese Infants. 3. Apolipoprotein E genotype and cerebral palsy.

READING LIST 97 – General Content Area: Treatments for HIV/AIDs–2011 1. Early Antiretroviral Therapy Reduces AIDS Progression/Death in Individuals with Acute Opportunistic Infections: A Multicenter Randomized Strategy Trial. 2. Asia can afford universal access for aids prevention and treatment. 3. Trends in reported aids defining illnesses (adis) among participants in a universal antiretroviral therapy program: an observational study.

READING LIST 98 – General Content Area: Myosin Light Chain Kinase (MYLK) Gene Mutation Affect in Smooth Muscle Cells–2012 1. Myosin light chain kinase is central to smooth muscle contraction and required for gastrointestinal motility in mice. 2. Mutation in myosin light chain kinase cause familial aortic dissections. 3. Chemical genetics of zipper-interacting protein kinase reveal myosin light chain as a bona fide substrate in permeabilized arterial smooth muscle.

READING LIST 99 – General Content Area: Chromosome 6 and Its Associated Diseases–2011 1. Novel Cleft Susceptibility Genes in Chromosome 6q. 2. A susceptibility locus on chromosome 6q greatly increases risk lung cancer risk among light and never smokers. 3. The identification of chromosomal translocation, t(4;6)(q22;q15), in prostate cancer.

READING LIST 100 – General Content Area: Early onset of autosomal dominant Alzheimer disease–2011

READING LIST 105 – General Content Area: Inflammasome Activation by Proteins–2011

1. Genetics of Alzheimer Disease. 2. New mutation in the PSEN1 (E120G) gene associated with early onset Alzheimer’s disease. 3. Evidence for Three Loci Modifying Ageat-Onset of Alzheimer’s Disease in EarlyOnset PSEN2 Families.

1. Activation of the NLRP3 inflammasome by islet amyloid polypeptide provides a mechanism for enhanced IL-1 2 in type 2 diabetes. 2. ER stress in Alzheimer’s disease: A novel neuronal trigger for inflammation and Alzheimer’s pathology. 3. The inflammasome: a caspase-1activation platform that regulates immune responses and disease pathogenesis.

READING LIST 101 – General Content Area: Multiplex PCR and Emerging Technologies for the Detection of Respiratory Pathogens–2011 1. A multiplex one-step real-time RT-PCR assay for influenza surveillance. 2. Taking New Tack, PrimeraDx Offers MDx Tech as Open Platform for Test Developers. 3. Comparison of Automated Microarray Detection with Real-Time PCR Assays for Detection of Respiratory Viruses in Specimens Obtained from Children.

READING LIST 102 – General Content Area: Single Nucleotide Polymorphism (SNP) Array Analysis–2011 1. A fast and accurate method to detect allelic genomic imbalances underlying mosaic rearrangements using SNP array data. 2. SAQC: SNP array quality control. 3. Calibrating the performance of SNP arrays for whole-genome association studies.

READING LIST 103 – General Content Area: Research of BRAF Gene Related to Cancer–2011 1. Kinase-Dead BRAF and Oncogenic RAS Cooperate to Drive Tumor Progression through CRAF. 2. Distinct patterns of DNA copy number alterations associate with BRAF mutations in melanomas and melanoma derived cell lines. 3. Pharmacodynamic Characterization of the Efficacy Signals Due to Selective BRAF Inhibition with PLX4032 in Malignant Melanoma.

READING LIST 104 – General Content Area: Microarray Single Nucleotide Polymorphism (SNP) Troubleshooting–2011 1. Model-based clustering of array CGH data. 2. Application of a target array comparative genomic hybridization to prenatal diagnosis. 3. A model-based circular binary segmentation algorithm for the analysis of array CGH data.

READING LIST 106 – General Content Area: DNA Barcoding–2011 1. Commercial Teas Highlight Plant DNA Barcode Identification Successes and Obstacles. 2. Mutational Patterns and DNA Barcode for Diagnosing Chikungunya Virus. 3. The Barcode of Life Data Portal: Bridging the Biodiversity Informatics Divide for DNA Barcoding.

READING LIST 107 – General Content Area: HERV-K and Its Correlation With Melanoma Cells–2011 1. Expression of human endogenous retrovirus K in melanomas and melanoma cell lines Cancer. 2. Expression of HERV-K correlates with status of MEK-ERK and p16INK4A-CDK4 pathways in melanoma cells cancer. 3. An endogenous retrovirus derived from human melanoma cells.

READING LIST 108 – General Content Area: Refractory Myeloma–2011 1. Pomalidomide plus low-dose dexamethasone in myeloma refractory to both bortezomib and lenalidomide: comparison of 2 dosing strategies in dual-refractory disease. 2. Relapse/Refractory Myeloma Patient: Potential Treatment Guidelines. 3. Emerging role of carfilzomib in treatment of relapsed and refractory lymphoid neoplasms and multiple myeloma.

READING LIST 109 – General Content Area: Short Tandem Repeat (STR) Technology in Forensic Community–2011 1. An integrated microdevice for highperformance short tandem repeat genotyping. 2. A comparison of the effects of PCR inhibition in quantitative PCR and forensic STR analysis. 3. Generating STR profile from "Touch DNA".

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Continuing Education Opportunities READING LIST 110 – General Content Area: Methods of Screening and Evaluation of Hepatitis C Virus–2011 1. Hepatitis c virus: prevention, screening, and interpretation of assays. 2. Serial follow-up of repeat voluntary blood donors reactive for anti-hcv elisa. 3. Comparison of fibrotest-actitest with histopathology in demonstrating fibrosis and necroinflammatory activity in chronic hepatitis b and c.

READING LIST 111 – General Content Area: Pharmacogenomics–2011 1. Pharmacogenomic testing: Relevance in medical practice: Why drugs work in some patients but not in others. 2. Clinical assessment incorporating a personal genome. 3. Genomics and drug response.

READING LIST 112 – General Content Area: Adrenoleukodystrophy–2011 1. Novel exon nucleotide deletion causes adrenoleukodystrophy in a Brazilian family. 2. X-linked adrenoleukodystrophy: ABCD1 de novo mutations and mosaicism. 3. Identification of novel SNPs of ABCD1, ABCD2, ABCD3, and ABCD4 genes in patients with Xlinked adrenoleukodystrophy (ALD) based on comprehensive resequencing and association studies with ALD phenotypes.

READING LIST 113 – General Content Area: Quality Assurance and Quality Control of Microarray Comparative Genomic Hybridization–2011 1. Customized oligonucleotide array-based comparative genomic hybridization as a clinical assay for genomic profiling of chronic lymphocytic leukemia. 2. Comparison of familial and sporadic chronic lymphocytic leukaemia using high resolution array comparative genomic hybridization. 3. Microarray-based comparative genomic hybridization.

READING LIST 114 – General Content Area: mFISH–2012 1. Human interphase chromosomes: a review of available molecular cytogenetic technologies. 2. Establishment of a new human pleomorphic malignant fibrous histiocytoma cell line, FU-MFH-2: molecular cytogenetic characterization by multicolor fluorescence in situ hybridization and comparative genomic hybridization. 3. CD5-negative Blastoid Variant Mantle Cell Lymphoma with Complex CCND1/ IGH and MYC Aberrations.

READING LIST 115 – General Content Area: Cystic Fibrosis - 2014 1. Rapid Detection of the ACMG/ACOGRecommended 23 CFTR DiseaseCausing Mutations Using Ion Torrent Semiconductor Sequencing 2. Long-Term Evaluation of Genetic Counseling Following False-Positive Newborn Screen for Cystic Fibrosis 3. Rapid Transport of Muco-Inert Nanoparticles in Cystic Fibrosis Sputum Treated with N-acetyl cysteine

READING LIST 116 – General Content Area: Autism - 2015 1. Intellectual disability and autism spectrum disorders: Causal genes and molecular mechanisms. 2. Aberrant tryptophan metabolism: the unifying biochemical basis for autism spectrum disorders? 3. Decreased tryptophan metabolism in patients with autism spectrum disorders

Copyright law prohibits AGT from supplying readers with the actual journal articles (electronically or otherwise). Availability of articles online does not imply the service is free. Some journals require a subscription or impose a fee. The web addresses are included for the convenience of those wishing to obtain the articles in this way.

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AGT Journal Club Question Order Form

To order the AGT Journal Club Questions, please fill in the requested information below. Make check or money order payable to AGT. Copyright law prohibits AGT from supplying readers with the actual journal articles (electronically or otherwise). Participants must obtain articles themselves. Discussion and Question Set for Reading List No. (Please enter the number of copies requested next to each Journal Club Number) ____54

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Visit our website at www.AGT-info.org Please allow 2-4 weeks for items to be shipped.

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Association Business

Letter from the President Change in inevitable. No matter our comfort level, or the gains and losses of the change, it will never be easy. Change is how we grow; as people, communities, and institutions. With that being stated, the Association of Genetic Technologists (AGT) is facing tremendous change and will need the help and support of all its members to continue to operate as successfully as we have in the past. High quality, professional business management services are expensive. AGT has utilized such services for over 20 years to help keep our best foot forward in the larger community of other genetic-based organizations. Increasing costs, declining membership and lesser interest in professional organizations have forced AGT to reevaluate our needs for a larger management company. In the summer/fall of 2016, the AGT Board of Directors and Council of Representatives held discussions on the reduction of operating costs while maximizing our investments into AGT. Tough phone conferences, followed by even tougher decisions, directed us down the path toward the cessation of the contract with our management company. On July 1st, 2017, we tendered our end-of-contract clause to be effective on December 31st, 2017. Many at AGT have worked with AMP Management Services/Kellen for many years and found this to be a very tough decision. However, this decision was needed to ensure AGT’s future survival. On the positive side of change, AGT has recruited Denise Juroske-Short, our current Secretary-Treasurer, to serve as our future Executive Director. Denise will work to fulfill the responsibilities of our previous management company to ensure there is no loss in service to AGT and its members. Denise is well-versed in all AGT processes and is currently learning all the finer details from our current management company as they transition out prior to the end of the year. Despite the uncertainty of change, AGT is still very optimistic about the health of our association and our relevance to all who work in genetics. We are continuing to expand biochemical and molecular genetic content in our annual meeting. This will allow us to reach a larger audience and give those in cytogenetics an educated look at the future of biochemical and molecular-based studies. Even though change is in process, AGT still needs even greater help from its membership. We need everyone to maintain their membership, recruit their co-workers and friends to AGT, and do their best to attend the AGT annual meeting. AGT is the professional representation of your work and enjoyment in the fields of biochemical genetics, cytogenetics, and molecular genetics. By staying involved, you not only promote your work in the field, you encourage all others who also work in genetics! AGT also has volunteer opportunities if you’re interested in becoming even further involved! I invite you, as a member, to volunteer to serve on a sub-committee and assist the board in association promotion and activity organization. This involvement can best prepare you for potential Board of Director or Council of Representative openings at AGT. We’d love to see everyone involved! I’d like to close with a list of the current AGT Board of Directors and Council of Representatives. These are

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the individuals who work tirelessly to ensure that the difficult decisions are made for the best interests of AGT. Their support and guidance has been critical in all the recent decisions, and I believe that they should know how important they have been to our association. Thank you to all who have helped to support AGT over the last year packed with difficult choices. AGT Board of Directors Patricia K. Dowling, PhD – Past President Denise Juroske-Short, PhD, MS, MB(ASCP) CM – Secretary-Treasurer / Incoming Executive Director Ephrem Chin, MBA, MB(ASCP) CM, QLC – Public Relations Director Sally J. Kochmar, MS, CG(ASCP) CM – Education Director Jennifer N. Sanmann, PhD – Meeting Director Christina S. Mendiola, BS, CG(ASCP) CM – Meeting Director AGT Council of Representatives Hilary E. Blair, BS, MS, CG(ASCP) CM – Representative to CCCLW Helen Bixenman, MBA/HCM, CHC, CG(ASCP)CM, DLMCM, QLC – Representative to ASCP BOC Amy R. Groszbach, BS, MB(ASCP) CM, MEd – Representative to ASCP BOC Peter C. Hu, PhD, MS, MLS(ASCP) CM, CGCM, MBCM – Representative to NAACLS Jun Gu, MD, PhD, CG(ASCP) CM – Representative to CAP / ACMG Lastly, I would like to again encourage membership and involvement in AGT. AGT is your professional organization. It’s yours to help drive and define, and I hope all members see the importance of involvement in the greater genetics community. If you want to see further improvements and change happen at AGT, please don’t hesitate to contact any member of the Board of Directors or Council of Representatives. We are all here to help the membership make AGT the finest professional genetics organization. However, the key to our success is you … AGT’s membership. Jason Yuhas, CG(ASCP) CM, President Association of Genetic Technologists

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Association of Genetic Technologists 42nd Annual Business Meeting Minutes Saturday, June 17, 2017 St. Louis Union Station Hotel St. Louis, MO 8:00 a.m. – 8:45 a.m.

AGT President Pat Dowling called the 42nd Annual Business Meeting to order at 8:10 a.m. on Saturday, June 17.

• President Pat Dowling invited Secretary/Treasurer, Denise Juroske-Short, to present a brief overview of the current financial condition of the association and of the approved 2017-2018 budget.

• President Pat Dowling thanked members for their participation and continued support.

• President Pat Dowling reported that the Bylaws revisions were sent to the membership in May for comment.

• The current, 2016-2017, AGT Board of Directors was introduced: President-Elect – Jason Yuhas, Secretary-Treasurer – Denise Juroske-Short, Education Director – Sally Kochmar, Public Relations Director – Ephrem Chin, Annual Meeting Director – Jennifer Sanmann and Annual Meeting Co-Director – Christina Mendiola.

o Members present at the business meeting RESOLVED to approve the bylaws changes as written. • Members were invited to ask questions about the Board initiatives, budget or any related organizations. No questions were asked.

• Members present at the business meeting RESOLVED to approve the 41st Annual Business Meeting Minutes.

• President Pat Dowling introduced the following Council of Representatives members and asked them to provide brief reports: o BOC – Representatives: Helen Bixenman, Amy Groszbach o NAACLS – Representative: Peter Hu o CAP – Representative: June Gu o CCCLW – Representative: Hilary Blair

• President Pat Dowling reported to the membership on the items completed in the past year and the initiatives taken at the Board of Directors Meeting. o AGT provided more webinars in 2016-2017 than ever before (eight). o AGT published the 4th edition of the Cytogenetics Laboratory Manual.

• President Pat Dowling recognized FGT and their work each year and introduced FGT President Robin Vandergon.

o AGT’s 2018 Annual Meeting planning will begin soon, and AGT will be surveying the membership for speaker and topic ideas.

• President-Elect Jason Yuhas presented President Pat Dowling with the presidential plaque for her service as President-Elect (2013-2015) and President (2015-2017).

o AGT will be transitioning to new management as of January 1, 2018 due to financial constraints.

• President Pat Dowling addressed the membership with some final remarks.

Denise Juroske-Short will become AGT’s administrative leader starting January 1, 2018.

■

There being no further business to come before the AGT membership, President Pat Dowling adjourned the 42nd Annual Business Meeting at 9:05 a.m. Central Time.

As no President-Elect candidate was nominated and then elected to the Board of Directors, Pat Dowling will continue on the AGT Board of Directors for one year as an Ex-Officio, Non-Voting member to assist with the transition.

■

Respectfully Submitted, Denise Juroske-Short Secretary/Treasurer

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AGT Annual Meeting Sponsors, Exhibitors and Volunteers AGT would like to acknowledge the following organizations for their support and assistance by providing sponsorship for speakers, sessions and conference materials for the AGT 42nd Annual Meeting. Their efforts helped to make this a very successful meeting. We would also like to thank the following exhibitors for their time, support and the valuable information they provided to enhance our Annual Meeting.

Platinum Sponsor

ADS Biotec Inc. Agilent Technologies Applied Spectral Imaging BioDot, Inc. Biological Industries USA, Inc. BioView Chroma Technology Enzo Life Sciences Foundation for Genetic Technology (FGT)

Gold Sponsors

Irvine Scientific Leica Biosystems Lumencor MetaSystems Oxford Gene Technology (OGT) Promega Corporation Rainbow Scientific, Inc./Genial Genetics SciGene STEMCELL Technologies Inc. Thermo Fisher Scientific

Silver Sponsor AGT would like to express a special thanks to the following individuals who spent countless hours of planning and hard work making this 42nd Annual Meeting a great success. Pat Dowling, AGT President

Bronze Sponsors

Jennifer Sanmann, Annual Meeting Director Christina Mendiola, Annual Meeting Co-Director

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AGT 42nd Annual Meeting

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Awards Presented at the AGT 42nd Annual Meeting

Mervat Ayad, Quest Diagnostics, this year’s Outstanding Achievement Award winner, pictured with Pat Dowling, AGT President.

Rhett Ketterling, Mayo Clinic, this year’s Gordon W. Dewald Lecturer, pictured with Pat Dowling, AGT President

Wenrui Yi, winner of the AGT Student Abstract Award, presented her submission, Comparison of Whole Genome Sequencing and Conventional Molecular Techniques for Genotyping Bacterial Isolates Causing Bacteremia in Cancer Patients, as a platform presentation during the meeting. She is pictured with AGT President, Pat Dowling.

Evan Roberts receives the New Horizons Award from Rainbow Scientific representative, Peter Mousseau. Rainbow Scientific sponsors this award.

Caleb Chu receives the Barbara J. Kaplan Scholarship from FGT Board Member, Bob Gasparini.

Farah Ladha receives the Outstanding Technologist Grant from FGT President, Robin Vandergon. This award is sponsored by Leica Biosystems.

The AGT Margaret Barch Memorial Workshop Award was presented to Marilu Nelson for her workshop, Grab Your Seat & Don’t Get Left Behind: Transitioning from Classical Cytogenetics to Molecular Genetics.

Lauren Wilson won the Joseph Waurin Excellence in Education Award. She is pictured here with Jim Waurin. Jim sponsors this award each year in honor of his father.

Christopher Sattler receives the Best Cytogenetics Platform Presentation Award for his abstract, Dual-Color, Break-Apart FISH Assay for PRKACA Rearrangements in Fibrolamellar Carcinoma

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Quyen Tran receives the EXCEL Award. This award is sponsored by Oxford Gene Technology.

The Best Poster Award goes to Automated Metaphase Finding and FISH Spot Counting Validation, presented to Stephanie Soewito, Quyen Tran, Justin Kee, Thong Vu, Qin Xu, Ayman Bakhit, Ming Zhao and Jun Gu, from the University of Texas, M.D. Anderson Cancer Center. Several authors are pictured here. The best poster award is sponsored by Irvine Scientific.

Aditi Khurana receives the Best Molecular Platform Presentation Award for her abstract, Application of a Novel Tumor Cell Isolation Platform.

Association Business

Oustanding Achievement Award Introduction Patricia Dowling, PhD, FACMG, Pathline Labs, Ramsey, NJ It is my heartfelt pleasure to present AGT’s Outstanding Achievement Award to a most deserving woman. A woman who I consider to be a great friend and colleague. Not only as a fellow AGT aficionado, but having worked for the same company, albeit on opposite sides of the country. Our award winner started her career as a med tech, but was recruited into the cytogenetics lab in 1985 to oversee the pre-clinical aspects of the lab. Her interest was piqued along the way by the work that was being done, and she moved into the darkroom. Her passion and work ethic were noticed, and she was recognized as a good candidate for cytogenetic technologist training. She completed her training, sat for and passed the NCA exam, became an AGT member, and took advantage of the CEU opportunities offered by AGT.

to supervisor, to manager of the cytogenetics lab, along the way helping to grow the lab from three techs to 120 techs today. In 2004 she initiated the technologist training program that has produced 450 graduates, 100 of whom are cytogenetic techs. Managing a lab of 120 techs wasn’t enough for Mervat, or for the powers that be in San Juan Capistrano (SJC)! In 2008, she was promoted to Director of Operations of all of SJC! It seems that the MBA she received in 1996 was working to her advantage. She is also passionate about women’s issues, and she joined the Women’s Leadership group in 2014. In 2015, she added oversight of the infectious disease branch to her list of responsibilities. It would have been so easy for Mervat to move on from AGT, but that didn’t happen. She is as passionate about cytogenetics, and operations, as she ever was, and it shows! She has provided countless expert speakers, and contributions and sponsorships, for the AGT annual meetings. In short, she loves AGT and is completely devoted to its continuing relevance and robustness, well into the future. And if this isn’t enough, Mervat is married to the love of her life and has raised two extremely successful daughters, one of whom is in medical school and one of whom just passed her bar exam. As for my feelings about Mervat, she is an inspiration to me, and I cherish her friendship.

The first AGT meeting she attended was in Monterey, California, and she hasn’t missed a meeting since. She began offering workshops that were very well attended, year after year. Her first stint on the Board of Directors was as the public relations director. She held other positions on the board and became president-elect in 2012 and president in 2014. Have you figured out who this person is yet? Let me tell you before I continue my tribute. The 2017 AGT Outstanding Achievement Award recipient is Mervat Ayad, from Quest Diagnostics Nichols Institute in San Juan Capistrano, California.

Please join me in congratulating Mervat as she receives the AGT’s Outstanding Achievement Award.

Mervat joined Quest Diagnostics in 1987 as a cytogenetic technologist. She was the third technologist in the lab. She quickly moved up through the different grades of cytogenetic technologist

– Pat Dowling, President of AGT

Outstanding Achievement Award Acceptance Mervat Ayad, BS, EMBA, CG(ASCP) CM, DLMCM, CCS, Director, Laboratory Operations, Quest Diagnostics, San Juan Capistrano, CA Thank you so much. It is truly an honor to be receiving this award and to be here with you this evening.

and I already messed up! So being the good friend she was, my friend came up with a master plan… we would share her clothes! There were three days in the conference. One day she would wear her top, and I would wear her bottoms and the next day we would switch! And for some reason, we thought we looked normal! Let’s just keep it that way! Even though my first AGT meeting ended up being a wardrobe catastrophe, I have been a proud AGT member ever since!

AGT has been a big part of my life for a long time. I remember all the way back to the very first meeting I attended in 1985. In fact, I have a very funny story about that meeting. This story happened when I was first starting my professional career in genetics. I wanted to be more knowledgeable about the field, and I was so excited to attend my very first meeting! My friend and I decided that we would take a road trip and drive to Monterey to be in attendance. We had a great time together until we got to Monterey and well… I realized that I had left my professional attire in my car… all the way back in Los Angeles! It was my first meeting,

I would like to thank the many people that have encouraged and supported me throughout these past years. Thank you Pat, Peggy, Bob, Jim, Deborah, Robin, all the Helens, Peter, Jun, Hilary, Denise, Amy, Jason, Christie and Marilyn, Helen and Margaret (God bless her soul), editors of the AGT manual. As I look back on these past 30 years, I am so grateful and honored to have been part of this wonderful organization. In the years to come, I cannot wait to see what the next generation will bring to AGT and the growth that will come from their contributions. Additionally, in return, I am looking forward to seeing how AGT helps this generation grow as it helped me throughout my journey. Again, thank you so much!

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2017 AGT Award Winners Outstanding Achievement Award Sponsored by Martha Keagle Mervat Ayad, Quest Diagnostics Nichols Institute, San Juan Capistrano, CA

Student Research Award Wenrui Ye, University of Texas, Houston, TX

AGT Margaret Barch Memorial Workshop Presentation Marilu Nelson, Human Genetics Laboratory, University of Nebraska Medical Center, Omaha, NE

Dr. Gordon W. Dewald Lecture Rhett Ketterling, Mayo Clinic, Rochester, MN

2017 FGT Award Winners Excel Award Sponsored by Oxford Gene Technology Quyen Tran, University of Texas, M.D. Anderson Cancer Center, Houston, TX

New Horizons Award Sponsored by Rainbow Scientific Evan Roberts, Human Genetics Laboratory, University of Nebraska Medical Center, Omaha, NE

Joseph Waurin Excellence in Education Award Sponsored by Jim Waurin Lauren Wilson, University of Connecticut, Storrs, CT

Barbara J. Kaplan Scholarship Caleb Chu, University of Texas, M.D. Anderson Cancer Center, Houston, TX

Outstanding Technologist Grant Sponsored by Leica Biosystems Farah Ladha, Baylor College of Medicine, Houston, TX

Best Poster Award Sponsored by Irvine Scientific Automated Metaphase Finding and FISH Spot Counting Validation – Stephanie Soewito; Quyen Tran; Justin Kee; Thong Vu; Qin Xu; Ayman Bakhit; Ming Zhao, MD, MS, CG(ASCP)CM; Jun Gu, MD, PhD, CG(ASCP)CM, University of Texas, M.D. Anderson Cancer Center, Houston, TX

Best Cytogenetics Platform Presentation Award Dual-Color, Break-Apart FISH Assay for PRKACA Rearrangements in Fibrolamellar Carcinoma – Christopher A. Sattler, CG(ASCP); Kathryn E. Pearce, MB(ASCP); Jason A. Yuhas, CG(ASCP); Rondell P. Graham, MBBS; Michael S. Torbenson, MD; Ivy M. Luoma, CG(ASCP); Tanya L. Despins, CG(ASCP); Patrick P. Bedroske, CG(ASCP); Darlene L. Knutson; Sara M. Kloft-Nelson; Patricia T. Greipp, DO, Mayo Clinic

Best Molecular Platform Presentation Award Application of a Novel Tumor Cell Isolation Platform – Aditi Khurana, CG, MB; Farideh Bischoff, Silicon Biosystems; Nicolò Manaresi, Silicon Biosystems; Mathew Moore, PacificDx; Phil Cotter, PacificDx; Cynthe Sims, PacificDx; Amanda Gerber, PacificDx; Valeria Sero, PacificDx; Samuel Koo, PacificDx; Marc Ting, PacificDx; Shelly Gunn, MD, PhD, PacificDx

Best Exhibitor Booth Award Promega

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Platform Abstracts, AGT 2017

AGT 42nd Annual Meeting June 15–17, 2017 St. Louis Union Station Hotel, St. Louis, MO

Platform, Poster and Student Poster Presentations from the 42nd Annual Meeting The Journal of the Association of Genetic Technologists proudly publishes the platform, poster and student poster abstracts presented at the 42nd AGT Annual Meeting in St. Louis, Missouri. We thank the authors for sharing their recent discoveries with us and hope the reader finds them stimulating, educational and interesting. The text of the abstracts is presented as it appeared in the 42nd Annual Meeting Final Program and has not been edited further. Please note: These abstracts have not been edited for grammar or spelling.

MOLECULAR PLATFORM PRESENTATIONS

Please note: These abstracts have not been edited for grammar or spelling.

M1 DIAGNOSTIC EFFICACY OF CHROMOSOMAL MICROARRAY ANALYSIS IN PRENATAL AND PRODUCTS OF CONCEPTION TESTING: A SINGLE INSTITUTION EXPERIENCE

Michael Evenson, CG(ASCP)CM, Cytogenetic Technologist, Washington University School of Medicine; Archana, Shenoy, MD, Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia; Vishwanathan, Hucthagowder, PhD, Clinical Cytogenetics at Molecular Pathology Laboratory Network, Inc.; Ina, Amarillo, PhD, Cytogenetics and Molecular Pathology, Department of Pathology and Immunology, Washington University School of Medicine; Katinka, Vigh-Conrad, PhD, Department of Pathology and Immunology, Washington University School of Medicine; Catherine, Cottrell, PhD, Institute for Genomic Medicine, Nationwide Children’s Hospital; Diana, Gray, MD, Department of Obstetrics and Gynecology, Washington University School of Medicine; Yoshiko, Mito, PhD, Cytogenetics and Molecular Pathology, Department of Pathology and Immunology, Washington University School of Medicine The advent of chromosomal microarray analysis (CMA) has enabled the identification of copy number variants (CNVs) and allelic imbalances that were not detectable by conventional cytogenetic analysis. Over the years, the resolution of the assay has improved, and in prenatal and reproductive care settings, CMA has become the recommended first-tier diagnostic test for pregnancies with ultrasonographic fetal abnormalities, fetal demise and stillbirth. Here we report our single-institution experience of CMA testing that was performed from 2013 to 2016 using cytogenomic microarrays containing ~2.6 million copy number and single-nucleotide polymorphism probes. Testing was successfully performed on 115 prenatal specimens (93 amniotic fluid (AF) and 22 chorionic villus sampling (CVS)), and 273 Products of conception (POC) specimens in this four-year period. The detection rates of genomic aberrations were 19.1% for prenatal cases (20.4% (AF) and 13.6% (CVS)), and 44.7% for POC cases, highlighting the diagnostic efficacy of CMA testing of the fetuses. A striking difference was noted in the detected abnormalities between the two groups: subchromosomal CNVs were predominant in prenatal testing (5/22 = 72.7%), whereas numerical abnormalities were predominant in POC testing (81/122=66.4%), with autosomal trisomies being the most common (68.4% of numerical abnormalities in POC), followed by Triploidy (17.3%), and sex chromosome aneuploidy (14.2%). These findings are consistent with the reported predominance of numerical aberration in POC, particularly autosomal trisomy. Nine percent of POC cases showed both subchromosomal and numerical aberrations. Regions of homozygosity (ROH) were reported in 1 prenatal (AF) and 3 POC cases. Review of clinical charts for a selection of abnormal cases lead to successful phenotype-genotype correlation. This retrospective study allowed us to illustrate the diagnostic efficacy of CMA testing for prenatal and POC specimens. CMA testing, in combination with conventional chromosome analysis and fluorescence in situ hybridization when necessary, will continue to provide crucial information to clinicians and families to aid management of pregnancies and evaluation of recurrence risks.

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Platform Abstracts, AGT 2017 M2 MEASUREMENT OF DISEASE SPECIFIC FREE OLIGOSACCHARIDES USING UPLC-MS/MS ANALYSIS IN PLASMA, DRIED BLOOD SPOTS AND CULTURED CELLS.

Allison Cason, , Biochemical Lab Technologist, Greenwood Genetics Center; Tim Wood, PhD FACMG, Greenwood Genetics Center; Laura Pollard, PhD FACMG, Greenwood Genetics Center; Rongrong Huang, PhD, Greenwood Genetics Center A group of lysosomal storage diseases (LSDs), primarily the glycoproteinoses, are characterized by the accumulation of specific free oligosaccharides (FOS) in tissues and other biological fluids as a result of one or several deficient enzymes that are involved in the breakdown of complex carbohydrate side-chains of glycoproteins. Historically, clinical laboratories have used thin-layer chromatography to detect the abnormal accumulation of FOS in urine; however, this method lacks both sensitivity and specificity. In an effort to improve the diagnosis of glycoproteinoses, our laboratory has developed a ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) assay for the detection of seven FOS following reducing-end labeling derivitization [Hex1HexNAc1Asn , Hex3HexNAc2Fuc1, Hex3HexNAc1, Hex1HexNAc1, Hex3HexNAc2, Hex2HexNAc3, and Neu5Ac1Hex3HexNAc2]. This analysis in urine has shown 100% sensitivity to detect patients with one of the eight different LSDs and we have clinically validated its use. Here we present data demonstrating this assay can also be applied to other biological specimens such as dried blood spots (DBS), plasma, leukocytes and fibroblasts, broadening its clinical utility. As part of assay validation, experiments were conducted in dried blood spots (DBS, n=24), plasma (n=15), leukocytes (n=14) and cultured fibroblasts (n=20) from a cohort of known patients as well as normal controls. The Hex3HexNAc1 level was elevated in all sample types from alpha-mannosidosis patients: average of 5-fold in DBS, 40-fold in plasma, 80-fold in leukocytes and 30-fold in fibroblasts. Hex3HexNAc2Fuc1 was detectable in DBS and plasma from alpha-fucosidosis patients, but not from unaffected controls or patients with other LSDs. Hex3HexNAc2 was elevated in all sample types for patients with beta-galactosidase deficiency: average of 6-fold in DBS, 30-fold in plasma, 10-fold in leukocytes and 100-fold in fibroblasts. Both Hex3HexNAc2 and Neu5Ac1Hex3HexNAc2 were elevated in DBS, plasma and leukocytes from one galactosialidosis patient, but not in fibroblasts. Hex1HexNAc1Asn was detectable in DBS, plasma and leukocytes from aspartylglucosaminuria patients, but not from unaffected controls or patients with other LSDs. Hex1HexNAc1 was elevated in DBS, plasma and leukocytes from one beta-mannosidosis patient. Interestingly, samples taken after a bone marrow transplant showed a 60-95% decrease, depending on the sample type. These results suggest the measurement of FOS for the diagnosis of the glycoproteinoses can be performed in a variety of samples types which may be useful in further diagnostic or therapeutic initiatives.

M3 DEFINING POLICIES TO WAIVE SANGER SEQUENCING FOR THE NGS-BASED DIAGNOSTIC WORKFLOW

Shelley Ordorica, MLT, Specialty Technologist, Children’s Hospital of Eastern Ontario; Kristin D. Kernohan, PhD; Caitlin Chisholm, MS, CGC; Ryan Potter, MLT; Mahdi Ghani, PhD; Hussein Daoud, PhD; Nasim Vasli, PhD; Amanda Smith, PhD, FCCMG; Olga Jarinova, PhD, FCCMG Implementation of Next-Generation sequencing (NGS)-based tests in the diagnostic laboratory requires development of efficient workflows that ensure test quality, effectiveness and anticipated turn-around time. The Children’s Hospital of Eastern Ontario (CHEO) Molecular Genetics laboratory offers a number of NGS-based tests conducted using targeted panels. Upon implementation of NGS-based assays, all reportable variants and coding regions of insufficient coverage were investigated by Sanger sequencing. This approach required significant time and resources for each patient sample, and represented a significant challenge for our hospital-based diagnostic laboratory. In response, our laboratory defined and validated “Sanger sequencing waiving” policies based on the previously advised New York State guidelines (https://www.wadsworth.org/sites/default/files/WebDoc/2080900015/Germline_NextGen_ Validation_Guidelines.pdf). In order to define the rules to waive Sanger sequencing, we used our cardiomyopathy panel as a model, and investigated data from over 300 completed assays with confirmed variants. All variants detected by NGS were validated by Sanger sequencing when the following criteria were applied: coverage is ≥ 100X; variant frequency is ≥ 35%; variant is a single nucleotide variant; and ≥ 10 unique variants have been Sanger validated in the gene of interest. Thus the rules to waive Sanger were defined. Any variant detected that does not meet all of the above criteria still require confirmation by Sanger sequencing. This policy was reconsidered for genes with high sequence homology, which will always be confirmed by Sanger sequencing, or those identified as NGS or Sanger “dead zones”, which are not confirmed (Mandelker et al, 2016). This policy has been in place for several months in our laboratory and resulted in significant improvement to analytical time and reporting turn-around times, without compromising patient care. In conclusion, strategic planning for Sanger validation of NGS detected variants can increase laboratory capacity, and optimize efficiency to provide the best possible care for patients.

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Platform Abstracts, AGT 2017 M4 DETECTING ACTIONAL MUTATIONS USING A 62-GENE NGS LEUKEMIA PANEL

Brittany Hennigan, MB (ASCP) CM, Molecular Technologist, Greenwood Genetic Center; Matthew Tedder, Clemson University and Greenwood Genetic Center; Julie Eggert, PhD, RN, FAAN, Clemson University; Fatima Abidi, PhD, Greenwood Genetic Center; Mike Friez, PhD, FACMG, Greenwood Genetic Center; Alka Chaubey, PhD, FACMG, Greenwood Genetic Center Leukemia is a malignant disease affecting the blood and bone marrow. It arises when immature white blood cells grow abnormally due to pathogenic variants in genes or environmental factors that confer a growth and/or survival advantage. It is a progressive disease and is fatal if untreated. Leukemia is classified by time of disease development, acute or chronic, as well as the type of cell affected, myelocyte or lymphocyte. It is mostly seen in adults (55+); however, certain types are seen more commonly in young adults and children. The disease can be treated through chemotherapy, radiation therapy, targeted therapy and bone marrow transplant. Molecular diagnostics has become a valuable resource in the care of leukemia patients by providing prognostic as well as therapeutic information. Pathogenic variants associated with leukemia have been identified as “actionable,” especially in cases for which no cytogenetic abnormalities are found. Actionable mutations are those that have a significant impact on diagnosis, prognosis, or treatment of the patient. In order to determine a genetic cause for patients diagnosed with leukemia and to identify any potential actionable mutations, such patients were tested using a 62-gene leukemia panel. This 62-gene leukemia panel uses the SmartChip NGS Target Enrichment System (WaferGen Biosystems, USA) for enriching the coding regions of 62 genes followed by sequencing using Illumina’s MiSeq (Illumina, USA) chemistry. Typically, eight samples are multiplexed to achieve appropriate coverage. Sequencing data is analyzed through our in-house bioinformatics pipeline, and the selected variants are confirmed by Sanger sequencing. To date, 23 patients have been tested resulting in the identification of nine pathogenic changes, one likely pathogenic alteration and 26 variants of uncertain clinical significance. Simultaneously, these patients were also tested on an assay that detects FLT3 internal tandem duplication (ITD) and the common FLT3-D835 mutation, both of which are considered actionable. Currently, two patients were identified to have actionable pathogenic variants in the FLT3 gene (FLT3-ITD and FLT3-D835H). Additionally, an actionable mutation was also detected in the NPM1gene (c.863_864insTCAG). As knowledge of genetic mutations in leukemia continues to grow, so too will the list of actionable mutations. This will lead to further integration of molecular diagnostics in the care of leukemia patients. From our analysis of 23 patients, the alterations mentioned above are considered actionable. These findings are expected to provide valuable prognostic and therapeutic information to the referring their physicians, resulting in better clinical management and care.

M5 APPLICATION OF A NOVEL TUMOR CELL ISOLATION PLATFORM

Aditi Khurana, CG, MB; Farideh Bischoff, Silicon Biosystems; Nicolò Manaresi, Silicon Biosystems; Mathew Moore, PacificDx; Phil Cotter, PacificDx; Cynthe Sims, PacificDx; Amanda Gerber, PacificDx; Valeria Sero, PacificDx; Samuel Koo, PacificDx; Marc Ting, PacificDx; Shelly Gunn, MD/PhD, PacificDx The DEPArray™ technology (Menarini Silicon Biosystems) is based on Dielectrophoresis (DEP); a non-uniform electric field exerting forces on neutral, polarizable particles, to trap cells in DEP “cages” and manipulate them individually. High quality image-based cell selection enables users to identify, isolate and recover intact specific individual rare cells of interest from complex, heterogeneous samples such as live or fixed cell suspensions, FFPEs, liquid biopsies and fine needle aspirates. The CellBrowserTM software allows user defined selection criteria for exposure and gating thresholds for maximum flexibility backed by image based morphological selection for maximum confidence. Here we demonstrate a workflow validation for application of molecular sequencing technologies and FISH downstream of rare cell recovery from 50µm FFPE scrolls and CTCs from CellSearchTM cartridges. FFPE scrolls are dissociated and stained with cell surface markers following protocol specific quality control. A minimum of 1500 cells from the enriched suspension are used to assess DNA integrity using a qPCR technique to predict amount of DNA required for successful downstream sequencing metrics. Cells from the suspension are processed through the DEPArray to recover stromal and tumor cell populations which are lysed and library prepared separately for a Next Generation Sequencing (NGS), Illumina platform based OncoSeek oncology panel. The panel allows simultaneous detection of SNVs, indels and CNAs from 63 oncology relevant actionable genes starting from very low amount of input DNA of 400pg. Alternatively the recovered stromal and tumor cell populations can be utilized for gene specific FISH investigation. For the validation CellSearch cartridges are processed through the DEPArray to isolate leukocytes as control, CTCs and atypical cells. This approach of single tumor cell isolation followed by low pass copy number determination on the Ion Torrent platform takes current CTC enumeration towards characterization. This may be used to inform treatment decisions and provide valuable prognostic information in reference to the patient’s leukocytes as control. It can be used to study cell- cell interactions, cellular response to drugs and identify potential drug candidates.

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Platform Abstracts, AGT 2017 M6 VERIFICATION OF NEXT GENERATION SEQUENCING AND MULTIPLEX LIGATION-DEPENDENT PROBE AMPLIFICATION FINDINGS IN THE PSEUDOGENE REGION OF PMS2; TECHNICAL CONCERNS AND ADJUNCT STUDIES UTILIZING OUR CLINICAL EXPERIENCE

Evan Roberts, BS, MB(ASCP)CM, Molecular Genetic Technologist II, UNMC Munroe Meyer Institute; Marilu Nelson, MS, MB(ASCP)CMCGCM, Human Genetics Laboratory, Munroe-Meyer Institute for Genetics and Rehabilitation, University of Nebraska Medical Center; Julie M. Carstens, MS, MB(ASCP)CMCGCM, Human Genetics Laboratory, Munroe-Meyer Institute for Genetics and Rehabilitation, University of Nebraska Medical Center; Janet E Williamson, BS, MLT(ASCP), MB(ASCP)CMCGCM, Human Genetics Laboratory, Munroe-Meyer Institute for Genetics and Rehabilitation, University of Nebraska Medical Center; Erin Kaspar, BS, MB(ASCP)CM, Human Genetics Laboratory, Munroe-Meyer Institute for Genetics and Rehabilitation, University of Nebraska Medical Center; Jacquelynn Evans, BS, MB(ASCP)CM, Human Genetics Laboratory, Munroe-Meyer Institute for Genetics and Rehabilitation, University of Nebraska Medical Center; Emily Rief, BS, MB(ASCP)CM, Human Genetics Laboratory, Munroe-Meyer Institute for Genetics and Rehabilitation, University of Nebraska Medical Center; Karissa Scott, BS, MB(ASCP)CM, Human Genetics Laboratory, Munroe-Meyer Institute for Genetics and Rehabilitation, University of Nebraska Medical Center; Bhavana J. Dave, PhD, FACMG, Human Genetics Laboratory, Munroe-Meyer Institute for Genetics and Rehabilitation, University of Nebraska Medical Center; Jennifer N. Sanmann, PhD, FACMG, Human Genetics Laboratory, MunroeMeyer Institute for Genetics and Rehabilitation, University of Nebraska Medical Center PMS2, located on the p-arm of chromosome 7, codes for the mismatch repair endonuclease PMS2 protein. This protein product is one of four key mismatch repair (MMR) proteins that together function to correct replication errors. Heterozygous germ line mutations in PMS2 have been associated with autosomal dominant Lynch Syndrome, which is characterized by an increased risk of colorectal and endometrial cancers as well as other cancers, including gastric and ovarian, along with tumor microsatellite instability in these tumors. PMS2 is also associated with autosomal recessive constitutional mismatch repair-deficiency (CMMRD), a childhood cancer predisposition syndrome, characterized by an increased risk for leukemia, lymphoma, and colorectal cancer. Affected individuals may also have a neurofibromatosis type 1 phenotype. PMS2 shares a considerable portion of its sequence with a pseudogene, PMS2CL, which encompasses exons 9 and 11-15 with extremely high sequence homology. This homology makes variant classification extremely difficult using next generation sequencing (NGS) techniques and conventional microarray deletion/ duplication analysis. Specifically, the short reads of most NGS technologies impede the accurate mapping to either the parent gene or the pseudogene, and microarray technologies cannot attribute copy number changes to either PMS2 or PMS2CL. To overcome this challenge, our laboratory has implemented the use of long range PCR utilizing three unique primer sets, with subsequent nesting of exons 9 and 11-15. This helps in accurate characterization of single nucleotide changes as well as small (<40bp) indels detected by NGS. For larger deletion and duplication detection, multiplex ligation-dependent probe amplification (MLPA) is used to supplement the limitations of microarray technologies. A review of 164 samples submitted to the laboratory from 2014 to present revealed only three unique PMS2 variants, classified as uncertain clinical significance or above, that were detected in the homologous region using either NGS or MLPA. Subsequent testing utilizing long range PCR and rare single nucleotide polymorphisms within the region(s) of interest confirmed the specificity of the long range products and, concurrently, the presence of the variant in either the parent gene or the pseudogene. Our study emphasizes that this use of long range PCR is an invaluable tool in verifying that reportable NGS and MLPA findings are in PMS2 and not in PMS2CL.

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Platform Abstracts, AGT 2017

CYTOGENETIC PLATFORM PRESENTATIONS

Please note: These abstracts have not been edited for grammar or spelling.

C1 COMPARISON OF INTERLEUKIN STIMULANTS ON CULTURED PLASMA CELLS TO INCREASE THE DETECTION OF CYTOGENETIC ABNORMALITIES

Natalie AaronsCooke, CG(ASCP), Clinical Laboratory Technician II, NeoGenomics; Erica Cessna, CG(ASCP)CM, NeoGenomics; Ayanna Walker, CG(ASCP)CM, NeoGenomics; Tiffany Chouinard, CG(ASCP)CMMBCM,HTLCM, NeoGenomics; John R. McGill, MS, PhD, FACMGG, NeoGenomics; Robert Gasparini, M.S., M.S., CG(ASCP)CM, DLM(ASCP)CM, NeoGenomics Plasma cells, a type of B-cell, are white blood cells that play a crucial role in antibody production. In the case of plasma cell neoplasms, plasma or myeloma cells build up in the bone marrow and form tumors. In many cases of plasma cell neoplasm, less than 10% of the cells found in the bone marrow are actually plasma cells. Flow cytometry results from patient samples received at our laboratory typically show less than two percent myeloma population. More precise plasma cell percentages can be obtained by CD138 antibody staining during morphologic analysis. As malignant plasma cells often have a low proliferation index, conventional cytogenetics frequently yields normal results. Interphase FISH studies increase the abnormality detection rate; especially with plasma cell enrichment; however, probes are limited to specific abnormalities. Stimulation of cytogenetic bone marrow cultures has been shown to increase the cytogenetic abnormality detection rate. Interleukin-6 (IL-6), Interleukin-4 (IL-4), Interleukin-3 (IL-3), and Interleukin-10 (IL-10) have all been shown to induce differentiation of myeloma cells into mature plasma cells (VM Lauta, Cancer 2003). Research conducted on plasma cell myeloma (R Catlett-Falcone, TH Landowski, MM Oshiro, et al, Immunity, 1999, Elsevier) suggests stimulation with IL-6 keeps myeloma cells from entering apoptosis. The American College of Medical Genetics and Genomics has recommended an IL-4 stimulated culture to improve detection of cytogenetic abnormalities that are otherwise undetectable. This is based on the understanding that IL-4 induces differentiation in naĂŻve helper T cells. Stimulation of bone marrow B cells with IL-3 and IL-10 have been show to differentiate into plasma cells (P Merville, J Dechanet, G Grouard et al, International, 1995, Jpn Soc Immunol). These stimulants are not typically used in the detection of plasma cell neoplasms by cytogenetic laboratories. The objective of this study is to evaluate the efficiency of IL-3/IL-10, IL-4 and IL-6 in detecting cytogenetic abnormalities in cases of plasma cell neoplasm compared with unstimulated cultures. All cytogenetic findings were correlated against flow cytometry and/or FISH findings. One hundred clinical cases were analyzed over a period of six months, with twenty cases per stimulant and a control group. Samples included in the study have indications of multiple myeloma, or other indications that suggest some sort of plasma cell neoplasm and had concurrent flow cytometry or FISH testing ordered. Incubation times ranged from 24 hours to 120 hours. Preliminary results indicate that cases with less a 2.5% plasma cell population, normal results were obtained via cytogenetics while both FISH and Flow cytometry were able to detect abnormalities or discrepancies. With higher populations of plasma cells, cytogenetic abnormalities were detected with or without stimulation. No correlation was seen in the abnormal cell population percentage in stimulated vs unstimulated. However, an impact on cell morphology and a decrease in the mitotic index of the cells cultured was noted. This observation has led to further testing in concentration and culture length with regards to cytotoxicity. Results of our study once completed may be beneficial for the clinical cytogenetics laboratories as new guidelines are recommending stimulation for plasma cell neoplasms when cytogenetics is ordered. Our findings may also be beneficial for patients as increased detection of cytogenetic abnormalities may lead to targeted and more effective therapy.

C2 VALIDATION OF A 17 PROBE FLUORESCENCE IN SITU HYBRIDIZATION (FISH) PANEL FOR DETECTION

OF T-LYMPHOBLASTIC LEUKEMIA/LYMPHOMA (T-ALL) ABNORMALITIES ON PARAFFIN-EMBEDDED SPECIMENS

Ivy M. Luoma, CG(ASCP), Development Technologist, Mayo Clinic; Kathryn E. Pearce, MB(ASCP), Development Technologist Coordinator, Mayo Clinic; Christopher A. Sattler, CG(ASCP), Development Technologist, Mayo Clinic; Tanya L. Despins, CG(ASCP), Development Technologist, Mayo Clinic; Ryan A. Knudson, CG(ASCP), Development Technologist, Mayo Clinic; Patrick P. Bedroske, CG(ASCP), Development Technologist, Mayo Clinic; Reid G. Meyer, CG(ASCP), Technical Specialist Coordinator, Mayo Clinic; Jason A. Yuhas, CG(ASCP), Education Specialist II, Mayo Clinic; David M. Menke, M.D., Consultant, Mayo Clinic; Christopher R. Conley, M.D., Consultant, Mayo Clinic; Rhett P. Ketterling, M.D., Consultant, Mayo Clinic Context: The diagnosis of pediatric/young adult lymphoblastic leukemia/lymphoma includes B-lymphoblastic leukemia/ lymphoma (B-ALL) in 85% and T-lymphoblastic leukemia/lymphoma (T-ALL) in 15%. The T-ALL subtype occurs most commonly in older children/young adults and often presents as a mediastinal mass with minimal or no bone marrow involvement. Since the standard methods for evaluating an ALL clone are bone marrow chromosome and FISH studies, the genetic evaluation of T-ALL has been problematic due to the frequent lack of bone marrow involvement. In addition, even if the bone marrow has sufficient involvement with T-ALL, the majority of common abnormalities are The Journal of the Association of Genetic Technologists 43 (3) 2017

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Platform Abstracts, AGT 2017 cytogenetically “cryptic”, which preclude easy identification into prognostic genetic subgroups. To more readily identify the common genetic abnormalities in T-ALL patients, we have validated a comprehensive 17 probe FISH panel on paraffin embedded specimens. Design: A combination of 17 commercial and laboratory developed FISH probe sets consisting of dual-color break-apart, enumeration, and dual-color dual-fusion probe strategies were utilized. The initial evaluation consists of 9 probe sets, including: TAL1/STIL, ABL1/BCR, TLX3(HOX11L2)/BCL11B, MLLT10/ PICALM, TP53/D17Z1, MLL(KMT2A), TRB, TRAD, and CDKN2A(p16)/D9Z1. Depending on the results of these initial probe sets, 8 additional “reflex probes” can be evaluated for further identification of translocation partners for the MLL(KMT2A), TRB, and TRAD genes. Following IRB approval, FISH analysis was performed by two technologists on 61 formalin-fixed paraffin-embedded specimens. These specimens included 36 T-ALL specimens and 25 normal tissue controls which were evaluated in a double-blinded method. Results: Upon study completion, the results were un-blinded and the normal tissue samples, which had all generated normal results, were used to calculate normal tissue cutoffs. Of the 36 T-ALL patient specimens, 24 (67%) had an abnormal result with one or more FISH probes. The abnormal results included 14 specimens with a CDKN2A(p16) deletion, 6 with TRB rearrangement, 5 with MLLTIO/PICALM fusion, 5 with TRAD rearrangement, 2 with TLX3(HOX11L2)/BCL11B fusion, 2 with TP53 deletion, 1 with ABL1 amplification and 1 with STIL/TAL separation. While the prognosis in T-ALL genetics is unclear for many abnormalities, the results obtained with this FISH panel include 5 patients with a favorable prognosis, 1 patient with an intermediate prognosis, and 8 patients with an unfavorable prognosis. Conclusion: We validated a 17 probe FISH panel for use in paraffin embedded tissue to help identify the common genetic abnormalities associated with T-ALL. The use of this FISH panel can help elucidate the prognostic genetic abnormalities in pediatric/young adult T-ALL patients that present with a mediastinal mass as the only sample available for genetic evaluation.

C3 DUAL-COLOR, BREAK-APART FISH ASSAY FOR PRKACA REARRANGEMENTS IN FIBROLAMELLAR CARCINOMA

Christopher A. Sattler, CG(ASCP), Development Technologist, Mayo Clinic; Kathryn E. Pearce, MB(ASCP), Development Technologist Coordinator, Mayo Clinic; Jason A. Yuhas, CG(ASCP), Education Specialist II, Mayo Clinic; Rondell P. Graham, M.B.B.S., Consultant, Mayo Clinic; Michael S. Torbenson, M.D., Consultant, Mayo Clinic; Ivy M. Luoma, CG(ASCP), Development Technologist, Mayo Clinic; Tanya L. Despins, CG(ASCP), Development Technologist, Mayo Clinic; Patrick P. Bedroske, CG(ASCP), Development Technologist, Mayo Clinic; Darlene L. Knutson, Technical Specialist I, Mayo Clinic; Sara M. Kloft-Nelson, Technical Specialist I, Mayo Clinic; Patricia T. Greipp, D.O., Consultant, Mayo Clinic Background: Fibrolamellar carcinoma is a liver cancer that provides a diagnostic challenge. It is a rare and distinct subtype of hepatocellular carcinoma that predominantly affects young patients without underlying cirrhosis. The treatment and prognosis significantly differ between classic hepatocellular carcinoma and fibrolamellar carcinoma; therefore, accurate diagnosis is critical. Recently, a unique genetic rearrangement was identified in fibrolamellar carcinoma which involves fusion of the DNAJB1-PRKACA genes(1.). A FISH assay would be an ideal tool to aid in the detection of this unique fusion event. Design: Since both genes reside within 500 kb of each other on chromosome 19, a dual fusion probe set is not feasible. Therefore, a break-apart strategy for detection of the PRKACA rearrangement that correlates with this fusion event was pursued. A dual-color, break-apart FISH probe was laboratory developed to detect the rearrangement of PRKACA. More specifically, it detects the loss of the 5’ region and retention the 3’ region of PRKACA at 19p13.1 associated with the DNAJB1-PRKACA fusion event in formalin-fixed paraffin-embedded tissues (SpectrumGreen 3’PRKACA, SpectrumOrange 5’PRKACA). FISH analysis was performed on 60 formalinfixed paraffin-embedded specimens including 10 fibrolamellar carcinoma tissue samples, 25 non-fibrolamellar carcinoma liver tumor samples, and 25 noncancerous control specimens in a blinded manner using standard sample processing techniques. Results: Of the 10 fibrolamellar carcinoma tissue samples, 10 demonstrated the classic loss of the 5’ region of PRKACA and retention of the 3’ region of PRKACA, associated with the DNAJB1-PRKACA fusion event. These 10 specimens were previously studied using RT-PCR and all 10 were found to be positive for the DNAJB1-PRKACA fusion transcript (2.); thus demonstrating 100% concordance. Of the 25 non-fibrolamellar carcinoma liver tumor samples, no PRKACA rearrangements associated with the typical abnormal patterns were observed. The 25 noncancerous control specimens had no PRKACA rearrangements and were utilized to generate normal cutoff values. Conclusions: We have developed a unique and robust FISH assay to detect PRKACA rearrangement associated with the DNAJB1-PRKACA fusion event. This probe set is available clinically and, to our knowledge, is the only such FISH test available to detect this rearrangement. This probe is valuable for accurate and timely diagnosis of fibrolamellar carcinoma. 1.Honeyman J, et al. Detection of a Recurrent DNAJB1-PRKACA Chimeric Transcript in Fibrolamellar Hepatocellular Carcinoma. Science. 2014 February 28; 343(6174): 1010-1014. Doi:10.1126/science.1249484. 2. Graham R, et al. DNAJB1-PRKACA is specific for fibrolamellar carcinoma. Mod Path. 2015, 28;822-829.

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Platform Abstracts, AGT 2017 C4 IMPLEMENTATION OF INTELLIFISH HYBRIDIZATION SOLUTION IMPROVES CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) FISH

Hong Xiao, PhD, Senior technologist, University of Michigan; Jiong Yang, PhD, University of Michigan; Carrie Landau, University of Michigan; Yang Zhang, University of Michigan; Leisa Stempek, CG(ASCP)CM, University of Michigan; Yinhong Shen, CG(ASCP)CM, University of Michigan; Weihong Zhao, CG(ASCP)CM, University of Michigan; Stacie Larabell, University of Michigan; Susette Miller, MB(ASCP)CM , University of Michigan; Lina Shao, MD, PhD, University of Michigan Chronic Lymphocytic Leukemia (CLL) is the most common form of adult leukemia in the Western world. Cytogenetic aberrations are detected in more than 80% of patients with previously untreated CLL. Currently, Fluorescence In Situ Hybridization (FISH) remains the standard method for detecting chromosomal aberrations that may have prognostic significance in CLL. Since implementing CLL FISH in 2010 using the Vysis CLL FISH Probe Kit as the clinical test, we have frequently observed weak signal strength and poor cell morphology when visualizing CLL FISH slides. As a result, the hybridization repeat rate for CLL panel probes has been significantly higher than those of other probes in our laboratory. In this study, we have developed a modified hybridization procedure that utilizes the Vysis IntelliFISH hybridization solution. The Vysis IntelliFISH hybridization solution is designed to reduce hybridization time while delivering equal or better quality results. We performed a parallel study in 12 CLL cases and observed equal or brighter signals, cleaner backgrounds, and better cell morphology with modified procedures than observed with routine method procedures. In addition, this new procedure has dropped the repeat rate for hybridization from 11.2% (12/107 cases) in May 2015-April 2016 to 0% (0/123 cases) in May 2016-December 2016; furthermore, the turnaround time (TAT) has been reduced from 9.27 +/-0.41 days to 6.26 +/- 0.24 days. Lastly, we have eliminated the lengthy slide pretreatment steps and the use of the toxic chemical formamide. In summary, the implementation of IntelliFISH hybridization solution has improved the quality and efficiency of CLL FISH, which ultimately translate into better and more effective patient care.

C5 PARTIAL TRISOMY 18 MOSAICISM DETECTED PRENATALLY: A CAUTIONARY CASE DESCRIBING THE IMPORTANCE OF FOLLOW-UP TESTING IN CVS SAMPLES

Matthew Noll, ASCP (CG), Technologist, LabCorp; James Harding, MD, Swedish Maternal and Fetal Specialty; Lauren Kesl, MS, CGC, Swedish Maternal and Fetal Specialty; Romela Pasion, MS, CGC, Laboratory Corporation of America速 Holdings, Center for Molecular Biology and Pathology, Department of Cytogenetics; Stuart Schwartz, PhD, Laboratory Corporation of America速 Holdings, Center for Molecular Biology and Pathology, Department of Cytogenetics; Jim Tepperberg, PhD, Laboratory Corporation of America速 Holdings, Center for Molecular Biology and Pathology, Department of Cytogenetics; Hiba Risheg, PhD, Laboratory Corporation of America速 Holdings/Dynacare, Department of Cytogenetics We describe a case of a 41 year old female referred for prenatal testing with advanced maternal age (AMA) and a circulating cell-free DNA (cfDNA) abnormal for trisomy 18. First trimester ultrasound at 11 weeks 3 days showed a singleton pregnancy with no apparent abnormalities. At 13 weeks gestation, the patient underwent chorionic villus sampling procedure. Fluorescence in situ hybridization (FISH) analysis of interphase cells derived from the cytotrophoblast were positive for an extra signal for chromosome 18. G-banded chromosome analysis of direct trophoblast and cultured villi were also positive for trisomy 18 in 20 metaphase cells examined. The patient was counseled for a fetus with trisomy 18 with a small chance of confined placental mosaicism (CPM). However, follow-up second trimester ultrasound at 17 weeks 3 days showed only an echogenic intracardiac focus and the patient elected for an amniocentesis at this time. FISH analysis of uncultured amniocytes did not show trisomy 18 nuclei within reporting criteria. G-banded chromosome analysis of all metaphase cells available in the in situ cultured amniocytes also showed a normal female karyotype. Due to the risk of CPM, weekly ultrasound monitoring of the pregnancy was performed. At 35 weeks gestation IUGR (growth at< 1 percentile) was present. The baby was born at 36 weeks gestation and presented with birth weight, head circumference, and body length at <3 percentile. Postnatal G-banded analysis of placental tissue showed trisomy 18 in all 20 metaphase cells examined consistent with prenatal CVS findings. Cytogenetic analysis of cord blood identified trisomy 18 in 2 of 50 metaphase cells examined. SNP microarray analysis performed on the cord blood identified extended regions of homozygosity (9.92 Mb and 6.75 Mb) observed exclusively at the terminal ends of chromosome 18. No presence of mosaicism was noted by microarray analysis suggesting the level of mosaicism seen in the blood was below the 10% detectable level of SNP array. These combined results suggest an early presence of trisomy 18 in the fetus with a trisomy correction resulting in the predominance of diploid uniparental disomy (UPD) 18 cells. There is a risk of clinical effects due to the low-level presence of trisomy 18. Although there are no confirmed imprinted genes reported on chromosome 18, genes within the homozygotic regions may be subject to autosomal recessive disorders due to pairing of recessive alleles. This case highlights the difficulties involved when interpreting prenatal cytogenetic results in identifying mosaic abnormalities, the importance of ultrasound findings, and a clear understanding of the limitations

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Platform Abstracts, AGT 2017 of different technologies. At present, there is no single technology that can give a definitive interpretation in mosaic cases, but used together a more accurate diagnosis can be made. Furthermore, this case highlights the importance of proper genetic counseling of patients when offering cytogenetic testing.

C6 HYPODIPLOIDY VS. HYPERDIPLOIDY IN B-CELL ACUTE LYMPHOBLASTIC LEUKEMIA

Sally Kochmar, MS, CG(ASCP), Laboratory Manager, MWH Med Genetics & Genomics Labs, University of Pittsburgh Medical Center; Susan Mann, MS, CG(ASCP), Laboratory Supervisor; MaryAnn West, BS, Lead Genetic Technologist; Zhishuo Ou, MD, Post-doctoral Fellow; Urvashi Surti, PhD, Cytogenetics Laboratory Director; Svetlana Yatsenko, MD, Cytogenetics Laboratory Associate Director B-cell precursor acute lymphoblastic leukemia (B-ALL) is the most common neoplasm in children and teenagers accounting for 70% of all infant and childhood leukemia. High hyperdiploidy (51+ chromosomes) is the most common abnormality pattern in childhood B-ALL, occurring in 25-30% of such cases. It is characterized cytogenetically by a nonrandom gain of chromosomes X, 4, 6, 10, 14, 17, 18 and 21 and is associated with a favorable prognosis. In contrast hypodiploidy resulted from a loss of multiple chromosomes is found in 5-8% of B-ALL cases. Low hypodiploidy with 31-39 chromosomes occurs in ~1% of cases and is associated with TP53 mutations and a poor prognosis. A clone constituting a duplication of the hypodiploidy stemline is frequently seen in these cases and may be mistaken for high hyperdiploid ALL. Whole genome analysis by classical karyotyping and targeted evaluation of critical loci by FISH play an important role in diagnosis, prognosis, and risk stratification, and are also used as biomarkers for disease monitoring and response to therapy. More recently, microarray techniques have been used to detect DNA copy number alterations in human cancers. We present two patients with apparently similar hyperdiploid karyotypes. Patient one (female) had an abnormal near-triploid clone with three X chromosomes and two copies of chromosomes 2, 13, 15, 16, 19, and 20; four copies of chromosomes 4, 8, 14, 18, 21, and 22; a deletion of 6q; a deletion of 11q and two copies of chromosome 1 with a duplication of the long arm. FISH studies supported the findings made by classical cytogenetics. Patient two (male) showed a hyperdiploid clone with an extra X, three copies of chromosomes 4, 5, 6, 9, 11, 14 and 19; four copies of chromosomes 1, 8, 18, 20, 21, and 22; and four copies of chromosome 10 including two abnormal chromosomes 10 with a deletion of most of 10q. FISH studies concurred with the classical cytogenetic findings and also detected monosomy 17. Microarray analysis in patient one revealed a gain in copy number consistent with trisomy, tetrasomy and structural aberrations detected by karyotype. SNP analysis showed a heterozygous pattern for all chromosomes. Thus patient one is considered to have a high hyperdiploid karyotype and a good prognosis. Microarray analysis for patient two showed a gain in copy number for all probes specific for chromosomes 1, 5,6, 8, 9, 11, 14, 18, 19, 20, 21, 22, and X along with gains for part of chromosome 4 and part of chromosomes 10. However, SNP analysis showed a loss of heterozygosity (LOH) for chromosomes 2, 3, 4pter-4p12, 4q12-4qter, 7, 10q11.22-qter, 12, 13, 15, 16, and 17, those that appear to have normal diploid state by karyotype. These findings are consistent with the presence of a near-triploid clone derived from a hypodiploid karyotype and this patient would therefore have a very poor prognosis. These two patients illustrate the utility of microarray analysis in distinguishing between true high hyperdiploidy and duplication of low hypodiploidy in patients with childhood ALL. This distinction is critical to the treatment and prognosis of these patients.

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POSTER ABSTRACTS

Please note: These abstracts have not been edited for grammar or spelling.

1 COMPARISON OF TWO METHODS FOR THE ISOLATION OF PERIPHERAL LEUKOCYTES FROM WHOLE BLOOD SAMPLES

Teresa Thompson, MB(ASCP)CM, Supervisor, Greenwood Genetic Center; Tim Wood, PhD, FACMG, Biochemical Laboratory Director; Laura Pollard, PhD, FACMG, Biochemical Laboratory Associate Director Isolation of peripheral leukocytes from whole blood samples is a standard protocol in many clinical laboratories. Once isolated, the resulting leukocyte pellet is used to measure various analytes or the activities of a large number of enzymes. Currently we use a dextran centrifugation method which stratifies the cell types based on a density gradient followed by several washes to remove any residual red blood cells or other debris. The procedure is difficult to automate and is therefore quite time-consuming, especially when processing a large number of samples. The Stemcell EasySepTM RBC Depletion Reagent, in conjunction with the EasySepTM magnet, allows for the magnetic removal of RBCs from whole blood. We wanted to determine if the Stemcell RBC depletion procedure could replace our current more laborious method for leukocyte isolation. The recommended Stemcell protocol includes two additions of the EasySepTM RBC Depletion Reagent with three five minute incubations in the EasySepTM magnet. We followed this with a single ten minute centrifugation to pellet the cells for future use. For an initial comparison we isolated leukocytes from peripheral blood samples collected from four healthy adult volunteers. Two tubes were collected from each volunteer and leukocytes were isolated using either the Stemcell or dextran centrifugation method using equal volumes of blood. The pellets were reconstituted in 1 mL deionized water and sonicated using a cell disruptor. The total protein concentration in each sonicate was measured using a modified Lowry method. The activities of ten different lysosomal enzymes were then measured using standard protocols already validated in our laboratory. The protein concentrations in the sonicates originating from the Stemcell method were significantly higher than those obtained from the dextran centrifugation method (t-test, P<0.005). The activities of five of the enzymes were significantly lower in cells isolated by the Stemcell method: heparan-N-sulfatase, beta-glucuronidase and alpha-fucosidase (P< 0.05), alpha- mannosidase (P<0.01) and N-acetyl-galactosamine-6-sulfatase (P<0.005). However, although the activities were lower using the Stemcell method, the activities remained within the previously established normal range for each enzyme; therefore the difference was not clinically significant. There was no statistical difference between the two isolation methods for the activities of alpha-galactosidase, alpha-iduronidase, beta- galactosidase, beta-glucosidase or beta-mannosidase. It is unclear why the leukocyte isolation method appears to impact the activities of some enzymes and not others. Enzyme activities are reported relative to the total protein concentration of the sonicate. Future experiments will evaluate whether the higher total protein concentration in the sonicates using the Stemcell method, which would impact enzyme activity calculations, is due to residual red blood cells or other cellular debris by adding a saline wash step after the initial centrifugation. The effect of this additional saline wash on enzyme activity will also be evaluated. Additional experiments are also planned to compare the leukocyte yields between the two methods using smaller volumes of blood, as this can negatively impact extraction efficiency. It is worth noting that with the Stemcell method the processing of 8 blood samples can be completed in just over one hour as compared to over three hours using the dextran centrifugation method. Additionally, the Stemcell method may also be amenable to automation. In summary, our preliminary data suggest that using the Stemcell RBC depletion reagents for the isolation of leukocytes is comparable to that of the more laborious commonly used dextran gradient methods and could save significant technologist time if implemented in a clinical laboratory setting.

2 THE APPLICATION OF A ONE-DAY HYBRIDISATION-BASED ENRICHMENT PROTOCOL FOR NEXT-

GENERATION SEQUENCING (NGS) INCORPORATING A RAPID (30 MINUTE) HYBRIDISATION STEP

Dr. Graham Speight, R&D Director, Oxford Gene Technology; Dr. Lyudmila Georgieva; Dr. Jacqueline Chan; Mr. Ezam Uddin; Prof. Nick Cross, National Genetics Reference Laboratory - Wessex, UK Although hybridisation-based enrichment protocols for NGS generate higher quality data (e.g. enhanced coverage uniformity, more complete coverage, and more accurate assessment of indels and internal tandem duplications [ITDs]), they are generally more time consuming than PCR-enrichment approaches. Here we present data from a rapid (30 minute) hybridisation protocol that enables Illumina sequencer- ready libraries to be generated from purified DNA in a single-day. This rapid one- day hybridisation protocol has been used with three different haematological tumour capture panels. Specifically these are the SureSeqâ&#x201E;˘ Core MPN, Myeloid and acute myeloid leukaemia (AML) panels. These panels range from a small (~1 Kb) hotspot panel, to a 25 gene (~53 Kb) panel. The quality of the data generated is comparable to the standard 4 hour hybridisation protocol and detects single nucleotide variants, indels as well as ITDs. Even typically difficult to sequence genes such as CALR, CEBPA and FLT3 are covered with high uniformity. The confidence in detecting low allele fractions was also confirmed using the JAK2 V617F Genotyping Sensitivity Panel from the National Institute for Biological Standards and Control (UK). This confirmed the accurate detection of The Journal of the Association of Genetic Technologists 43 (3) 2017

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Poster Abstracts, AGT 2017 low level variants down to a 1% variant allele fraction at a read depth of >1000x. This protocol therefore offers a similar turn-around time to amplicon-based NGS enrichment protocols, without the associated disadvantages, such as PCR bias, allelic bias (indels) and drop-outs, as well as poor uniformity of coverage.

3 T HE APPLICATION

OF A HYBRIDISATION-BASED NEXT- GENER ATION SEQUENCING (NGS) ENRICHMENT PANEL FOR THE ANALYSIS OF SOMATIC VARIANTS IN BREAST TUMOUR SAMPLES AND THE ASSESSMENT OF PERFORMANCE USING A REFERENCE STANDARD Dr. Graham Speight, R&D Director, Oxford Gene Technology; Jacqueline Chan; Lyudmila Georgieva; Sabine Eckert

Solid tissue tumours biopsies like breast are typically archived as formalin-fixed, paraffin embedded (FFPE) blocks. This approach unfortunately can significantly compromise the quality of the nucleic acids. To overcome these issues, we have used the SureSeq™ FFPE DNA Repair Mix, in combination with hybridisation-based NGS custom enrichment panels. The first panel is the SureSeq™ Ovarian Cancer Panel which targets key breast cancer-related genes (BRCA1, BRCA2, ATM, TP53, ATR, NF1 and PTEN). The second panel we use is a custom panel from the SureSeq myPanel™ NGS Custom Cancer Panel range that includes the relevant genes for the identification of a broad range of variants present in a commercially available multiplex reference standard. We compare the uniformity and mean target coverage between a PCR amplification-based and a hybridisation-based enrichment approach for BRCA1 and BRCA2. The improved performance observed with the utilisation of the FFPE DNA Repair Mix and the hybridisationbased enrichment approach enables the generation of highly uniform coverage, permitting the detection of low allele fraction single nucleotide variants as well as insertions and deletions. The uniformity of the coverage for most samples is greater than 99% of bases covered at 20% of the mean, ensuring that all bases within the panel can be assessed. We have also assessed the performance of a custom panel using the Structural Multiplex Reference Standard from Horizon which contains a broad range of variant types and allele frequencies. We report on the allele frequency, the concordance and detection accuracy.

4 CHARACTERIZATION OF CHROMOSOME ABNORMALITIES IN 747 MYELODYSPLASTIC SYNDROME (MDS) CASES

Farah A. Ladha, BS, CG (ASCP), Cytogenetic Technologist II, Baylor Genetics, Department of Molecular and Human Genetics, Baylor College of Medicine; Janice L., Smith, PhD, FACMG, Baylor College of Medicine Myelodysplastic syndrome (MDS) is a group of hematological diseases classified by ineffective hematopoiesis. The World Health Organization classifies MDS according to degree of dysplasia, percentages of blast cells, and genetic information. The well- described cytogenetic features of myelodysplastic syndrome involve -5/deletion (del) 5q, -7/ del 7q, +8, 11q aberrations, del 20q, and loss of the Y chromosome. While there is an increased emphasis on using a standard fluorescence in-situ hybridization (FISH) MDS panel to solely detect those listed chromosome regions in interphase cells, it is important to understand the diagnostic value of whole chromosome analysis using the conventional G-banding technique. This study consists of bone marrow cases with the clinical indication of MDS, as stated by the physician. All of these cases were referred to the diagnostic laboratory for oncology chromosome analysis over a period of 6 years. Using the standard G-banding technique, bone marrow analysis was performed on unstimulated 24-hour and 48-hour cell cultures. From the 747 cases that were studied, 227 (30.4%) reported abnormal chromosome results. The focus was on the commonly described cytogenetic features displayed in MDS. The resulting abnormalities were then investigated to determine the incidence of these aberrations as either simple isolated abnormalities or with involvement in complex karyotypes. In this particular study, karyotypes are described as complex when involving 3 or more chromosome abnormalities. Out of the 60 cases that reported a del 5q, 13 (21.6%) cases were an isolated abnormality and 40 (66.6%) cases reported a complex karyotype. The entire loss of chromosome 5 was found in 11 cases, none of which were isolated. From the 50 cases that reported del 7q/-7, isolated abnormalities were found in 14 (28%) cases and 32 (64%) cases were of complex karyotypes. Out of 35 cases that reported +8, 12 (34.4%) cases presented it solely and 20 (57.1%) cases involved complex karyotypes. The deletion of 11q was found in 17 cases, 8 (47%) of which were sole abnormalities and 8 (47%) were involved in complex abnormalities. Rearrangements of 11q were found in 11 cases, 2 (18%) of which were sole abnormalities and 9 (81.8%) of which presented with complex abnormalities. Del 20q was reported in 18 cases, 12 (66.6%) of which were isolated and 5 (27.7%) cases involved complex karyotypes. Loss of the Y chromosome was observed in 43 (10.4%) of the 415 male cases, with –Y solely in 30 (69.7%) cases. Upon assessment of the categorized isolated chromosome aberrations compared to the involvement of complex karyotypes, the relevance of standard chromosome analysis is evident in determining clonal nature of such aberrations. This study investigates the established diagnostic implications of each listed abnormality as it may differ between sole anomalies and complex chromosome involvement. Utilizing conventional whole chromosome analysis provides valuable prognostic information that can aid in interpreting complex rearrangements found under the MDS spectrum.

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Poster Abstracts, AGT 2017 5

DYNAMIC MOSAICISM OF AN X RING CHROMOSOME

Carlos Alonso Muñoz, BS,CG(ASCP)CM, Director, Laboratorios Mendel; Karla Nathalie Gaytán Nares; Víctor Alfredo Pérez Contreras; Carlos Cortés Penagos Introduction. A ring chromosome is a structural rearrangement formed from the deletion of distal regions, followed by a fusion of these ends forming a new circular structure or ring (r). This type of chromosomal abnormality is low frequency (1 / 30,000 to 1 / 60,000) and can occur on any of the human chromosomes. The phenotype is diverse, correlated with the size and instability of the ring chromosome. [2] Among the posible clinical features observed are: developmental delay, dysmorphic signs, low height, microcephaly, intellectual deficit, cardiac defects, low psychomotor development and infertility. In some cases, a ring chromosome can be present without clinical consequences other than infertility. [3] [1] The instability of the ring chromosome originates a dynamic mosaicism that is the presence of diverse cell lines due to the effects of the exchange of sister chromatids at the mitosis stage, among them are: double ring formation, complex ring changes, ring doubling, opening ring or loss of ring chromosome (monosomy). [3] Main objective: To present evidence of dynamic mosaicism on the formation of an X ring chromosome. Material. Female peripheral blood in sodium heparin, patient with presumptive diagnosis of dysmorphic syndrome. Methods. Conventional karyotype (GTG bands) and FISH analysis were performed using the WCPX and single centromeric sequence probe DXZ1, Xp11.1-q11.1 from CYTOCELL. Cytogenetic analysis of 200 metaphases showed a karyotype: mos45, X [31] / 46, X, r (X) (p21q12)? [138] / 46, X, r (X) (p22.1q22)? [30] /47, X, r (X) (p22.1q22)? X2 [1].In order to confirm the origin of the rings obtained by Cytogenetics, an analysis of FISH (painted X chromosome) was carried out. 30 metaphases were analyzed, 6 presented a signal (wcpX) and 24 metaphases two signals (wcpX), signals that correspond to the X chromosome. They presented rings of different size, which correlates with what is observed in the conventional karyotype (figure 2). Centromeres were detected by FISH analysis (DXZ1 probe) observing a signal at 80 interfaces and 3 metaphases, two signals at 193 interfaces and 15 metaphases. The different cell lines are expressed as: mos45, X [40] / 46, X, r (X) (p21q12)? [168] / 46, X, r (X) (p22.1q22)? [38] / 46, X, r (X) (p22.3q28)? [1] / 47, X, r (X) (p22.1q22)? X2 [1] .r (X) (wcpX +, DXZ1 +). Conclusions. Dynamic mosaicism of the X ring chromosome is demonstrated. The X ring chromosome presents instability at the cell division (mitosis) generating mosaics or different cell lines in the same person, a consequence of the instability is the presence of aneuploidies. The deficient exchange of material between sister chromatids generates rearrangements like dicentric rings, interlaced rings, which form unbalanced rings in different sizes with consequent genetic ruptures. The instability of the ring infers on the variability of the phenotype. We will pursue to verify the breakpoints with CGH Array, to get to know the genes involved in the different breakpoints. Bibliography. 1. Pooja C, Sushil K, Anjali R, Amit K. 2016. JARG, 1-8. 2. Caba L, Rusu C, Pl ia u V, Gug G, Gr mescu M, et al. 2012. BJMG 15/2, 35-46. 3. Guilherme1 R, Ayres V, Kim C, Pellegrino R, Takeno S, et al. 2011. BMC Medical Genetics 12:171.

ROLE OF DIFFERENT B-CELL MITOGENS IN DETECTION OF ABNORMALITIES IN B LYMPHOCYTES

Emily Kruse, BS, CG(ASCP)CM, Cytogenetic Technologist, The Ohio State University Wexner Medical Center; Rakia Reed, BS, CG(ASCP)CM; Lapo Alinari, MD, PhD; Gerard Lozanski, MD; Jennifer Woyach, MD; Jodi Hanna, BS, MT(ASCP)CM; Nanette Kendall, BS; Rachel Lippello, MPH, MLS(ASCP)CM; Christie Plickert, BS, MT(ASCP) CGCM; Lynn V. Abruzzo, MD, PhD, ABMG; Nyla A. Heerema, PhD, FACMG, ABMG A 52 year old male patient was transferred to the James Cancer Hospital at the Ohio State University Wexner Medical Center in November of 2016 for evaluation of leukocytosis with lymphocytosis. Immunophenotypic results were consistent with a B cell lymphoproliferative disorder, in particular a variant form of chronic lymphocytic leukemia (CLL). In addition, IGHV mutation analysis showed unmutated status which indicates a poor prognosis. In our laboratory, CLL samples are cultured for 72 hours and one culture is stimulated with pokeweed mitogen (PWM), phorbol myristate acetate (PMA), and CpG oligodeoxynucleotides (ODN), and a second culture is stimulated with CpG ODN only. In contrast, B cell lymphoma samples are cultured for 72 hours and stimulated with CpG ODN only. Conventional cytogenetic analysis on a peripheral blood sample showed two unrelated clones. One clone of cells was distinguished by a t(2;5)(q33;q31) and a t(14;19)(q32;q13) and was predominantly seen in the culture stimulated with PWM, PMA and CpG ODN. While this t(2;5) is not known to be associated with a specific disease, a t(14;19) is a recurring abnormality in CLL. However, the second unrelated clone of cells contained a t(11:14) (q13;q32) as the sole abnormality and was detected only in the culture stimulated with CpG ODN only. This translocation is typically found in mantle cell lymphoma. FISH analysis on the PWM, PMA, CpG culture with the dual fusion IGH-CCND1 probes (Abbott Molecular), was positive for a t(11;14) (3.8%) and also showed three copies of IGH (48.6%) in different cells, consistent with the second IGH rearrangement present in the cytogenetic analysis, the t(14;19). It is interesting to note that, based on conventional cytogenetics and FISH analysis there are two distinct populations of cells possibly suggesting two different diseases, while immunophenotypic results describe only one population of cells. This may be because the cells analyzed by cytogenetics and FISH were cultured with two different mitogens which may stimulate different cell populations. CpG oligodeoxynucleotides are synthetic DNA molecules that stimulate the immune system and are

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Poster Abstracts, AGT 2017 recognized by a B cell receptor. The introduction of the PWM and PKW, also B-cell mitogens, in addition to the CpGs appeared to stimulate growth of the t(14;19) cells that were seen predominately in that culture, but not the t(11;14) cells which were seen predominately in the CpG culture only. This supports our hypothesis that different culture conditions influence which cells are stimulated to divide and can yield different cytogenetic results.

7 WHEN TRISOMY 13 IS NOT TRISOMY 13: THE POWER OF INTEGRATED FISH, MICROARRAY, AND CHROMOSOME ANALYSIS

Sean Houde, CG(ASCP)CM, Cytogenetics Senior Technologist, LabCorp; M. Katharine Rudd, PhD FACMG Interphase fluorescence in-situ hybridization (FISH) is commonly used to detect chromosome aneuploidies in fetal and newborn specimens. It is a valuable diagnostic tool to obtain rapid results without the need to culture cells. However, FISH is limited by the specific probes in each assay, and FISH does not provide information on the structure of chromosome abnormalities. Here we present a prenatal case tested at 20 weeks gestation after detection of multiple fetal anomalies and a positive maternal screen result. FISH analysis of uncultured amniocytes using the RB1 probe from chromosome 13q14.2 revealed 3 signals consistent with trisomy 13 in all cells. Surprisingly, chromosome banding revealed 46 chromosomes including one normal and one abnormal 13 that appeared to have an inverted duplication and terminal deletion of the long arm. According to microarray analysis, there was a normal copy number in proximal 13q, a 44-Mb interstitial duplication of 13q12.11-q21.31, and a 50.8-Mb terminal deletion at 13q21.31. Together, these results clarified a complex chromosomal rearrangement in which only half of the long arm of chromosome 13 was trisomic, consistent with the three RB1 signals. Chromosome analysis determined the orientation of the trisomic segment was inverted, and confirmed the duplication and deletion were part of a single derivative chromosome. In some cases, inverted duplications can disrupt gene(s) at the duplication breakpoint, leading to a more deleterious effect than tandem direct duplications. However, this rearrangement does not appear to disrupt genes at the distal duplication breakpoint. Inverted duplication with terminal deletion is a relatively common copy number variation that leads to partial trisomy and monosomy of the same chromosome arm. The monosomic and disomic segments in this case were very large, and are likely to be associated with a severe phenotype. In our study, each cytogenetic test revealed a different piece of information necessary to understand the full structure of the patientâ&#x20AC;&#x2122;s chromosome rearrangement. This example points out the caveats of FISH testing and the need for careful follow-up studies.

8 CLINICAL UTILITY OF CYTOGENETIC CONFIRMATORY TESTING FOLLOWING ABNORMAL

NON-INVASIVE PRENATAL SCREENING FINDINGS FOR FETAL ANEUPLOIDY: A STUDY OF 63 CONSECUTIVE CASES

Megan Hillen, BS, CG(ASCP)CM, MHA, Cytogenetic Technologist, Cytogenetics and Molecular Pathology, Department of Pathology and Immunology, Washington University School of Medicine; Yoshiko Mito, PhD, Assistant Professor of Pathology and Immunology; Associate Director, Cytogenetics and Molecular Pathology; Katinka Vigh- Conrad, PhD; Amie Stanley, MS, Division of Maternal-Fetal Medicine, Ultrasound and Genetics, Department of Obstetrics and Gynecology, Washington University School of Medicine; Diana Gray, MD, Division of Maternal-Fetal Medicine, Ultrasound and Genetics Fetal aneuploidy is a common genetic anomaly, detected in up to one-third of pregnancy losses, and in approximately 1/300 of all live births, with the most frequent being Trisomy 21 (causative of Down syndrome). Prenatal detection of aneuploidy has been commonplace for the last 40 years, and as chorionic villus sampling (CVS) and amniocentesis provides diagnosis. In order to minimize the number of invasive procedures, effective use of prenatal screening algorithms with high sensitivity and specificity is crucial. Conventional maternal serum screening algorithms utilizing a combination of maternal age, ultrasound findings, and measurement of maternal serum analytes are well-established and widely-used with moderate to high sensitivity. Over the last decade, another noninvasive screening approach has been introduced, which quantifies circulating cell-free fetal DNA (ccffDNA) in maternal serum by next- generation sequencing or microarray analyses (ccffDNA screening or noninvasive prenatal screening (NIPS)). This latest approach showed promise for improved sensitivity and specificity, particularly in earlier studies for high-risk women. In order to test the performance of ccffDNA screening, we reviewed 63 consecutive cases that were received at our laboratory for cytogenetic analysis following abnormal ccffDNA screening findings during a 42 months period from 2013 to 2016 (amniotic fluid (AF) n=30; CVS n=22; postnatal peripheral blood (PB) n=11). Additionally, we reviewed 39 cases received in our laboratory during the same period for cytogenetic analysis with the indication of increased risk of aneuploidy determined by maternal serum screening (AF n=34; CVS n=2; PB n=3). Concordance between the reported abnormal screening results and the subsequent diagnostic cytogenetic results was examined. Analysis of ccffDNA-positive cases showed that 46 (73%) of 63 were confirmed by cytogenetic analysis, whereas only 2 (5%) of 39 cases with increased risk by maternal serum screen were confirmed. Analysis also demonstrated high concordance rates for cases with indication of Trisomy 21 (88%; n=33) and Trisomy 18 (89%; n=9), but lower concordance rates (<50%; n=21) for indications of Trisomy 13, Monosomy X, or other sex chromosome abnormalities. This trend

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Poster Abstracts, AGT 2017 was true for all specimen types. Results of the above analysis suggest that non-invasive prenatal screening utilizing ccffDNA has higher positive predictive value than maternal serum analyte screenings. However, confirmatory cytogenetic testing is crucial following NIPS to rule out false-positives, especially for aneuploidies other than trisomy 18 or 21. Improvements in non-invasive detection methods offer patients more consistent prenatal diagnostic results than previously experienced.

9 A RARE COMPLEX REARRANGEMENT REPRESENTING A VARIANT OF T(1;22)(P13;Q13) IN A PEDIATRIC NON-DOWN SYNDROME ACUTE MEGAKARYOBLASTIC LEUKEMIA

Damian Bridges, BS, CG(ASCP), Lead Technologist, Childrenâ&#x20AC;&#x2122;s Mercy Hospital; Michael Tiller BS, CG(ASCP); Linda Cooley, MD, MBA; Weijie Li, MD; Lei Zhang, PhD Pediatric Acute Megakaryoblastic Leukemia (AMKL) is a rare subtype of AML (FAB M7) diagnosed mostly in myeloid leukemia of Down syndrome (DS). In pediatric non-DS AML, AMKL accounts for ~10% of cases. The t(1;22)(p13;q13) with RBM15/MKL1 fusion was the earliest recognized recurrent abnormality in pediatric AMKL and accounts for ~10% of cases. Although early reports suggested a poor prognosis for AML with t(1;22), recent studies have shown that patients with t(1;22) respond to intensive AML therapy with long disease-free survival. Here we report an AMKL case with complex rearrangements, which involve chromosomes 1p, 16p and 22. That the rearrangements were balanced was confirmed with microarray analysis. FISH analysis using probes for RBM15 and MKL1 confirmed RBM15/ MKL1 fusion. The complex karyotype in this case represents a variant of the typical t(1;22)(q13;q13). Our patient, a 14 month old female, presented with fever, acute otitis media and petechial hemorrhages. Peripheral blood hemogram showed a WBC count in the normal range (6.92K/mcL), low hemoglobin (8.4gm/dL), a critically low platelet count (26K/mcL), and ~11% circulating blasts. The bone marrow biopsy was packed with medium to large immature mononuclear cells in a fibrotic stroma. Abnormal cells were diffusely positive for CD61, CD31 and partially positive for CD4, CD45, and CD117. These results led to a pathological diagnosis of acute myeloid leukemia with megakaryoblastic differentiation. Chromosome analysis revealed structural rearrangements of 1p, 16p and both 22â&#x20AC;&#x2122;s. Banded chromosomes and metaphase FISH analysis showed bands 1p13.3p31.2 inserted into chromosome 22 at 22q13.1; the 16p terminal band translocated to the terminus of the derivative chromosome 22; bands 16p12p13.3 inserted into chromosome 1 at 1p13.3; and the terminal 22q band translocated to the derivative chromosome 16 short arm terminus. The second copy of chromosome 22 was a variably sized ring, with duplication of bands 22q12.1q13.2, confirmed by microarray and FISH analyses. The nomenclature for the karyotype is: 46,XX,der(1)del(1)(p13.3p31.2) ins(1;16)(1p13.3;p12p13.3),der(16) t(16;22)(p12;q13.3), der(22)ins(22;1) (p13.1p31.2)t(16;22)(p13.3;q13.3),r(22)(p11.2q13.3)dup(22)(q12.1q13.2) [14] .ish der(1)(MYH11+),der(16)(D16S3400-,SHANK3+,CBFB+,16qtel48+), der(22)(TBX1+,SMARCB1+,NF2+,PDGFB+,D16 S3400+,SHANK3-), r(22) (TBX1+,SMARCB1+,NF2++,PDGFB++,SHANK3+) This case illustrates the merit of using conventional cytogenetic, microarray and FISH methodologies in combination to provide an accurate assessment of clonal aberrations, which have impact on patient diagnosis and therapeutics.

PLAG1 REARRANGEMENTS IN SALIVARY GLAND PLEOMORPHIC ADENOMA

Melinda A. Claydon, BA, CG(ASCP)CM, Clinical Laboratory Technologist, Roswell Park Cancer Institute; A. W. Block, PhD; S. N. J. Sait, PhD Pleomorphic adenoma is the most common salivary gland tumor of all the sites in both adults and children. About 75-80% of parotid gland tumors and 60% of all salivary gland tumors are pleomorphic adenomas. Pleomorphic adenomas are morphologically characterized by a biphasic pattern of differentiation illustrated by the coexistence of epithelial and mesenchymal areas. Numerous studies have shown that approximately 70% of pleomorphic adenomas are cytogenetically abnormal. Chromosome abnormalities include 3p21, 8q12, 12q12-15 rearrangements, and trisomy 8. Among these abnormalities, 8q12 rearrangements constitute the largest cytogenetic subgroup (40% of pleomorphic adenomas). Abnormalities of the PLAG1 locus (8q12.1) have been reported in salivary gland tumors and are found mostly in benign pleomorphic adenomas as well as in carcinoma ex-pleomorphic adenomas arising in them. Rearrangements of PLAG1 and tissue overexpression have also been reported in a large percentage of pleomorphic adenomas in cases without cytogenetically detectable 8q12 aberrations. We report 5 patients with a surgical pathology diagnosis of benign mixed tumor or pleomorphic adenoma. The patients included 1 male and 4 females with a median age of 52 years. All 5 patients had a cytogenetic abnormality involving chromosome 8q12. 4 of 5 patients showed a rearrangement of the PLAG1 locus by fluorescence in-situ hybridization (FISH) analysis. Further characterization of these cases will potentially shed light into the mechanisms behind these rearrangements which might have prognostic and therapeutic implications.

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Poster Abstracts, AGT 2017 11 TETRAPLOIDY AND NEAR TETRAPLOIDY IN ACUTE MYELOCYTIC LEUKEMIA AND MYELODYSPLASTIC SYNDROME - A REPORT OF SEVEN NEW CASES

Susan T. Nedumakel, CG(ASCP), Laboratory Supervisor, Clinical Cytogenomics Laboratory, Department of Pathology, Anatomy, and Cell Biology, Thomas Jefferson University; Dorothy Foster; Julie Best; Patricia Norton; Danielle Fortuna; Guldeep Uppal; Jerald Gong; Zi-Xuan Wang; Stephen Peiper; Jinglan Liu Tetraploidy and near tetraploidy (T/NT) (81–103 chromosomes) karyogram with or without numerical and structural abnormalities is rare in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Only single case or small case series have been reported to date. The clinical significance is not well understood mainly due to the rarity of the entity. Here we report the clinical, morphological, cytogenetics, molecular genetics and prognostic features in seven cases of T/NT-AML/MDS. The seven cases studied resulted from the search in the database of the Clinical Cytogenetics Laboratory at The Thomas Jefferson University Hospital between 2009- 2016. Chromosomal abnormalities were documented using the International System for Human Cytogenetic Nomenclature (2016). A T/NT clone with ｳ3 chromosome abnormalities were classified to have a complex T/NT karyogram, and the others as a non-complex T/NT karyogram. Available clinical data was reviewed, including age, gender, presentation, treatment, response and outcomes. Peripheral blood smears, bone marrow aspirate and core biopsy results were assessed. Bone marrow cellularity, blast morphology and percentage, background dysplasia were particularly studied. Immunophenotype by flow cytometry and/or immunohistochemistry and laboratory data were also collected. After review of the related data, a total of seven patients (6 with AML and 1 with MDS) with a T/NT karyogram were identified, comprising 0.47% of all AML and MDS patients during this seven-year interval at The Thomas Jefferson University Hospital. Four patients were male and three were female. Age ranged from 54–81 years (median, 72 years). T/NT karyogram was detected at initial diagnosis of AML/MDS in five patients, and acquired during the course of disease in two patients. Three patients had a non-complex and four had a complex T/NT karyogram. Other than chromosomal gains, del 7q/7 was the most common structural abnormality in our series (n = 4) followed by del 5q/5 (n=2) and del 17p (n=2). Recurrent translocations seen in AML/MDS which were also present in our series were i(17)(q10) and t(1;7). All patients exhibited medium to large sized blasts, frequently with irregular nuclear contours and prominent nucleoli as well as various levels of cytoplasmic vacuoles and/or inclusions. Auer rods were not common. Mutation tests for FLT3 internal tandem duplication (ITD) and IDH1 codon 132 on two patients were negative. Next-generation sequencing (NGS) technology based hematological malignancy gene panel test was performed on one case and revealed a pathogenic mutation in the gene SRSF2. In summary, T/NT karyogram is an infrequent abnormality in AML and MDS with distinctive morphologic features for blasts. Seven new cases were reported. The prognostic significance and possible mechanism of a T/NT karyogram are discussed. This is also believed to be the first time that NGS panel test was utilized to characterize this unique entity.

12 CYTOGENOMIC CHARACTERIZATION OF AN INTERSTITIAL DUPLICATION/ TRIPLICATION OF THE 7Q35-Q36.1 REGION IN AN INFANT WITH MULTIPLE CONGENITAL ANOMALIES

Christine Sulym, BS, Medical Laboratory Technician II, Duke University Health System- Clinical Labs; Christina Bradberry, CG(ASCP)MB; Kimberly Gobac, CG(ASCP); Marc Delos Angeles, CG(ASCP); Gloria Haskell, PhD; Kristen Deak, PhD, FACMG Partial chromosome triplications are rare in the literature and the mechanisms of these types of rearrangements are not well understood. Here, we report the case of an infant girl with a severe phenotype that has an interstitial triplication/duplication of distal chromosome 7. At birth, the infant presented with respiratory distress and was found to have cardiac abnormalities such as a peripheral pulmonic stenosis and patent foramen ovale, requiring ventilation. She also had dysmorphic facial features including low set small ears, downslanting and wide-spaced eyes, flattened nose and micrognathia. Additionally, brain MRI showed gray matter heterotopia and possible delayed myelination. SNP chromosomal microarray showed an interstitial gain involving the 7q35-q36.1 region; there is a triplication (4 copies) of approximately 6.08 Mb involving the proximal region, while the more distal region is a duplication (3 copies) of approximately 2.5 Mb. Chromosome analysis confirmed that the duplicated material is present on 7q. The abnormality is presumed de novo, however, parental testing is pending. Previously, Lehnen et al. (Genome Res. 2009;125(3):24852) reported a child with terminal triplication of a similar region of chromosome 7, involving approximately 13.5 Mb of distal 7q. These two cases have similar dysmorphic facial features and both have abnormalities of the cardiovascular and neurological systems. The duplicated region in our case partially overlaps with the duplicated region previously reported, and although smaller, this case is more complex with both triplicated and duplicated regions. Of note, the PRKAG2 gene, which was proposed to play a role in the cardiac phenotype of the patient with the triplication, is located in the duplicated region of this case. Although the mechanism underlying this complex rearrangement is not known, copy number changes of 7q36.1 have been suggested to be segmental duplication-mediated. Additionally, pending FISH analysis may be useful in determining the orientation and location of the duplicated material within chromosome 7. This case adds to the limited medical genetics knowledge of complex chromosome rearrangements and the clinical features associated with gains of 7q, and potentially suggests a critical region for common abnormalities.

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Poster Abstracts, AGT 2017 13 AN UNUSUAL CASE OF HYPERTETRAPLOIDY IN PRODUCTS OF CONCEPTION FROM

FEMALE WITH RECURRENT MOLAR PREGNANCIES AND SUBSEQUENT DIAGNOSIS OF CHORIOCARCINOMA Sheri Hedrick, CG(ASCP), Genetic Technologist, Nationwide Chilrdens”s Hospital; Elizabeth Barrie, PhD; Elizabeth Hamelberg, CG(ASCP)CM; Theodora Jacobson, MS, LGC; Matthew Meleski; Danielle Mouhlas, MS, LGC; Ruthann Pfau, PhD, FACMG; Christopher R. Pierson, MD, PhD; Caroline Astbury, PhD, FACMG Gestational choriocarcinoma is a neoplasm that arises from transformation of trophoblastic elements of the placenta, with about 50% occurring after a molar pregnancy. Complete hydatidiform moles are typically diploid, while partial moles are often triploid. We report on an unusual case of a hypertetraploid karyotype of 97,XXXX,+6,+8,+12,+13,+14 from cultured products of conception. The patient, a 32 year old G3P0Ab3 female, had two previous losses, both of which were partial molar pregnancies. She presented with a third partial molar pregnancy which terminated at 8 weeks gestation and was referred for cytogenetic analysis. A sample of cystic villi described by the pathologist as dysmorphic villi with cisterns was cultured, resulting in the aforementioned hypertetraploid karyotype. Subsequent diagnosis of choriocarcinoma prompted additional interest in this case, including potential predisposition to molar pregnancy. The gene NLRP7 affects genomic imprinting of the egg and may prevent fertilization by multiple sperm. NLRP7 is mutated in 48- 80% of patients with ｳ2 molar pregnancies, however sequencing of this gene by an outside laboratory did not reveal any pathogenic variants in our patient. The unusual karyotype result prompted further investigation, and was particularly interesting in light of the patient’s subsequent diagnosis of gestational choriocarcinoma. We describe additional workup available for cases with multiple molar pregnancies with or without choriocarcinoma.

14 PRENATAL DETERMINATION OF TRUE CHIMERISM IN A POSTNATAL HEALTHY FEMALE

Elizabeth Hamelberg, CG(ASCP)CM, Supervisor of Cytogenetics, Nationwide Children’s Hospital; Elizabeth S. Barrie, PhD; Carol Deeg, CG(ASCP); Linda Erdman, CG(ASCP); Cecelia Green-Geer, CG(ASCP); Sayaka Hashimoto, MS, LCG; Sheri Hedrick, CG(ASCP); Matt Meleski; Don Roman, CG(ASCP); Ruthann Pfau, PhD, FACMG; Caroline Astbury, PhD, FACMG A chimera is defined as a single organism composed of cells from different zygotes and is extremely rare. This can result in a variety of clinical presentations. We report a case of suspected XX/XY chimerism that was confirmed by prenatal and postnatal studies. A sixteen year old female with a gestational age of 20 weeks 6 days had an amniocentesis to rule out a partial molar pregnancy. AneuVysion results identified 94% female and 6% male cells. Subsequent chromosome analysis yielded a karyotype of chi 46,XY[2]/46,XX[19], consistent with the AneuVysion findings. An apparently female fetus was delivered at 39 weeks 2 days. Post-delivery chromosome studies were performed on two sources of placental tissue as well as a peripheral blood sample. The placental tissue yielded a karyotype of 46,XX in all cells while the peripheral blood confirmed the presence of chimerism with a chi 46,XY[8]/46,XX[12] karyotype. FISH was performed on peripheral blood metaphases using dual color probes for the X centromere and the SRY locus at Yp11.31. FISH results revealed the presence of two X chromosome centromeres in 55% (11/20) of the metaphases, with the remaining metaphases showing a single X chromosome centromere and SRY signal. The final karyotype reported for the peripheral blood sample was chi 46,XY[8]. ish Yp11.31(SRYx1)/46,XX[12]. The presence of both XX and XY cell lines within the peripheral blood supports the diagnosis of true XX/XY chimerism. Additional evaluation of the proband is pending.

15 OLIGODEOXYNUCLEOTIDE AND INTERLEUKIN-2 IMPROVE DETECTION OF CHROMOSOMAL ABNORMALITIES IN MATURE B-CELL NEOPLASMS

Colin Keegan, CG(ASCP), Lead Technologist, Dartmouth Hitchcock Medical Center; James Stevens; Liming Bao Cytogenetics plays an important role in diagnosis, prognostication and treatment guidance of hematologic neoplasms. Mature B-cell neoplasm including chronic lymphoblastic leukemia and lymphomas are common hematologic malignancies in adults. Karyotyping is an effective tool for comprehensive profiling cancer genome at chromosome levels. However, karyotyping analysis of mature B-cell neoplasms has been hampered by low analyzable metaphase chromosomes from cytogenetics preparations due to low proliferation of mature B-cells in the neoplasms. In this study, we explored oligodeoxynucleotide and interleukin-2 (ODN) as B-cell mitogens to increase detection of chromosome abnormalities in mature B-cell neoplasms. Unstimulated and ODN-stimulated cultures were used in parallel in cytogenetic preparations of 324 mature B-cell neoplasms, followed by conventional karyotyping analysis. Fluorescent in-situ hybridization (FISH) for common cytogenetic aberrations was concurrently performed on most of the cases. Clonal abnormalities were observed in 109 of 324 (34%) cases. Of these 109 cases with chromosomal abnormalities, 42 (39%) were seen only in ODN cultures, and 24 (22%) cases had abnormalities that were not identified in the concurrent FISH study. Our results demonstrate that oligodeoxynucleotide and interleukin-2 as B-cell mitogens would greatly improve detection of clonal chromosomal abnormalities in mature B-cell malignancies.

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Poster Abstracts, AGT 2017 16 CLONAL EVOLUTION PATHWAYS AND GENETIC PROGRESSION SCORES BACKTRACK SPECIFIC

GENETIC EVENTS AND OUTLINE UNIQUE RECURRENT CHROMOSOME ABERRATIONS THAT PREDICT CELL OF ORIGIN AND CLINICAL OUTCOME IN TRANSFORMED FOLLICULAR LYMPHOMA

Rolando Garcia, PhD, Clinical Fellow in Cytogenetics, UT Southwestern; Berni Elias, CG(ASCP); Sangeeta Patel, CG (ASCP); Prasad Koduru, PhD Follicular lymphoma is the most common form of indolent lymphoma, with the median survival of newly diagnosed patients of more than ten years. The cytogenetic hallmark of follicular lymphoma is the (14;18) translocation, juxtaposing the BCL2 gene at 18q21 to the heavy chain immunoglobulin (IGH) gene at 14q32. The rearrangement leads to constitutive overexpression of BCL2 allowing cells to slowly proliferate, and most importantly; to bypass apoptosis. The outcome of FL is varied and approximately 2-3% of cases per year transform to a more aggressive lymphoma mainly diffuse large B-cell lymphoma. In this study, we explore cytogenetic data to outline evolution pathways, establish the genetic status of tumor samples by determining the genetic progression score (GPS) derived from clonal evolution, assess cell of origin (COO) from karyotype evolution and predict outcome of transformed follicular lymphoma (t-FL). Tumor samples were selected from an institutional database and from the reported literature. All tumor samples contained histological evidence of transformation. To construct evolution pathways, cytogenetic data from tumor samples was parsed using a text analysis (tm library in the R statistical package). The translational Oncology package was then used to generate tumor progression pathways and the Rtreemix library was used to generate GPS of tumor samples (a GPS > 0.8 was considered a high score). Karyotypic pathways of clonal evolution served to assess COO assignment and a Kaplain-Meier survival curve was plotted to determine outcome. All p-values < 0.05 were considered significant. A total of 24 t-FL tumor samples including thirteen institutional cases with histological proof of transformation and eleven samples of documented t-FL from the literature were identified. Forty recurrent cytogenetics aberrations (RCAs) were generated from the text analysis. These RCAs were applied to a tumor evolution pathway analysis, but only 20 were used to determine GPS. There were six major pathways, including MYC, add(8)q24, 1qL, +18, +12 and 1p36L. These evolution pathways generated 25 distinct subclones. GPS intervals ranged from a low score of 0.4 to a high score of 1 and a GPS > 0.8 was significantly associated with shorter survival (p = .0003). Patients with MYC rearrangements showed an evident trend in adverse outcome (p = 0.14). RCAs present in patients with high GPS and shorter survival included: +X, MYC, 1q21 rearrangements, 1qL, +5, del(6)q, +7, +8, 9pL,+12, del(15)q and +18. Of these, only MYC, +12 and +18 represented major entry points to clonal pathways. Based on patterns of evolution, RCAs may also be used to assign COO in tFL (e.g. +18, 1p36L, -19, 13qL, -22, +2 to a non- germinal center t-FL and +12, +10, +21, +7 to a germinal center tFL group). Our analysis suggests a small number of cytogenetic entry points to evolution pathways; however, common and side by side clonal expansion results in a heterogeneous set of subclones in t-FL. In this regard, presence of unique RCAs to assess COO and the use of GPS, as a relevant prognostic factor, may be used to meaningfully categorize these tumors and select appropriate therapy for patients. Key words: t-follicular lymphoma, evolution pathways, GPS

A CAUTIONARY TALE: INTERPHASE DOES NOT EQUAL METAPHASE

Jessica J. Sikes, BS, CT(ASCP), CG(ASCP), Cytogenetic Technologist, Univerisity of Mississippi Medical Center; Beth Barnett, MS, CG(ASCP); S. K. Hall, BS, CT(ASCP), CG(ASCP); H. Huang, BS, CG(ASCP); S. L. Hurley, MS, CG(ASCP); A. Meloni-Ehrig, PhD, DSc, ABMGG; T.R. Dennis, PhD; H. H. Hobart, PhD, FACMG Here we report a case of acute myeloid leukemia (AML) with dual amplification of MYC and KMT2A (formerly MLL). This particular sample was processed independently by two laboratories and reported out with similar but not congruent results. Upon review of G-banded chromosomes, double minutes were observed which led to FISH testing. Extrachromosomal double minutes (dmin) and intrachromosomal homogeneously staining regions (hsr) are physical manifestations of gene amplification1. The amplified bits of chromosomal material vary in size from 0.5 MB to approximately 8 MB and are sometimes cryptic to G-band analysis2. Though their specific role in leukemogenesis is unknown, dmin and hsr are usually associated with rapid progression of disease and thus poorer prognosis. In this case, the indication for study suggested possible involvement of MYC or KMT2A, two commonly amplified genes in AML as well as in high-grade myelodysplasia. Both laboratories ran FISH tests using these probes. One lab initially relied upon interphase cells for their interpretation and the patterns observed suggested the presence of hsr that hybridized with the MYC probe. They also saw patterns that were consistent with double minutes containing KMT2A. The other lab focused mainly on metaphase cells for their analysis. They found that both KMT2A and MYC hybridized to double minutes and observed no hsr. Although this discrepancy is not clinically significant, it shows how caution should be used when interpreting FISH observations of interphase cells. Sikic B., p. 656 in Molecular Basis of Cancer, 4th. Ed. (2015), Mendelsohn et al eds., Elsevier, Philadelphia. Volkert et al, Amplification of EVI1 on cytogenetically cryptic double minutes as new mechanism for increased expression of EVI1. (2014), Cancer Genetics 207:103.

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Poster Abstracts, AGT 2017 18 METHOD VALIDATION FOR NOVEL IMAGE AND CELL SURFACE MARKER BASED ENRICHMENT

FOR BREAST AND GASTROESOPHAGEAL FFPE BIOPSIES WITH DOWNSTREAM FLUORESCENCE IN-SITU HYBRIDIZATION APPLICATION

Aditi Khurana, CG, MB (ASCP), Clinical Project Manager, PacificDx; Marc Ting; Samuel Koo, MSc; Valeria Sero, MSc; Amanda Gerber; Cynthe Sims, PhD, HCLD (ABB); Philip Cotter, PhD, FACMG, FFSc(RCPA); Mathew Moore, PhD, Principal, PacificDx; Farideh Bischoff, PhD, Chief Clinical Development Officer, Silicon Biosystems; Chiara Bolognesi, Menarini Silicon Biosystems; Nicolò Manaresi, PhD, CSO, Silicon Biosystems The American Society of Clinical Oncology and College of American Pathologists recommend testing to determine HER2 status in all patients with invasive breast cancer or ancillary cancers of breast origin or metastasis. HER2 targeted therapies are recommended based on results if determined clinically appropriate. Current FFPE based FISH testing results in 10 to 15% of equivocal cases even after repetitive FISH, IHC, FISH alternative chromosome or microarray testing. Confounding issues in average HER2 status include an already unstable CEP17 control skewed by truncation artifacts, overlapping cells and genome wide dilution of HER2 gene gains. Here we present a method validation of 40 IHC/FISH HER2 negative and 40 IHC/ FISH positive samples processed through the DepArrayTM (Menarini Silicon Biosystems, San Diego,CA) to isolate stromal (Keratin-,Vimentin+) and tumor (Keratin+, Vimentin-) populations. Establishing a patient specific baseline aiding in determination of true HER2 status based on single cell evaluation of the tumor cells without dilution with non- tumor or heterogenic tumor HER2 cells especially in negative and equivocal cases.

CASE STUDY: AN APL CASE WITHOUT A DETECTABLE RARA GENE REARRANGMENT

Siu Fung Lee, CG(ASCP)CM, Clinical Laboratory Technologist II, NeoGenomics Laboratories; Renan, Mota, MS, CG(ASCP)CM; Felix, De La Cruz, CG(ASCP)CM; Tiffany, Chouinard, CG(ASCP)CM,MB(ASCP)CM,HTL(ASCP)CM; David, Morgan, MD; Robert, Gasparini, MS, CG(ASCP)CM, DLM(ASCP)CM Acute Promyelocytic Leukemia (APL or APML) is a subtype of Acute Myeloid Leukemia (AML). APL is well characterized by its genetic abnormality, a reciprocal translocation between chromosomes 15 and 17 (i.e., t(15;17)(q22;q12)). This translocation results in the expression of the fusion gene, PML-RARA which eventually activates the translation of an oncoprotein blocking differentiation of the myeloid cell. Just like the symptoms of AML, APL patients usually experience anemia, pancytopenia, fever, and bleeding. Although the median survival of untreated APL is less than 30 days, the introduction of ATRA (all trans retinoic acid) therapy combined with chemotherapy, results in complete remission (CR) rates of approximately 80 to 95% in as little as 38 to 44 days. In addition, the longer term “cure rate” is as high as 80%, making APL the most curable subtype of AML. The most common tests that accurately detect the t(15;17) include cytogenetics, FISH, and RT-PCR on both bone marrow and peripheral blood. The main reason for the high accuracy of these tests is that 92% of APL cases have an exact breakpoint in RARA (on chromosome 17) with only three breakpoint cluster regions on PML (chromosome 15). A RARA translocation to any of the three breakpoints on PML would not affect the diagnosis or the effectiveness of ATRA therapy. Therefore, the ability to detect the (15;17) translocation makes the three tests listed above the preferred method of testing. However, there is a subset of APL patients in the 8-9% range that are more complicated with slightly different treatments based on the actual diagnosis. In general, these remaining APL cases can be separated into three groups: 1) a small insertion of RARA into PML (4%, cryptic by cytogenetics); 2) complex translocations involved in chromosome 15 and 17 (2%); and 3) one of the seven alternative RARA fusion partners from chromosomes 4, 5, 11, 16, 17 and X (less than 2%). We highlight a case where our patient, a 22 year-old female presented to her physician’s office with the following findings. A bone marrow evaluation was ordered as part of her work-up and came back suspicious for APL; however, both FISH and cytogenetics were reportedly normal. The physician suspected that the patient’s symptoms were related to APL and sent a part of the bone marrow sample for repeat testing to our laboratory. Based on a limited remaining sample that was already 5 days old, the physician prioritized the repeat testing in the following order: PML/RARA dual fusion probe by FISH, RARA break apart probe by FISH, cytogenetics, and RT-PCR for the PML-RARA transcript. Due to the limited amount of bone marrow received, only the FISH and cytogenetic tests were able to be performed. Cytogenetics appeared normal as did the RARA break apart probe set by FISH. However, the PML/RARA FISH test revealed an atypical, abnormal fusion signal pattern of 2R1G1F in just over 84% of the interphase nuclei scored. Further investigation and close examination with metaphase FISH (M-FISH) using this dual color, dual fusion PML/RARA probe set revealed a small red signal on top of one of the green signals, forming a fusion. This M-FISH finding confirmed a small insertion of PML from the long arm of chromosome 15 into the long arm of chromosome 17, juxtaposing the PML and RARA genes. There have been reports in the literature that have suggested different probe designs can affect the interpretation of atypical PML-RARA gene rearrangements. To follow-up on this, our lab tested the dual color, dual fusion t(15;17) probe sets from 4 different companies including DAKO, MetaSystems, Abbott Molecular, and CytoCell. Three of the four probe sets resulted in an abnormal, atypical FISH signal pattern, interestingly with different intensities of the fused red signal. However, one probe set revealed a normal FISH result. Such a finding confirms that although 92% of APL patients have exactly the same break point, some atypical APL cases may not be detectable based on The Journal of the Association of Genetic Technologists 43 (3) 2017

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Poster Abstracts, AGT 2017 probe set design. PML RARA (or any other probe set) designs are not standardized, and are viewed as proprietary by many companies. This lack of standardization results in probes of different sizes, colors, intensities and hybridization patterns, all of which have the potential to lead to a false negative result. Using this case as an example, we hope to raise awareness of a potential pitfall in PML RARA gene rearrangement studies utilizing FISH especially for those 8% of atypical APL cases.

20 COMPARING DUAL COLOR DELETION PROBE WITH TRI-COLOR MIXTURE OF ENUMERATION AND DELETION PROBE IN DEL(7Q)/-7 FISH TEST

Kenian Liu, Moffitt Cancer Center; Tania Quintana; Angie Root; Asha Grover; John Ross Background: Del(7q)/-7 is a common cytogenetic aberration in hematological malignancies, especially in myeloid neoplasms. There are different sets of commercial probes available for del(7q)/-7 FISH test. In this study, we compare test results using dual color probe set with tri-color probe set (Cytocell, Cambridge, UK) to provide information about advantage or disadvantage of each probe set for FISH test. Materials and Methods: 20 cases with positive FISH results detected using dual color deletion 7q probe were assayed with tri-color enumeration and deletion 7q/-7 probe mixture. Dual color deletion 7q probe set consists of two probes, which targets 7q22.1-22.2 and 7q31.2. The 7q22.1-22.2 probe, labeled in red, covers a 396kb region including the telomeric end of the RELN gene and extending beyond the marker D7S658. The 7q31.2 probe, labeled in green, covers a 203kb region including the TES gene. Tri-color deletion 7q/-7 probe set has a mixture of three probes, CEP7, CUX1, and EZH2. CEP7 probe labeled in aqua targets alpha satellite sequence of chromosome 7 centromere. The CUX1 (7q22.1) probe mix, labeled in green, consists of two probes (152kb and 273kb) covering the telomeric end of the CUX1 gene, including markers SHGC-58179 and D7S543E. CUX1 gene is 1.36M bp centromeric to RELN gene in 7q22.1-22.2 probe. The EZH2 (7q36.1) probe, labeled in red, covers a 305kb region, including the EZH2 and CUL1 genes and the D7S2419 marker. Schematic diagram of both probe sets are indicated in Figure 1. Results: Table 1: summarize test results using dual color and tri-color probe sets. Discussion and Conclusion: Monosomy 7 occurs in nearly 50% of cases with chromosome 7 abnormality, which cannot be detected in dual color probe set. It has been reported that patients who have MDS with isolated del(7q) had some distinct clinical-pathologic characteristics as well as better survival than patients with MDS with isolated monosomy 7. High incidence of co-deletion of two probe target regions, which have been described as common deletion region is observed in dual color probe test. Based on test results during recent 5 years, deletion involves only 7q21.1-q21.2 or 7q31.2 is rarely observed. Deletion in 7q21.1-q21.2 probe was observed in all 20 cases using dual color probe but no deletion of 7q21.1 was found in 4 in 20 cases using tri-color probe test. This indicates that there is a common breakpoint between two probe target regions.

21 A PATIENT WITH UNTREATED CHRONIC LYMPHOCYTIC LEUKEMIA/SMALL LYMPHOCYTIC

LYMPHOMA (CLL/SLL) WITH A SUBSEQUENT DIAGNOSIS OF CHRONIC MYELOGENOUS LEUKEMIA (CML)

Padma Doddapaneni, Cytogenetics Laboratory, Department of Pathology, NewYork Presbyterian Hospital/Weill Cornell Medicine; Xiaowei Sun; Govinda Hancock; Amy Chadburn; Susan Mathew A 79 year-old woman diagnosed at an outside institution with, but untreated for CLL/ SLL (CD19+, dim CD20+, CD5+, CD23+) in 2000, presented in the fall of 2012 with worsening anemia. A CBC in September 2012 showed a white blood cell (WBC) count of 113.7K/µL, hemoglobin of 8.9g/dL and platelet count of 190K/µL. Interphase fluorescence in-situ hybridization (FISH) on peripheral blood for CLL/ SLL-related (trisomy 12, deletions of 13q14.3 and ATM and TP53), and MDS-related (monosomy 5, monosomy 7, trisomy 8, and deletions of EGR1, 7q31 and 20q12) alterations were negative. However, the karyotype showed 46,XX,t(9;22)(q34;q11.2) [7]46,XX[18]. In December 2012, the patient was transferred to our institution. The peripheral blood WBC count was 119K/µL with 97% lymphocytes, 1% neutrophils and 2% basophils. Lactate dehydrogenase (LDH) was elevated at 288 IU/L. Flow cytometry on bone marrow aspirate showed a population of cytoplasmic lambda immunoglobulin light chain restricted B-cells (44% of total cells) positive for CD19, dim CD20, CD5 and CD23 and negative for CD10 and FMC-7 consistent with CLL/SLL. The bone marrow core biopsy showed that the lymphoid infiltrate occupied ~50% of the intertrabecular space with the remaining cellularity showing granulopoiesis, decreased erythropoiesis and many small, hypolobated megakaryocytes without an increase in blasts. FISH was performed on the bone marrow sample to evaluate for CLL/SLL and CML abnormalities. Two abnormal clones corresponding to the diagnosis of two neoplasms, CLL/SLL and CML were identified. Heterozygous (41%) and homozygous (44%) deletions of the 13q14.3 region were observed in this sample. FISH was negative for trisomy 12, and deletions of MYB, ATM and TP53 genes. BCR- ABL1 gene rearrangements were observed in 13% of 200 cells analyzed indicating the presence of a t(9;22) translocation. Thus, in conjunction with FISH, a diagnosis of CLL/SLL (with extensive multinodular and diffuse involvement) and chronic myelogenous leukemia, BCR-ABL1 posiThe Journal of the Association of Genetic Technologists 43 (3) 2017

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Poster Abstracts, AGT 2017 tive, was made. In January, 2013 after the diagnosis of CML, the patient started treatment with Gleevec (Imatinib). The patient responded well to Gleevec but in July 2013 the bone marrow showed increased involvement by CLL/ SLL. Dasatinib was introduced to target CLL/SLL as well as CML. In October, 2014 due to rising leukocytosis (WBC 358K/ µL), worsening anemia (hemoglobin 7.8g/ dL) and thrombocytopenia (platelets 89K/µL), dasatinib was discontinued. The patient was started with ibrutinib as FISH became negative for BCR-ABL1 in May 2014, with the persistence of the CLL/SLL clone based on the presence of the 13q14.3 deletion. Both dasatinib and ibrutinib were discontinued in 2015 due to weight loss and low WBC count. In July 2016, ibrutinib was started for rising lymphocytosis. In addition, the patient developed neutrophilia with circulating blasts. Flow cytometry on peripheral blood showed an increase in immature myeloid cells concerning for accelerated phase of CML. A bone marrow was performed which was 100% cellular composed of ~50% CLL/SLL cells and ~50% CML cells, but showed no evidence of large cell transformation of CLL/SLL or blast transformation of CML. Flow cytometry again identified the patient’s CLL/SLL population but also identified an abnormal myeloid blast population (3% of analyzed cells) which was CD34, CD117, CD7 dim, and CD33 positive. Conventional cytogenetic analysis showed the following karyotype: 46,XX,t(9;22)(q34;q11.2)[4]/47,idem,+8[8]. FISH showed BCR-ABL1 rearrangement in 46.5% of cells. As seen in previous studies, heterozygous (22%) and homozygous (52.5%) deletions of 13q14.3 region were observed. Currently, the patient is on dasatinib for her CML as well as for its activity in CLL/SLL. The patient is tolerating the therapy well with no significant complications. The coexistence of CLL/ SLL has been described with many solid tumors, but is not as frequently reported with other hematological disorders. Only a few reports in the literature have documented the coexistence of CLL/SLL with myeloproliferative neoplasms including CML. However, these patients had previously received CLL/SLL treatment and thus the emergence of CML was attributed to prior therapy. We describe a patient who was initially diagnosed with CLL/SLL developed CML without any prior treatment. Although the underlying etiology of CML in our patient cannot definitively be established, one of the contributing factors may be the immunodeficiency commonly seen in CLL/SLL patients.

22 DOUBLE MINUTES IN MYELOID NEOPLASMS: IDENTIFICATION OF MYB AND MYC AMPLIFICATIONS BY FLUORESCENCE IN-SITU HYBRIDIZATION

Shelia Rackers, MT, CG(ASCP), Senior Cytogenetic Technologist, Washington University in St. Louis School of Medicine; Katinka Vigh-Conrad, PhD; Yoshiko Mito, PhD Double minutes (dmin) are small, circular, paired chromatin bodies that lack a centromere and contain amplified copies of a particular DNA segment. Dmin is a cytogenetic hallmark of gene amplification in cancer, observed relatively frequently in solid tumors, but rarely seen in hematological neoplasms. Limited reports are available with identification of amplified genes and the prognostic value of dmin in hematological neoplasms remains uncertain. In the current study, we present the cytogenetic and clinicopathologic features of two patients with myeloid neoplasms exhibiting dmin. The first patient is a 69-year old male diagnosed with myelodysplastic syndrome, refractory anemia excess blasts-2. Based on his cytogenetic history of trisomy 6, fluorescence in-situ hybridization (FISH) analysis for MYB gene locus at 6q21 was performed on a bone marrow specimen, which incidentally identified MYB gene amplification in 48% of the cells analyzed. Subsequent chromosome analysis revealed the presence of three to twenty double minutes per metaphase in 80% of the cells with or without trisomy 6 (46~47,XY,+6,3~20dmin[cp17]/46,XY[3]), suggesting that double minutes are the source of MYB gene amplification. Literature review revealed that MYB gene amplification is a common finding in BRCA1-associated breast tumors, but extremely rare in myeloid neoplasms. The second patient is a 71-year old male diagnosed with acute myeloid leukemia that evolved from a previously established myelodysplastic syndrome. Chromosome analysis of bone marrow specimen revealed a complex karyotype containing monosomy 5 and 7 and multiple structural aberrations in all twenty metaphase cells analyzed. In addition, two of twenty metaphase cells showed more than twenty double minutes in each cell. Subsequent FISH analysis revealed a MYC gene amplification, which is one of the most commonly amplified genes in myeloid neoplasms. The current study highlights the crucial role of fluorescence in-situ hybridization in identification of amplified genes in dmin. We have identified MYB gene amplification, which is an extremely rare finding in myeloid neoplasms. Characterization of double minutes in more cases in hematological neoplasms will lead to better understanding of its clinical significance and prognostic value.

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Poster Abstracts, AGT 2017 23 UTILITY OF SEQUENTIAL METAPHASE FISH IN DETECTING ATYPICAL RUNX1-RUNX1T1

REARRANGEMENT IN A AML PATIENT WITH A THREE- WAY T(8;14;21) AND AN ADVERSE OUTCOME

Bonita Brooks, CG(ASCP)CM, Senior Cytogenetic Technologist, Washington University in St. Louis School of Medicine; Ina E. Amarillo, PhD Acute myeloid leukemia with t(8;21)(q22;q22) has a frequency of ~5% of all AML cases and confers good prognosis. This translocation results in 5’-RUNX1-RUNX1T1-3’ chimeric fusion on pathogenic derivative chromosome 8 (and 5’-RUNX1T1-RUNX1-3’ on der 21). In rare cases, a complex rearrangement results in a single fusion FISH signal pattern. To date, the prognostic implication of this atypical rearrangement remains unclear. Here we report a 59-year-old Caucasian woman presented in our BMT clinic with a three-week history of shortness of breath and fatigue, anemia and thrombocytopenia, and initial blast count of 12%. Bone marrow (BM) samples were sent for surgical pathology and cytogenetics studies with a clinical indication of new leukemia. BM pathology revealed a markedly increased blast count of 42% with rare Auer rods and cytoplasmic granules, and this leukemia was given a diagnosis of AML with myelodysplasia-related changes (WHO) or AML with maturation (FAB M2). Initial karyotype analysis revealed a three-way translocation involving chromosomes 8, 14 and 21. Reflex FISH studies revealed an atypical rearrangement in 91.5% of interphase cells examined; a single fusion, two RUNX1 (green) and two RUNX1T1 (red) signals. Sequential metaphase FISH studies mapped the single fusion on der(8q), one green signal on der(21q) (no fusion), and one red signal on der(14q).The karyotype was revised to 46,XX,t(8;14;21)(q22;q24;q22) [20].ish t(8;14;21)(RUNX1T1 con RUNX1; RUNX1T1+; RUNX1+)[5]. She underwent a series of chemotherapy and bone marrow transplant, and clinical and cytogenetic remission-relapse cycle with an evolution of a complex karyotype within 20 months before expiring. This patient report highlights the importance of identifying the location of a pathogenic RUNX1-RUNX1T1 fusion by sequential metaphase FISH studies in monitoring AML cases with complex karyotype and atypical t(8;21).

24 ROUTINE UTILIZATION OF A SNP MICROARRAY FOR CHRONIC LYMPHOCYTIC LEUKEMIA: EFFICACY AND INFORMATIVE FINDINGS

Zhinous Hosseini, Cytogenetic Technologist, LabCorp; Savanna Schepis MB(ASCP)CM; Peter Papenhausen, PhD; Stuart Schwartz, PhD Chronic lymphocytic leukemia is the most common form of adult-onset leukemia in the Western world. It is typically an indolent disease, but can evolve into a more dangerous and lethal disease. The standard method of analysis is to utilize fluorescence in-situ hybridization (FISH); however, microarray analysis has the potential to identify significant genetic alterations that would remain uncharacterized not targeted by the DNA probes utilized in routine FISH analysis. It will also better define existing abnormalities, substantially increasing the abnormal clonal detection rate. A total of 612 patients who were confirmed to have CLL were studied by microarray analysis. 21.1% of the 612 patients were normal according to FISH analysis; however, the array studies demonstrated that 55.4% of this FISH negative group were abnormal. Overall, array analysis detected abnormalities in over 90% of the patients. The array also detected additional abnormalities in 65% patients where there was a FISH abnormality. By FISH analysis, genome complexity (³ 3 abnormalities) was detected in only ~9% of the patients. By utilizing array analysis genome complexity could be detected in ~46% of the patients suggesting a more progressive disease. Copy-neutral loss of heterozygosity was detected in 17.9% of the patients. The most common regions involved in CN-LOH include 1p, 11q, 13q, 17p, and 20q, several of which are associated with specific gene mutations. In conclusion, the array is clearly more efficacious in detecting genome aberrations compared to FISH analysis. Although the array may be slightly less sensitive than FISH, it could pick up abnormalities present in at least 10% of the cells. The array delineated genome complexity in ~46% of the cases while FISH was not as effective in detecting this complexity. Specifically, the array analysis also subdivided 13q deletions into two groups; one with the more expected good prognosis (only a 13q deletion present) and the other with complexity and a higher chance of being an aggressive disease. Because the array analyzes the entire genome it has successfully detected disorders such as myelodysplasia (in this cohort) that would not otherwise be seen by CLL FISH analysis. It has also detected anomalies such as chromothripsis and CN-LOH that could not be delineated by FISH, but are likely associated with a more aggressive disease.

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Poster Abstracts, AGT 2017 25 PRENATAL DIAGNOSIS AND MOLECULAR CYTOGENETIC CHARACTERIZATION OF RING

CHROMOSOME 13 BY CHROMOSOMAL MICROARRAY AND CHROMOSOME ANALYSES IN A FETUS WITH MULTIPLE CONGENITAL ANOMALIES

Kate E. Stehl, CG(ASCP), Cytogenetics Technologist, Cytogenetics and Molecular Pathology, Department of Pathology and Immunology, Washington University School of Medicine; Michael J. Evenson, CG(ASCP); Katinka Vigh-Conrad, PhD; Yoshiko Mito, PhD Chromosomal microarray analysis (CMA) is a robust diagnostic tool. CMA can identify major chromosomal aberrations as well as submicroscopic copy number variations that are not detectable by chromosome (karyotype) analysis. CMA is currently recommended as a first-tier clinical diagnostic test for individuals with developmental disabilities or congenital anomalies, as well as in pregnancies showing one or more major fetal abnormalities by an ultrasound survey. Here we describe cytogenetic and molecular cytogenetic characterization of a fetus with multiple structural anomalies. Ultrasound survey at 20-weeks gestational age revealed intrauterine growth restriction, holoprosencephaly, fetal hydrops, and ventricular septal defect. Microarray analysis on the amniotic fluid using Affymetrix CytoScan HD array revealed a 20 Mb terminal loss on chromosome 13 with breakpoint at band q32.1 as well as a 76 Mb mosaic loss on chromosome 13 from band q12.11 to band q32.11, suggestive of mosaic monosomy 13. Fluorescence in-situ hybridization (FISH) studies confirmed both the terminal deletion and mosaic monosomy 13. To elucidate the structural basis of the 13q aberration, chromosome analysis was performed, and a ring chromosome 13 was identified in metaphase cells from a short-term culture (46,XX,r(13)(p13q32)). Additional cytogenetic analysis of a long-term culture revealed a near-tetraploid karyotype with the ring chromosome 13 and loss of one chromosome 13 (91,XXXX,- 13,r(13)). Tetraploidy is a well-described artifact of long-term culture of amniocytes, and since loss of chromosome 13 was not observed in a short-term culture, it was considered as a long-term culture artifact. Consistent with this, tetraploid cells or loss of chromosome 13 was not observed in 200 nuclei analyzed by FISH studies on directly prepared cells without cell-culture. Prenatal finding of ring chromosome 13 is extremely rare. Review of limited literature of ring chromosomes 13 resulting in 13q terminal deletions in prenatal settings showed a striking overlap of ultrasound findings with the present case, with findings of intrauterine growth restriction, neural tube defects, brain malformations, and facial dysmorphisms. The present case highlights that a combination of cytogenetic and molecular cytogenetic approaches is essential for successful characterization of genomic structural abnormalities. CMA enables the high- resolution analysis of chromosomal aberrations, and therefore aids in identifying novel associations between a genomic alteration and clinical phenotype. Key words: Prenatal diagnosis, chromosomal microarray analysis, cytogenetic, ring chromosome, karyotype

PRENATAL CHROMOSOMAL MICROARRAY - VALUABLE AID TO KARYOTYPING

Leena Gole, PhD, FACMG, ABMG, MHGSA, Dip.Gen.Couns., Dr, National University Hospital, Singapore; Constance Chua, MSc, CG(ASCP), MHGSA, NUH; Sok Peng, Chua, MSc, NUH; Shirley Chan, MSc, NUH; Lin Lin Su, MBBS, MRCOG, MMED, NUH; Arjit Biswas, MBBS, MD, MRCOG, FRCOG, FAMS, NUH Chromosomal microarray analysis is the primary diagnostic tool for children with developmental delay and/or structural malformation. Despite ACOG recommendations, in prenatal diagnosis, this technology has not been readily accepted for fear of obtaining extraneous information. However, our experience with prenatal samples during the validation process yielded surprising results. Only patients with high risk ultrasound findings were included in this study. DNA from cultured amniotic fluid was run on the Affymetrix CytoScan 750K SNP chip, with concurrent karyotyping done. Analysis was done using the Affymetrix ChAS software with the following cutoffs – DNA copy number loss of ³ 1 Mb; gain of ³ 2 Mb; UPD of ³ 15 Mb interstitially or ³ 10 Mb telomerically. Here we report 6 cases where microarray provided helpful information to the patients. Case 1. Abnormal ultrasound findings, A=18. Karyotype of 46,XY,del(5) (p13p15.1).ish 5p15.2(D5S23,D5S721) x2. Microarray showed the deletion of NIPBL gene, with no involvement of Cri-du Chat region, which can result Cornelia De Lange syndrome 1. Case 2. Fetus with cardiac echogenic foci and echogenic bowels, A=22+6. Karyotype of 47,XY,+mar. ish 15q11.2q13(D15S11-,D15Z1-). Microarray showed no gain or loss, indicating that the marker contained no significant genomic content. Case 3. Missed abortion, gestation not provided. Karyotype of 46,XX,add(6)(p24). Microarray refined the abnormality as dup(3p) and del(6p), leading to the investigation of parents as possible translocation carriers. Case 4. Suspected Dandy-Walker malformation, A=23+1. Karyotype of 46,XX. Microarray showed a deletion at 4p, which can result in Weyers acrofacial dysostosis or Ellis-van Crevald syndrome. Case 5. Fetus showed symmetrical IUGR, A=21+3. Karyotype of 46,XX,del(13)(q12q14). Microarray showed that RB1 gene was not involved, but that the deletion results in developmental delay and other significant developmental or morphological phenotypes. BRCA2 gene was also deleted. Case 6. Twins. Lower fetus with IUGR, CVS and VSD, A=19+3. Karyotype of 46,XX,der(21)ins(21;?)(q11.2;?). Microarray showed no gain or loss of genomic content. In conclusion, microarray testing has proved to be very useful to parents in a prenatal setting as in these cases, the added information was helpful in the decision making process. With proper genetic counselling, and with precise guidelines in place regarding incidental findings, microarray testing for prenatal samples is beneficial.

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Poster Abstracts, AGT 2017 27 ISOLATION AND LOW PASS GENOME WIDE SEQUENCING FOR CLINICALLY RELEVANT COPY NUMBER ANALYSIS IN CIRCULATING TUMOR CELLS

Cynthe Sims, PhD, HCLD(ABB), Supervisor, PacificDx; Aditi Khurana, CG, MB(ASCP), PacificDx; Samuel Koo, MS, PacificDx; Marc Ting, BS, PacificDx; Judy Webb, PhD, ResearchDx; Kyle Horvath, PhD, ResearchDx; Suman Verma, PhD, ResearchDx; Valeria Sero, Menarini Silicon BioSystems; Alberto Ferrarini, Menarini Silicon BioSystems; Genny Buson, PhD, Menarini Silicon BioSystems; Farideh Bischoff, PhD, Menarini Silicon BioSystems Recent advances allowed for the robust and routine detection of Circulating Tumor Cells from liquid biopsies in the clinic. The presence of circulating tumor cells (CTCs) has prognostic value in multiple malignancies. Current CTC enrichment methods rely on epithelial and leukocyte markers. Here we describe an alternative methodology incorporating both cell surface markers and morphology to evaluate individual candidate CTCs, atypical cells lacking the classic CTC profile and CTCs with additional markers followed by low pass genomic analysis. This approach provides information regarding somatic and germ-line Clinically Actionable Copy Number Alterations (CACNAs) that can assist physicians in selecting appropriate targeted therapies (e.g. HER2 amplification and Herceptin administration) and aid in prognostic determination (e.g. p53 loss and MDM2 gain in prostate). We demonstrate the validity of single cell whole genome amplification and low-pass whole genome sequencing approach on CTCs, atypical cells and leukocytes sorted from 20 Veridex cartridges using the Menarini SilconBiosystems DEPArray platform. Additional samples for low pass copy number analysis included single cells from well characterized cell lines with known copy number alterations. This approach, single cell isolation followed by low pass copy number determination takes current CTC enumeration through a logical analysis step. This technique may be used in the clinic to inform treatment decisions and provide valuable prognostic information about a patientâ&#x20AC;&#x2122;s disease.

28 DETECTION OF MOSAIC PATHOGENIC ALTERATIONS CAUSING A MECP2- RELATED DISORDER IN TWO UNRELATED MALE PATIENTS

Jessica A. Cooley, BS, MB(ASCP)CM, Lab Manager, Molecular Diagnostic Laboratory, Greenwood Genetic Center; Catherine J. Spellicy, PhD; Renee Bend, PhD; Raymond J. Louie, PhD; Jennifer L. Stallworth, MS; Jessica Worthington, BS; David B. Everman, MD; Steven A. Skinner, MD; Michael J. Friez, PhD, FACMG; Jennifer A. Lee, PhD, FACMG Rett syndrome (MIM:312750) is a neurodevelopmental disorder caused by pathogenic alterations in the MECP2 gene. It is characterized by normal development until 6 to 18 months of age followed by brief developmental stagnation, rapid regression and deterioration of higher brain function, followed by stabilization with persistent neurodevelopmental and physical disabilities. The regression is a critical component of the clinical diagnosis and especially involves loss of hand use (which is replaced with repetitive hand movements), loss of language and motor skills, and abnormal gait. Additional findings may include bruxism, apnea, sleep impairment, fits of screaming, scoliosis, growth retardation, and abnormal muscle tone. Rett syndrome is an X-linked dominant disorder that usually only affects females, with an incidence of 1 in 10,000-15,000 female births. In the hemizygous state, the same pathogenic alterations in MECP2 that cause Rett syndrome in females were initially thought to be lethal in males. In recent years, alterations in the MECP2 gene have been identified in males who have the more severe phenotype of MECP2-related severe neonatal encephalopathy (MIM:300673). These male patients do not typically survive past the age of two years without aggressive medical support. Interestingly, however, there have also been male cases reported due to X-chromosome aneuploidy or mosaicism involving a pathogenic alteration in MECP2. Here, we report two mosaic pathogenic alterations in MECP2 in two unrelated male patients. Although these patients have many of the overlapping phenotypic features of Rett syndrome, they have been diagnosed as having a MECP2-related disorder based on the 2010 revised clinical consensus criteria for Rett syndrome (Neul J.L. et al.). Typically, sequence analysis of the MECP2 gene identifies >95% of the causative alterations in patients with classic Rett syndrome, versus around 65% in patients with non-classic Rett syndrome. Both of the mosaic cases presented here were detected by sequencing, the first by Next-Generation Sequencing (NGS), (specifically, whole exome sequencing, or WES) and the second by Sanger sequencing. WES has been effective in identifying the causative variant(s) in patients who have complex phenotypes that may not correspond to a specific disorder, or in patients who have had targeted genetic testing in the past and still lack a clear diagnosis. With the advancement of NGS technology and its decreasing price, WES is being performed more routinely in the clinical laboratory. The deep coverage often afforded by NGS allows for the detection of low level mosaic alterations that are sometimes difficult to detect by Sanger sequencing, which has been the long considered gold-standard sequencing method. Patient 1 was run on the Illumina NextSeq500 Sequencing System. A c.730C>T (p.Gln244*) nonsense variant was found with 181X read depth, but only 19.3% minor allele frequency, and was confirmed as a mosaic variant by Sanger sequencing. Patient 2 was analyzed by Sanger re-sequencing of exon 4 of MECP2 after another laboratory reported a hemizygous c.397C>T (p.R133C) variant detected via Sanger sequencing. However, our data indicated that this variant was likely a mosaic alteration, given the presence of both the wild type and variant alleles at the c.397 nucleotide position. RNA studies were subsequently performed for Patient 2,

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Poster Abstracts, AGT 2017 which confirmed the mosaicism at the transcript level. Both male patients had a normal 46,XY karyotype, and therefore X-chromosome aneuploidy was ruled out. Although mosaicism can be difficult to detect, both Sanger sequencing and NGS have proven to be effective methods for detecting mosaic alterations upon careful analysis of the data. For the two patients presented here, their phenotypes were consistent with a MECP2-related disorder, and identification of these mosaic alterations has provided a molecular diagnosis.

29 NAVIGATING THE JOURNEY OF THE VARIANT OF UNCERTAIN CLINICAL SIGNIFICANCE: IDENTIFICATION AND SEGREGATION OF A RARE MYLK VARIANT WITH FAMILIAL AORTOPATHY

Jacquelynn Evans, BS, MB,CG(ASCP)CM, Molecular Genetic Technologist III, UNMC, Munroe-Meyer Institute, Human Genetics Laboratory; Jennifer Sanmann, PhD, FACMG; Anji Yetman, MD, Division of Pediatric Cardiology, Children’s Hospital and Medical Center; Bhavana Dave, PhD, FACMG; Marilu Nelson, MS, MB,CG(ASCP)CM; Janet Williamson, BS, MLT, MB,CG(ASCP)CM; Erin Kaspar, BS, BA, MB(ASCP)CM; Emily Rief, BS, MB(ASCP)CM; Hope Chipman, MS, CGC; Lois Starr, MD, FAAP, FACMG As next-generation sequencing (NGS) gene panels continue to expand and eventually give way to whole exome sequencing, a growing number of cases are yielding complex results involving numerous variants of uncertain clinical significance (UCS) in genes associated with disorders that have closely overlapping features. Following through with segregation studies is difficult in a clinical setting for many reasons and may, or may not, ultimately impact the variant classification, depending on the other available supporting pieces of evidence for classification. In the setting of familial thoracic aortic aneurysmal dissection (FTAAD) disorders, understanding the etiology can make a significant difference in the patient’s medical and surgical care. To illustrate this algorithm and its challenges, we present a family with a history of thoracic aortic dissection. A two year old female was evaluated for developmental delay, joint laxity, mild hypotonia, and family history of thoracic aortic dissection. Echocardiogram revealed mild dilation of the ascending aorta. Her mother was also discovered to have mild dilation of the aorta, as was the maternal grandfather. The maternal great uncle suffered a dissection of the ascending aorta and, thus, raised concern for an autosomal dominant aortopathy. NGS was performed for the proband utilizing an NGS connective tissue disorder panel including 50 genes associated with Marfan and Marfan-like syndromes, Ehlers-Danlos syndrome (vascular and non-vascular types), Stickler syndrome, Loeys-Dietz syndrome, cutis laxa, and other disorders which can contribute to disorders with FTAADs. Three heterozygous variants, initially classified as having uncertain clinical significance in accordance with established classification criteria, were identified; these variants were found in COL3A1, MYLK, and NOTCH1. In order to better define the potential clinical significance of these results, targeted Sanger sequencing was performed for the healthy father, the affected mother, and the maternal great uncle who survived a dissection of the ascending aorta. The MYLK variant, c. 3824G>A (p.Arg1275Gln), seemed clinically to be the best fit with the family’s presentation and segregated three degrees of genetic separation to the maternal great uncle. The information currently available regarding this MYLK variant is extremely limited, which makes formal reclassification of the variant difficult from the laboratory perspective, and the uncertain but concerning impact that this variant may have on phenotype makes medical management challenging for the family as well.

30 TARGETED FAMILIAL FOLLOW-UP STUDIES FOR NEXT-GENERATION SEQUENCING VARIANTS OF UNCERTAIN CLINICAL SIGNIFICANCE RESULT IN LIMITED VARIANT RECLASSIFICATION

Karissa M. Scott, BS, MB(ASCP)CM, Molecular Genetic Technologist II, Human Genetics Laboratory, Munroe-Meyer Institute for Genetics and Rehabilitation, University of Nebraska Medical Center; Marilu Nelson, MS, MB(ASCP) CM CGCM; Janet E. Williamson, BS, MLT(ASCP), MB(ASCP)CM CGCM; Evan M. Roberts, BS, MB(ASCP)CM; Jacquelynn J. Evans, BS, MB(ASCP)CM CGCM; Erin E. Kaspar, BS, MB(ASCP)CM; Emily J. Rief, BS, MB(ASCP)CM; Julie M. Carstens, MS, MB(ASCP)CM CGCM; Bhavana J. Dave, PhD, FACMG; Jennifer N. Sanmann, PhD, FACMG Sequencing variants, when classified as having uncertain clinical significance (UCS), put the onus on the physician to interpret how the genetic results may, or may not, alter medical management and treatment. Targeted familial follow-up studies may aid in further clarification of variant classification based on whether the variants occur de novo or whether the variants segregate with disease and, therefore, may assist the physician with management decisions. However, segregation of the variant in a small number of family members is often insufficient evidence for formal variant reclassification. Our laboratory offers and bears the cost of targeted follow-up studies in relatives of probands with UCS variants. To evaluate the cost for the laboratory versus the benefit in patient care, we initiated a retrospective review of the results over a 13-month period. Indiscriminate follow-up testing of 81 UCS variants resulted in the altered classification of five variants, all of which were downgraded from UCS to UCS- likely benign due to the detection of the variant in unaffected relatives in conjunction with other classification evidence. Familial

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Poster Abstracts, AGT 2017 testing did not provide adequate evidence for reclassification of any variants from UCS to UCS-likely pathogenic or to pathogenic. Parental follow-up studies were performed in the majority (80%) of cases; however, some studies included distant relatives (4%) of the proband, such as aunts, uncles or cousins. To increase the likelihood of generating impactful results, it may be beneficial for laboratories to limit complementary follow-up testing of UCS variants to only genes that are implicated in autosomal dominant disease that align, at least modestly, with the patientâ&#x20AC;&#x2122;s phenotype. Additionally, differentiation between UCS variants classified as such due to a paucity of available information and those classified as such due to concerning but insufficient evidence for pathogenicity may also be a useful consideration prior to familial testing. A specific and focused approach to familial follow-up studies may increase the success rate of reclassifying UCS variants and may enhance the utility of information returned to the physician for management and treatment decisions.

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Student Poster Abstracts, AGT 2017

STUDENT ABSTRACTS S2 IMPROVING CERVICAL CANCER OUTCOMES IN RURAL HONDURAS WITH LOW COST HPV SCREENING

Courtney M. Studwell, Diagnostic Genetic Sciences, University of Connecticut, Storrs, CT; Aaron Atkinson, PhD, Laboratory for Clinical Genomics and Advanced Technology, Department of Pathology and Laboratory Medicine, Dartmouth-Hitchcock Medical Center, Geisel School of Medicine; Linda Kennedy, MEd, Norris Cotton Cancer Center, Dartmouth-Hitchcock Medical Center, Geisel School of Medicine at Dartmouth; Suyapa Bejarano, MD, La Liga Contra el Cancer, HondurasJorge, Arturo Plata Espinal, MD, La Liga Contra el Cancer, Honduras; Ethan P.M., LaRochelle, Thayer School of Engineering, Dartmouth College; Kathleen Doyle Lyons, ScD, Norris Cotton Cancer Center, Dartmouth-Hitchcock Medical Center, Geisel School of Medicine; Gregory J. Tsongalis, PhD, Laboratory for Clinical Genomics and Advanced Technology, Department of Pathology and Laboratory Medicine, Dartmouth-Hitchcock Medical Center, Geisel School of Medicine Introduction: Cervical cancer rates in low- and middle-income countries (LMICs) are 2 to 3 times higher than those in developed countries. In such areas of Honduras, cervical cancer is the most common cancer affecting women, accounting for approximately 417 deaths annually. As the majority of cervical cancers are caused by human papillomavirus (HPV) infection, implementing a low-cost, rapid, near patient HPV screen would greatly improve cervical cancer outcomes for the women of Honduras. We have adapted HPV tests from QuanDx® for both high and low-risk HPV to provide rapid results that can be used in collaboration with La Liga Contra el Cancer in Honduras to influence treatment in underserved communities. In addition, we sought to provide insight on the prevalence of HPV infection in the population by identifying the high-risk and low-risk genotypes present. Methods: We used real- time PCR followed by melt-curve analysis and the MeltPro High Risk and Low Risk HPV Genotyping assays (QuanDx/Zeesan Biotech, San Jose, CA) to detect HPV subtypes. Melt-curve analysis was automated and had enough resolution to detect 14 high-risk genotypes and 14 low-risk genotypes in two separate assays. DNA was previously extracted by a boiling alkaline lysis method from cervical swabs collected from 111 women in La Mosquitia, Honduras. Isolated DNA samples were added to the lyophilized PCR reagents, amplified on the SLAN96 real time PCR instrument (QuanDx/Zeesan Biotech, San Jose, CA), and analyzed for HPV genotypes. DNA samples that failed to yield a melt-curve were re-extracted with the Lab-Aid® 824 and re-analyzed. The results were reported to physicians in Honduras to provide follow up care for women at risk for cervical cancer from HPV infection. Results: Approximately 35% of the population examined had at least one HPV infection; 27 samples tested positive for high-risk HPV strains and 18 samples were positive for low-risk HPV strains. The most prevalent high-risk HPV genotypes were HPV-52 and HPV-16 representing 29% and 19% of high-risk infections, respectfully. The most prevalent low-risk HPV genotype was HPV-72 representing 18% of low-risk infections. Re-extraction of DNA samples with the Lab-Aid® 824 increased the ability to detect HPV resulting in the discovery of 9 additional high-risk HPV infections and 3 additional low-risk HPV infections reducing the “invalid” rate from 10-13% to 0% while maintaining workflows adapted to previous field experiments in Honduras. Conclusion: HPV infections are prevalent in the La Mosquitia region of Honduras and genotype distribution differs from that of developed countries and less isolated villages. In the future, the implementation of a cervical screening program utilizing molecular HPV testing would greatly improve the identification of at risk individuals and help reduce the cervical cancer rate in Honduras while remaining cost-effective. The MeltPro High Risk and Low Risk HPV Genotyping assays (QuanDx/Zeesan Biotech, San Jose, CA), in combination with the SLAN-96 real time PCR instrument (QuanDx/Zeesan Biotech, San Jose, CA), are rapid-assays that have the ability to identify individuals in the population who are at risk of cervical cancer at lower costs compared to other commercially available methods. In addition, the Lab-Aid® 824 is a useful and cost-effective addition to the screening workflow for samples requiring further purification.

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Student Poster Abstracts, AGT 2017 S3 ADVANCING NON-INVASIVE PRENATAL TESTING UTILIZING INTACT SINGLE FETAL CELLS FROM MATERNAL CIRCULATION: FROM WHOLE GENOME AMPLIFICATION OF SINGLE CELLS TO COPY NUMBER ANALYSIS USING NEXT-GENERATION SEQUENCING

Adriel Kim, BS, CG(ASCP), MB(ASCP), University of Texas MD Anderson Cancer Center; Liesbeth Vossaert, PhD, Baylor College of Medicine; Roseen Salman, BS, MB(ASCP), Baylor College of MedicineSadeem Qdaisat, BS, CG(ASCP), MB(ASCP), Baylor College of Medicine; Qun Wang, PhD, Baylor College of Medicine; Xinming Zhuo, PhD, Baylor College of Medicine; Chad Shaw, PhD, Baylor College of Medicine; David Henke, MPH, Baylor College of Medicine; Jennifer Chow, PhD, RareCyte Inc.; Lance U’Ren, PhD, RareCyte Inc. Background: Single cell genomics has rapidly evolved becoming a valuable tool for both basic and clinical research. Such progress can be attributed to advances in molecular technologies, including whole genome amplification (WGA) and next- generation sequencing (NGS). Several WGA methods have been developed to amplify human genomic DNA from single cells, providing DNA quantities suitable for various downstream molecular assays. Through collaborative efforts, our lab has demonstrated reliable recovery of Intact fetal cells from maternal circulation, suggesting a promising alternative for non-invasive prenatal testing (NIPT). To this end, our lab proposes that copy number analysis using NGS is feasible for cell-based NIPT. Research Objectives: Two specific aims were made: (1) assess the efficiency and reliability of commercially available WGA methods to produce DNA from single cells that is suitable for NGS copy number analysis, and (2) conduct a cohort study to evaluate the accuracy of single cell-based NIPT compared to invasive procedure prenatal testing. Materials and Methods: Ampli1™, GenomePlex®, and PicoPlex® commercial WGA methods were selected to amplify genomic DNA from single cells. The total DNA yields, reaction failure rates, quality control multiplex qPCR, and NGS data from selected cells were evaluated to assess the efficiency and reliability of each WGA method. Peripheral blood samples from pregnant women undergoing invasive procedures were collected for the cohort study. Fetal cells were recovered using a cocktail of antibodies to identify fetal cells among maternal cells and amplified by PicoPlex®. Each amplified fetal genomic DNA was genotyped and sequenced on Illumina HiSeq 2000 platform for copy number analysis. The NGS data were then compared to the results of conventional karyotype, fluorescence in-situ hybridization, and/or microarray obtained through invasive procedure. Results: The cells amplified by PicoPlex® method had 0% reaction failure rate, compared to 62.5% and 41.7% for Ampli1™ and GenomePlex®, respectively, with highest resulting mean yield. The multiplex qPCR using 8 primer sets also showed that PicoPlex® performed more efficiently than the other two methods, resulting in the median of 5 positive amplicons out of expected 8. Out of 49 clinical samples from which at least one fetal cell was recovered, the average recovery of fetal cells per sample was 3.88 with median of 3 cells. 84.1% of genotyped single fetal cells were confirmed to be fetal origin. The copy number data was consistent with diagnostic data in all cases, where high quality results were available, including several cases of aneuploidy and two microdeletion cases. Conclusion: Our lab has demonstrated that genomic DNA from single circulating fetal cells can be amplified, producing DNA that is suitable for downstream NGS for genotyping and copy number analysis. These data suggest a feasible alternative for invasive prenatal testing to detect aneuploidies and submicroscopic copy number variants and may eventually be extended to detect point mutations in the future.

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Student Poster Abstracts, AGT 2017 S4 COMPARISON OF THE EPICENTRE MASTERPURE DNA PURIFICATION KIT AND THE QIAAMP DNA MINIKIT FOR ISOLATION OF GENOMIC DNA FROM BLOOD

Emily A. Welebob, University of Texas MD Anderson Cancer Center; Brett Hannigan; Cheng Peng; Mehrdad Rajaei; Stephanie Zalles; Wenrui Ye, PhD; Mary Coolbaugh Murphy, PhD, MB(ASCP)CM; Peter C. Hu, PhD, FACSc; Awdhesh Kalia, PhD, MB(ASCP)CM, FACSc Background: Isolation of high quality genomic DNA (gDNA) is often the first step in molecular diagnostic procedures. PCR is a major component in molecular diagnostic testing, and its success is greatly dependent on the quality of gDNA preparation. The PCR product is critical for further downstream applications in techniques including, but not limited to, next-generation sequencing (NGS). With multiple kits available, it is critical to identify a reproducible, cost-effective method for isolation of high quality and quantity gDNA from blood. Methods: Here, we compare the Epicentre MasterpureTM DNA Purification Kit (catalogue no. MB711400) and the QIAampÒ DNA Mini Kit (catalogue no. 51104) for isolation of purified gDNA from blood. Six trainee technologists evaluated both kits. Each trainee extracted gDNA using 200 µL of fresh whole blood samples (n = 5) using both kits. Collectively, a total of 30 gDNA extractions were performed using each kit. Concentration and purity of extracted gDNA samples were determined with the NanoDropTM spectrophotometer (Thermo-Scientific), and integrity of extracted gDNA was observed using agarose gel electrophoresis. PCRs targeting human RN18S1, ACTB, HBB, GAPDH housekeeping genes were performed to assess suitability for downstream enzymatic applications. Statistical analysis of results was conducted using t-test and ANOVA models. Results: Average gDNA yield using the MasterpureTM kit was 8.629ug ±7.759. While the QIAampÒ kit had a lower average yield of 4.026±1.130, it demonstrated less variation between yields obtained by technologists. When compared using 95% confidence intervals, the yields of gDNA extracted by trainee technologists were not significantly different across the two kits. NanoDropTM generated A260/ A280 ratios ranged from 1.8-2.0 for all samples, indicating an acceptable level of purity. MasterpureTM produced a single high molecular weight gDNA band upon electrophoresis, while gDNA bands from QIAampÒ included smears indicating degradation. Both gDNA preparations were suitable for PCR amplifications suggesting adequate gDNA for downstream enzymatic applications. MasterpureTM allowed for use of different initial blood volume, whereas the QIAampÒ spin column was limited to an initial volume of 200 µL. MasterpureTM was less expensive at $442 for 400 mL of whole blood, but QIAampÒ had a faster turnaround time and has the possibility of being automated. Conclusion: We conclude that higher gDNA yields were obtained using the Epicentre MasterpureTM kit. However, gDNA from both kits produced comparable PCR results. Therefore the QIAampÒ DNA Blood Mini Kit proved to be a more reliable method for gDNA extraction with consistent yields and faster processing time.

S5 SPECIFIC GENETIC SIGNATURES ASSOCIATED WITH BREAST CANCER AMONG AFRICAN AMERICAN AND CAUCASIANS

Maha El Naofal, MS, University of Texas MD Anderson Cancer Center; Jose Thaiparambil, PhD, Houston Methodist Research Institute; Jun Gu, MD, PhD, University of Texas MD Anderson Cancer Center; Peter Hu, PhD University of Texas MD Anderson Cancer Center; Randa El-Zein, MD PhD, Houston Methodist Research Institute Introduction: Breast cancer is the second leading cause of cancer mortality among women in the U.S. Significant disparities exist in breast cancer among African American (AA) women compared to Caucasian (CA). AA women are often diagnosed with large, aggressive and high grade tumors, leading to a poorer prognosis and a higher mortality rate in comparison to CA women. Socioeconomic disparities with reduced access to health care has been proposed as a factor that contributes to the higher breast cancer mortality observed in AA. However, after the adjustment for the socioeconomic factor, the poor prognosis was found to be independent from the access to health care and socioeconomic status. This indicates that underlying biological factors may play a crucial role in breast cancer differences observed between ethnic groups. One of the key biological factors contributing to tumorigenesis is genetic instability and telomere dysfunction. Longer leukocyte telomeres were reported in AA at birth as compare to CA, however a high rate of telomere attrition was observed. Previous studies have examined an association between telomere length and the risk of cancers, but so far no studies have reported in association with telomere length among AA and Caucasian breast cancer patients. Here, we hypothesize that overall telomere length could play a crucial role in disease presentation and aggressiveness of breast cancer among AAs as compared to Caucasians. Methods: A total of 30 AA and 51 CA patients’ formalin fixed paraffin embedded (FFPE) breast cancer blocks were obtained from Methodist Hospital tissue bank. Genomic DNA was extracted using Ambion™ RecoverAll™ Total Nucleic Acid Isolation Kit. Telomere length was determined using Real-Time PCR detection system (Bio-Rad) and CFX Manager™ software to estimate absolute telomere length. Two standard curves and the overall telomere length

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Student Poster Abstracts, AGT 2017 were measured using the methods established by Oâ&#x20AC;&#x2122;Callaghan et al (2011). Results: Variation in absolute telomere length was observed within and among the different ethnic groups by age and different subtypes. Prospective Utilization/ Implication of Results: Telomere lengths assessment may be potentially useful in prediction of the patients with poorer outcomes. In addition, a better understanding of the underlying biology associated with aggressive disease, may lead to identification of therapeutic targets that are ethnically appropriate.

CHROMOSOME 8p12-21 ALTERATION IN NON-SMALL CELL LUNG CANCER (NSCLC)

Mohamed Hady M. Baity, University of Texas MD Anderson Cancer Center; Xiaoshan Zhang; Li Wang; Ming Zhao; Jun Gu; Peter Hu; Bingliang Fang BACKGROUND The mortality rate of non-small cell lung cancer (NSCLC) is high because of its late diagnosis and resistant to many anticancer treatments. Molecularly, lung cancers are highly heterogenous. Different genetic alterations underlying lung cancer may affect treatment outcomes, in part due to their influence on the metabolic or signal transduction pathways of the affected cells. Chromosome 8p rearrangement is an example of a genetic aberration, which is detected in many types of cancer such as breast cancer, colon cancer, and lung cancer. Within this region, there are many genes associated with cancer cell metabolism. One of these genes is glutathione disulfide reductase (GSR), which contributes importantly to redux hemostasis and cellular defense against oxidative stress. Radiotherapy and chemotherapy with cisplatin and paclitaxel are anticancer treatments that induce oxidative stress. We hypothesized that the GSR gene can be altered by either deletion or amplification in tumor cells. The GSR gene alteration may affect anticancer treatment response. The aim of this study is to develop a fluorescence in-situ hybridization (FISH)-based method to rapidly detect deletion or amplification of GSR. METHODS Samples from NSLC patients (n = 70) were collected using the touch print technique. We designed a home-brewed probe (here named DG_mbaity_1), labeled with spectrum orange, and targeting GSR (chromosomal location: 8p12p21) bacterial artificial chromosome clone (RP11- 411F21). A CEP8 probe labeled with spectrum green was used as the control probe (Abbott Molecular) to determine the specificity of DG_mbaity_1. The ratio of GSR to CEP8 and GSR to PERK signals were calculated from FISH and qPCR, respectively data. RESULTS Of the 70 NSLC touch print samples 20 (29%) showed trisomy or aneuploidy with GSR deletion by FISH. GSR-to-CEP8 signal ratios indicated a strong correlation (r2 = 0.9464; and (p=0.05) to the molecular GSR/CEP8 ratio from the same cases. CONCLUSION Our results suggest that the GSR FISH with PCR test might be a rapid screening test for NSCLC patients.

S7 LOSS OF CHROMOSOME 1p36 AND GAIN OF CHROMOSOME 12p ARE THE FREQUENT GENETICS CHANGES IN PEDIATRIC GERM CELL TUMORS

Hui Yi Yon, MSc, University of Texas MD Anderson Cancer Center; Vishnupriya Borra, Baylor College of Medicine; Yi-Jue Zhao, Baylor College of Medicine; Moez Dawood, Baylor College of Medicine; Jun Gu, University of Texas MD Anderson Cancer Center; John Hicks, Baylor College of Medicine; Murali Chintagumpala, Baylor College of Medicine; Jodi Muscal, Baylor College of Medicine; Dolores Lopez- Terrada, Baylor College of Medicine; Pulivarthi Rao, Baylor College of Medicine Pediatric germ cell tumors (GCTs) are relatively uncommon, accounting for approximately 3% of all childhood malignancies. These tumors occur in both gonadal and extragonadal sites and are more frequent in girls than boys. Pediatric GCTs differ from those in adults in histology, primary sites, cytogenetics, and age distribution. Gain of chromosome 12p, most of the times in the form of an isochromosome 12p, is typical and occurs in 80% of adult malignant GCTs. However, gains of 12p are seen less frequently in pediatric malignant GCTs especially in very young children. Loss of 1p36 has been reported in malignant ovarian GCTs and yolk sac tumors. To understand the genetic basis of pediatric GCTs, we performed G-banding in combination with multicolor spectral karyotyping (SKY), fluorescence insitu hybridization (FISH) on 65 GCTs with various histological sites and array CGH on 19 cases. Combined SKY and G-banding analysis identified several recurrent breakpoint clusters -1p36, 1p13, 1q10, 1q21, 2q13, 8q24, 9p13, 12p13, 12q10, 14q32, 16q24 and 12 non-recurrent reciprocal translocations. Of these translocations, chromosome 8q24 band was involved with three different partner chromosomes. Frequent chromosomal aberrations involving chromosomes 1p36 and an isochromosome 12p [i(12)(p10)] were found in 32 cases (49%) and near triploidy/tetraploidy, gain of isochromosome 12p and frequent loss of chromosomes 4, 5, 10, 11, 13, and 18 were observed in 19 malignant mixed GCTs. We are evaluating the clinical significance of these copy number aberrations (CNAs). Molecular characterization of chromosomal regions of 1p36 and 12p utilizing the currently available genomic technologies will provide new insights into the biology and clinical behavior of pediatric GCTs.

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Student Poster Abstracts, AGT 2017 S8

CLINICAL VALIDATION OF A DUAL-COLOR FISH PROBE ASSAY FOR IRF4/ DUSP22

Pei Zhao, University of Texas, MD Anderson Cancer Center; Helen Mata; Ashley Haden; Ruizhi Duan; Jun Gu; Hatice Deniz Gur; Pei Lin; Ming Zhao; Peter Hu In healthy individuals the interferon regulatory factor 4 (IRF4) and the dual specificity protein phosphatase 22 (DUSP22) genes are juxtaposed to each other located on Chromosome 6p25 region. Rearrangements of these genes have been implicated in Anaplastic lymphoma kinase (ALK)-negative anaplastic large cell lymphoma(ALCL). Cytomolecular techniques such as FISH can be used to evaluate these rearrangements and therefore, may become a significant prognostic index for ALK-negative ALCL patients. In this study, we used an IRF4/DUSP22 (6p25) dual-color break probe, KI-10613 developed by Leica Biosystems, to look at random T-cell lymphoma tissue samples. We have analyzed 8 different patient tissue samples of which 3 were positive for the rearrangement. This is consistent to what has been reported in the literature of ~30%. Additional samples are currently being tested.

AUTOMATED METAPHASE FINDING AND FISH SPOT COUNTING VALIDATION

Stephanie Soewito, University of Texas MD Anderson Cancer Center; Quyen Tran; Justin Kee; Thong Vu; Qin Xu; Ayman Bakhit; Ming Zhao, MD, MS, CG(ASCP)CM; Jun Gu, MD, PhD, CG(ASCP)CM Efficient productivity and turnaround times are important metrics for clinical laboratories as a faster diagnosis can lead to faster treatment. Conventional cytogenetic techniques involve G-banded karyotyping and fluorescent in-situ hybridization (FISH). Diagnosis confirmation relies on analyzing a large sample of cells while trained technologists manually evaluate chromosomal abnormalities. However, with the increased demand of cytogenetic testing, manual analysis becomes error-prone and highly time consuming. Manual evaluation also requires at least two trained technologists which can vary in results due to subjective FISH signal observation in close proximity. This may hinder findings for a true positive patient diagnosis and prognosis interpretation. Technological advancements for a fully-automated metaphase finder and FISH spot counting utilize a system that improves the workflow of these techniques for robust, large scale and efficient analysis. The goal of this study was to assess the efficiency and accuracy of automated metaphase finding and FISH counting over manual operation using the GSL-10 scanner and CytoVision software. This study will put forth a standardized method that can be utilized by other cytogenetic laboratories to generate consistent results and validate automation of metaphase scanning and FISH spot counting. We screened normal cases of G-banded samples and CEP X/Y FISH stained samples manually and automatically. The Cytovision software is equipped with an automated sample acquisition component that generates digital images that facilitates quality review, reclassification, and documentation. Samples were also timed in a series of phases and scored manually and automatically using CytoVision. Our results indicated a significant decrease in time to automatically locate and capture the same number of metaphase cells compared to manual operation. FISH signal capture and counting time for 200 interphase cells was considerably faster for automated processing versus manual processing, despite the need for additional human interaction to filter through the images. Validation of automated reading is a major technical breakthrough that allows us to consider the routine use of this technique. Automated scanning and analysis can potentially improve the quality control and productivity of the clinical cytogenetic laboratories.

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Association Business

AGT, The Organization for Cytogenetic & Molecular Professionals AGT, originally founded in 1975 as the Association of Cytogenetic Technologists, serves to: • promote the scientific and professional development of all areas of genetics; • foster the exchange of information between those interested in genetics; • encourage cooperation between those persons actively or formerly engaged in genetics; and • stimulate interest in genetics as a career. AGT has approximately 1,000 members. Membership is open to all who are employed or interested in genetics. All regular members are entitled to hold office, vote in elections, attend all AGT meetings, and receive The Journal of the Association of Genetic Technologists and access the AGT International Online Membership Directory.

Board of Directors Officers President Jason A. Yuhas, BS, CG(ASCP)CM Mayo Clinic Division of Laboratory Genetics Cytogenetics Lab 200 First St. SW Rochester, MN 55905 507-538-7634 yuhas.jason@mayo.edu Secretary-Treasurer Denise Juroske Short, MSFS, MB(ASCP)CM 219 Timberland Trail Lane. Lake City, TN 37769 dmj4565@gmail.com Public Relations Director Ephrem Chin MBA, MB(ASCP)CM, QLC 1907 Woodlawn Terrace Ct. Sugar Land TX 77479 404-579-9995 nzelfman@gmail.com Education Director Sally J. Kochmar, MS, CG(ASCP)CM Magee-Womens Hospital Pittsburgh Cytogenetics Lab 300 Halket St., Room 1233 Pittsburgh, PA 15213 412-641-4882 skochmar@upmc.edu Annual Meeting Director Jennifer N. Sanmann, PhD, FACMG UNMC Human Genetics Laboratory 985440 NE Med. Center Omaha, NE 68198-5440 402-559-3145 jsanmann@unmc.edu

Annual Meeting Co-Director Christina Mendiola, BS, CG(ASCP)CM University of Texas Health Science Center – San Antonio 7703 Floyd Curl Dr. San Antonio, TX 78229 210-567-4050 mendiolac@uthscsa.edu

Representative to NAACLS Term: 9/12 – 9/20 Peter C. Hu, PhD, MS, MLS(ASCP)CM, CGCM, MBCM University of Texas M.D. Anderson Cancer Center School of Health Sciences 1515 Holcomb Blvd., Box 2 Houston, TX 77030 713-563-3095 pchu@mdanderson.org

Ex-Officio Member Patricia K. Dowling, PhD Pathline Labs 535 E. Crescent Ave. Ramsey, NJ 07446 PDowling@pathlinelabs.com

Representative to CAP/ACMG Term: 1/16 – 12/21 Jun Gu, MD, PhD, CG(ASCP)CM University of Texas MD Anderson Cancer Center School of Health Professions Cytogenetic Technology Program 1515 Holcombe Boulevard, Unit 2 Houston, TX 77030 (713) 563-3094 jungu@mdanderson.org

Council of Representatives Representative to CCCLW Term: 7/14 – 6/20 Hilary E. Blair, BS, MS, CG(ASCP)CM Mayo Clinic 200 First St. SW Rochester, MN 55905 507-255-4385 blair.hilary@mayo.edu

Publications

Representatives to BOC Term: 10/12 – 9/18 Helen Bixenman, MBA/HCM, CHC, CG(ASCP)CM, DLMCM, QLC San Diego Blood Bank 3636 Gateway Ctr. Ave., Ste. 100 San Diego, CA 92102 619-400-8254 hbixenman@sandiegobloodbank.org

AGT Journal Editor Mark D. Terry 1264 Keble Lane Oxford, MI 48371 586-805-9407 markterry@charter.net

Term: 10/11 – 9/20 Amy R. Groszbach, MEd, MLT(ASCP) CM, MBCM Mayo Clinic Molecular Genetics Laboratory – Hilton 920 200 First St. SW Rochester, MN 55905 507-284-1229 groszbach.amy@mayo.edu

Other Contacts Liaison to ASCLS Governmental Affairs Committee Jennifer Alvares Oxford Gene Technology 832 W. 36th St. #3 Chicago, IL 60609 312-714-0932 jen.crawford34@gmail.com FGT Board of Trustees President Robin Vandergon, CG(ASCP)CM, DLMCM NeoGenomics 30 E. River Park Place West, Ste. 400 Fresno, CA 93720 559-392- 0512 rvandergon@neogenomics com

Executive Office AGT 4400 College Blvd, Suite 220 Overland Park, KS 66211 913-222-8665 913-222-8606 FAX agt-info@kellencompany.com www.AGT-info.org

Staff Contacts: Christie Ross, Interim Executive Director/Education Program Coordinator 913-222-8626 cbross@kellencompany.com Diane Northup, Administrative Assistant 913-222-8630 agt@kellencompany.com

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Association Business

ABSTRACT SUBMISSION DEADLINE: Friday, February 2, 2018

AGT 2018 Call for Abstracts

Dialogue and the sharing of ideas is critical to the success of our field, so we want to hear about the work being done in your laboratories. The 43rd Annual Meeting Program Committee invites all interested persons to submit abstracts for the AGT 43rd Annual Meeting. Returning in 2018: • A significant increase in the number of submissions selected for a platform presentation • Multiple platform presentation sessions throughout the scientific meeting • Focused platform presentation sessions, such as cytogenetics and molecular genetics • Expanded areas of interest, to include topics such as clinical genetics, genetic counseling and regulatory affairs Abstracts will be printed in the Final Program Book and in the 2018 Third Quarter issue of the Journal of the Association of Genetic Technologists if they are presented at the meeting. All abstracts must contain: • a description of the study design, • a statement of results (including data), and • an informative conclusion. Abstracts will be assigned to platform or poster presentations by a Review Committee. The Committee will consider the author’s preference and the time constraints of the meeting.

Poster Presentations

Submission Requirements

Poster presentations are scheduled for Friday, June 1 and Saturday, June 2.

Abstracts must be submitted electronically through AGT’s online submission site. If you are submitting more than one abstract, you must submit each abstract separately. You may submit your abstract here: https://www.surveymonkey.com/r/AGT2018Abstracts.

Please Note: First authors on posters must be AGT members at the time of submission in order to be eligible to win the Best Poster Award.

It is important that you follow all instructions carefully. Abstracts submitted incorrectly will not be considered for presentation.

Platform Presentations Platform presentations are limited to 15 minutes each: twelve minutes for the presentation and three minutes for questions/discussion. If AGT accepts your abstract for platform presentation, you will receive notification by March 31, 2018. All platform presentations presented by AGT members are eligible to win the Best Platform Award at the Annual Meeting. Abstracts not accepted for platform presentation may be accepted for poster presentation.

ABSTRACTS MUST BE RECEIVED BY FRIDAY, February 2, 2018.

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Association Business

AGT 2018 Call for Student Abstracts & Student Research Award Entries ABSTRACT DUE DATE: Friday, March 16, 2018 Student members enrolled in an accredited Cytogenetics or Diagnostic Molecular Science program are invited to submit student abstracts for presentation and as an entry the 2018 Student Research Award. The award will be presented to the recipient at the AGT 43rd Annual Meeting in Tampa, Florida, May 31–June 2, 2018. The entries are submitted to the AGT Executive Office, which then forwards the abstracts to a panel of Association members. The winner is selected by this panel based on a scored evaluation of the submitted abstracts. All other participants will be invited to present a poster at the Annual Meeting. Eligibility:

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Criteria

Individuals/students eligible to submit abstracts for the award are:  Those enrolled in a NAACLS “Serious Applicant Status” or “Accredited” undergraduate or certificate program in Cytogenetics or Diagnostic Molecular Science (U.S. Programs).  Those enrolled in a Canadian Society of Laboratory Technology Approved undergraduate or certificate program in Cytogenetics or Molecular Biology (Canadian programs).  Those attending formal undergraduate or certificate programs outside of the United States and Canada if the program is recognized at the national level of the country in which the program is located. Applicants MUST be enrolled in the program between March 1, 2017 and March 4, 2018 Individuals/students graduated prior to March 1, 2017 are not eligible. Applicants must be members of the Association of Genetic Technologists at the time of application to be eligible to win the Student Research Award, but nonmembers may submit an abstract in the student category. Information regarding AGT membership is available from your program director or the AGT Executive Office. The applicant will be required to be the first author on the abstract. The applicant and a program official will be required to validate that: the research was conducted during enrollment and completed prior to graduation from the program, the applicant was the primary investigator, and the abstract submitted is the work of the applicant. Applications may be submitted throughout the year in order to be considered for the 2018 Student Research Award. All entries received after Friday, March 16, 2018 will be considered for the 2019 Student Research Award.

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A purpose for the experiment or investigation where the scientific merit or contribution is stated. A hypothesis, which indicates scientific expectations of the investigation and should be appropriate for the purpose. Scientific methods which are appropriate to the investigation, concise and organized. Data resulting from the investigation/experiment should be concise, specific and organized. The interpretation should be consistent with the data. The conclusions should be consistent with the data and interpretation. They may be intertwined with the interpretation, should support or refute the hypothesis, and should include the need for further investigation or suggest how other variables may influence the results. References should be used when appropriate. The institution name or any other identifying information should not be included in the abstract.

Submission Requirements

Abstracts must be submitted electronically at: https://www.surveymonkey.com/r/AGT2018StudentAbstracts. If you are submitting more than one abstract, you must submit each abstract separately. It is important that you follow all instructions within the online submission site carefully. Abstracts submitted incorrectly will not be considered for presentation. Student Research Award:

• The recipient will be notified by April 27, 2018. • The winner will receive complimentary 2018 Annual Meeting registration and expenses to travel to the meeting. • The recipient will be invited to present his/her research in a 10-minute platform presentation. • All other submissions will be considered for poster presentation.

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Association Business Association Business

The Foundation for Genetic Technology (FGT) was organized exclusively for scientific and educational purposes. It is a non-profit organization whose funds and assets are used to promote education in genetic technology and provide professional opportunities for The Foundation for Genetic Technology (FGT) was organized exclusively for scientific and educational purposes. It is a non-profit training through grants, scholarships organization whose funds and assets and are awards. used to promote education in genetic technology and provide professional opportunities for As always, behind the scenes, the dedicated members of the Foundation work throughout the year to strive to continue the mission of training through grants, scholarships and awards. the FGT. Along with support from AGT, the Foundation is able to fund the scholarships and grants that were presented at the Annual AsMeeting. always, behind thethis scenes, dedicated members of the Foundation, work throughout year to strive to the continue the mission AGT None of wouldthe have been possible without the help of the donors, vendorthe sponsorships and members-at-large of the FGT. Along with support from AGT, the Foundation is able to fund the scholarships and grants that were presented at the of AGT. Once again, the Foundation is fiscally solvent this year, which is a tribute to our many hard-working and dedicated members. Annual Meeting. None of this would have been possible without the help of the donors, vendor sponsorships and the members-at-large major source of income for FGTiscomes the this sale year, of thewhich studyisguides for to theour Cytogenetic and Molecular exams. These are of A AGT. Once again, the Foundation fiscallyfrom solvent a tribute many hard-working and dedicated members. available year-round and can be purchased by visiting the AGT website, and accessing FGT from the Resources tab. Another financial A major sourceregional of income for FGT comes from the The sale of the Coast study meeting guides foris the Cytogenetic These are resource has been meetings sponsored by FGT. West usually in March,and andMolecular the East exams. Coast meeting is available year-round and Please can berefer purchased visiting AGTmeeting website,information. and accessing FGT from the Resources tab. Another financial scheduled in September. to the by website forthe further resource has been regional meetings sponsored by FGT. The West Coast meeting is usually in March, and the East Coast meeting is The Silent Auction atPlease the AGT Meeting a tremendous success, with over $600 of donated items. This event has scheduled in September. refer Annual to the website for was further meeting information. proven to be a great way for the FGT to raise money and also promote interaction among the attendees. Thank you to everyone who participated…those of you whoAGT donated items, the winners of our auction and those who stopped by the FGT booth tofor inquire about The Silent Auction at the Annual Meeting was a tremendous success. This event has proven to be a great way the FGT to raise money also promote among thetoattendees. Thank you to everyone of youPat whoLeMay donated us. Your inputand is important, andinteraction your interest is vital keep FGT opportunities available.who Forparticipated…those more information, email at items, the winners of our auction and those who stopped by the FGT booth to inquire about us. Your input is important, and your plemay1945@aol.com. interest is vital to keep FGT opportunities available. For more information, email Robin Vandergon at rvandergon@neogenomics.com.

Do You Know Someone…? Having just come off a very successful AGT Annual Meeting in St. Louis, DO YOU SOMEONE …? Missouri, it is timeKNOW to look ahead to 2018. Having justAGT comeand off aour very successful AGT Annual Meeting infor Orange Along with corporate sponsors, the Foundation Genetic County, California, is time to look 2017. Technology has many itscholarships and ahead awardstothat we present at the AGT Annual Meeting every year. A complete list, along sponsors, with requirements, application forms and Along with AGT and our corporate the Foundation for Genetic submission deadlines is listed in the FGT section on that the AGT website.atThere is a wide Technology has many scholarships and awards we present the AGT range of awards available something for every member consider…from Annual Meeting everyand year. A complete list,AGT along with to requirements, the newly certified to the career-oriented professional and theon seasoned application formstechnologist and submission deadlines is listed in the FGT section the veteran technologist. encourage each AGT member consider nominating AGT website. There We is a wide range of awards available andtosomething for every someone for thesetoawards. It is a wonderful waycertified to acknowledge the accomplishments AGT member consider…from the newly technologist to the careerand the dedication of those individuals we know work so We hardencourage for the field oriented professional and the seasonedwho veteran technologist. ofeach genetics. aretomany qualified candidates, manyfor of these whomawards. work inIt small AGTThere member consider nominating someone is laboratories feel out of the but each application reviewed ofby a a wonderfulthat waymay to acknowledge theloop, accomplishments and the is dedication committee, and is not a popular vote. those individuals who we know work so hard for the field of genetics. There are many qualified candidates, many of whom work in small laboratories that Youfeel oweout it of to the yourselves, AGTapplication members, istoreviewed recognize may loop, butaseach byoutstanding a committee,members and ofisour organization. If we fail to promote ourselves, then no one is notprofessional a popular vote. goingYou to do us. So, if you knowmembers, an AGTtomember who demonstrates passion, oweititfor to yourselves, as AGT recognize outstanding members knowledge and a commitment to genetics, please check out the various of our professional organization. If we fail to promote ourselves, thenawards no onethat our is organization going to do sponsors. it for us. So, if you know an AGT member who demonstrates passion, knowledge and a commitment to genetics, please check out the various awards that our organization sponsors.

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Association Business Foundation for Genetic Technology

4400 College Boulevard, Suite 220 Overland Park, KS 66211 FGT website: http://www.agt-info.org/Pages/fgt.aspx

nology (FGT) was organized exclusively for scientific and educational purposes. It is a non-profit 2018 Grants & Awards Deadline: Friday, March 30, 2018 are used to promote education in genetic technology and provide professional opportunities for and awards. For more information, guidelines, criteria and application forms for the grant and awards listed below visit the FGT website at

http://www.agt-info.org/Pages/fgt.aspx. dedicated members of the Foundation work throughout the year to strive to continue the mission of GT, the Foundation is able to fund the scholarships and grants that were presented at the Annual Outstanding Technologist Grant Honorsand an the outstanding AGT member technologist who is BOC-certified with ave been possible without the help of the donors, vendor($500) sponsorships members-at-large three or more years of work experience in the genetic industry. Sponsored by Leica Microsystems. is fiscally solvent this year, which is a tribute to our many hard-working and dedicated members.

T comes from the sale of the study guides for the Award Cytogenetic and Molecular exams. Florence Dowling Genome ($500) Acknowledges andThese honorsareoutstanding technologists in cytogenetics and hased by visitingmolecular the AGT genetic website,technology. and accessing from the Resources tab.toAnother financial ThisFGT award program contributes the growth of genetic technology as a profession by recognizing sponsored by FGT. The West Coast meeting is usually in March, and the EastbyCoast meeting is individuals with superior professional commitment. Sponsored Patricia Dowling. to the website for further meeting information. New Award ($750) newly certified items. AGT members whohas submit an essay about their genetic experience Annual Meeting was Horizons a tremendous success, with Honors over $600 of donated This event to attendinteraction the AGT Annual Meeting. Sponsored Rainbow Scientific. T to raise moneyand anddesire also promote among the attendees. Thankby you to everyone who

ted items, the winners of our auction and those who stopped by the FGT booth to inquire about EXCEL Award ($500) AGT members enrolled in a formal university/hospital or laboratory-based program in diagnostic interest is vital to keep FGT opportunities available. For more information, email Pat LeMay at molecular technology or a NAACLS-accredited certificate or undergraduate cytogenetics or molecular program may be eligible to compete for free student registration to the AGT 43rd Annual Meeting, as well as one full-day or two half-day workshops. Sponsored by Oxford Gene Technology. Barbara J. Kaplan Scholarship ($1,000) AGT members enrolled in a formal training program, including university/ hospital, laboratory-based or a NAACLS-accredited program for molecular genetics or cytogenetics may be eligible for the $1,000 scholarship. Program directors may visit the FGT website for more information. Sponsored by FGT. Joseph Waurin Excellence in Education Award ($500) Honors an outstanding AGT member educator in the genetic community who is BOC-certified and has a minimum of five years of teaching experience. Sponsored by James Waurin.

Best Poster Awardâ&#x20AC;Ś? ($300) All AGT members attending the AGT Annual Meeting may vote for the poster that fits OU KNOW SOMEONE

the winning criteria (i.e., interesting and informative topic, well-organized, clear and concise data, best illustrations, clinical/ correlation cutting-edge g just come off laboratory a very successful AGTand Annual Meetingtechnology) in Orange submitted by an AGT member by the abstract deadline. Sponsored by Irvine California, it is time to Scientific. look ahead to 2017. with AGT and our corporate sponsors, the Foundation for Genetic Best Platform Presentation Awardat($500) All AGT members attending the platform presentations at the AGT Annual gy has many scholarships and awards that we present the AGT vote forlist, the presentation best fits the winning criteria (i.e., presentation must be given by a technologist, be an Meeting every Meeting year. Amay complete along with that requirements, interesting and informative havesection clinical/laboratory correlation and present cutting-edge technology). An AGT member on forms and submission deadlines is listed intopic, the FGT on the must be among the authors of the abstract submitted by the abstract deadline. Sponsored by FGT. bsite. There is a wide range of awards available and something for every mber to considerâ&#x20AC;Śfrom the newly certified technologist to the careerBoothtechnologist. Award professional andBest the Exhibitor seasoned veteran We encourage Honors exhibitors at the AGT Annual T member to consider nominating someone for theseMeeting. awards. AGT It is members will vote by ballot on the following criteria: 1. Best interaction (quality of interaction) ful way to acknowledge the accomplishments and the dedication of with AGT membership, including availability to meeting participants answering with detail. ividuals who we know work and so hard for thequestions field of genetics. There qualified candidates, many of whom work in small laboratories 2. Most valuable technical informationthat or product information, including presentation of literature available. out of the loop, but each application is reviewed by a committee, and 3. Best overall booth design, including appearance of exhibit and visual impact of the display. opular vote. Most innovative formembers attendees. we it to yourselves, as AGT4. members, to recognizegifts/raffles outstanding ofessional organization. If5. we Creativity. fail to promote ourselves, then no one to do it for us. So, if you know an AGT member who demonstrates 6. Only exhibitors listed in the final program will be eligible. knowledge and a commitment to genetics, please check out the various hat our organization sponsors. Please refer to the website listed above for the details and

the application forms for all of these awards, grants and scholarships.

The Journal of the Association of Genetic Technologists 43 (3) 2017 The Journal of the Association of Genetic Technologists 42 (3) 2016

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Association Business

Foundation for Genetic Technology 2017-2018 Board of Trustees Voting Members President Robin Vandergon NeoGenomics 30 E. River Park Place West, Ste. 400 Fresno, CA 93720 559-392-0512 rvandergon@neogenomics.com Vice President, AGT Representative, Awards & Scholarship Chair Patricia K. Dowling Pathline Labs 535 E. Crescent Ave. Ramsey, NJ 07446 201-393-5578 pdowling@pathlinelabs.com Secretary DeNesha Criswell NeoGenomics 618 Grassmere Park Drive, Unit 20 Nashville, TN 37211 239-768-0600 x2107 dcriswell@neogenomics.com

Treasurer, Chair Capital ManagementÂ Committee Bob Gasparini NeoGenomics Laboratories 12701 Commonwealth Dr. Ft. Myers, FL 33913 239-357-4237 bgasparini@neogenomics.com AGT Representative, Grants Committee Chair Peter C . Hu University of Texas M.D. Anderson Cancer Center School of Health Sciences 1515 Holcomb Blvd., Box 2 Houston, TX 77030 713-563-3095 pchu@mdanderson.org Public Member, FGT Fundraising Chair Jeff Sanford MetaSystems Group, Inc. 70 Bridge St., Ste. 100 Newton, MA 02458 617-924-9950 jsanford@metasystems.org

Compliance Officer Helen Jenks 1506 Arroyo Grande Dr. Sacramento, CA 95864 helenjenks@sbcglobal.net

Non Voting Members Advisor, AGT President Jason Yuhas Mayo Clinic 200 First St. SW Rochester, MN 55905 507-538-7634 yuhas.jason@mayo.edu Ex-Officio, AGT Education Director Sally J. Kochmar Magee-Womens Hospital Pittsburgh Cytogenetics Lab 300 Halket St., Room 1233 Pittsburgh, PA 15213 412-641-4882 skochmar@upmc.edu

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PRODUCT ORDER FORM Item Description

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The AGT Cytogenetics Laboratory Manual, 3rd Edition

[Please note: Note: The 4th Edition of AGT's Cytogenetics Laboratory Manual is only available through the publisher or Amazon. Click here to order publication now through the publisher.

The Cytogenetics Symposia, 2nd Edition

The AGT Molecular Biology Techniques Review Guide Select method of delivery:

 Dropbox  Secure Document Hyperlink The Dynamics of Chromosome Spreading Video – CD featuring Jack Spurbeck

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Please allow 2-4 weeks for shipping.

Regular Members. Regular membership shall be available to persons who are professionally interested in the field of genetics.

New Membership Application Please check the appropriate membership category. If you are applying for a collaborative membership, please indicate the related organization and your member ID number:

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Student Members. Student membership shall be available to persons who are pursuing a full or part-time course of study at an educational institution or school and who are interested in pursuing a career in the field of genetics. Emeritus Members. Emeritus membership shall be available to persons who are retired from or inactive in the field of genetics. Collaborative Members. Collaborative membership shall be available to persons who currently hold membership in any other health-related national organization and who have never been members of ACT/AGT.

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 Director  Head (Lead, Core) Technologist

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 Biochemical  Cytogenetics  Molecular  Other Appropriate years experience in Genetics:  under 2  2-4  5-7  8-10  11-15  16-20  21-30 Principal area of Genetics: (check one)

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Please note: AGT does not accept purchase orders and does not bill/invoice for services. Mail application form and appropriate fee for membership in correct U.S currency. Money order or check in U.S. funds drawn on a U.S. bank only. CHECKS DRAWN ON INTERNATIONAL BANKS WILL NOT BE ACCEPTED. Make checks payable to Association of Genetic Technologists. For your convenience, you may pay by credit card. Applications received after September 15 are applied toward the next membership year. NOTE: Membership expires on December 31 of each year.

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SEND APPLICATION AND FEE TO: Association of Genetic Technologists 4400 College Boulevard, Suite 220 Overland Park, KS 66211 Phone: 913-222-8665 FAX: 913-222-8606

[NOTE: Submission and acceptance of this membership application authorizes the AGT Executive Office the right and privilege to email you as a member. AGT does not sell or distribute in any other manner its member email address list.] 9/14

Association Business

How does AGT membership benefit you? TAY INFORMED

AGT’s publications, educational offerings and research reports deliver the news and information most relevant to your role so you’re prepared to tackle any challenge

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CCESS TO PEOPLE

Increase your value to your organization. AGT’s directories of members and labs, educational offerings, research reports and our flagship Journal can help you realize greater success.

Connect with thousands of cytogenetics, molecular and biochemical genetics professionals. Become a leadership volunteer, contribute content or participate in committees.

EACH NEW HEIGHTS

Between our in-person and online education opportunities (many free to members!) and online resources, we will help get you there. Learn from industry leaders and colleagues alike.

Are you ready to be a S.T.A.R? Join today!

How does AGT membership benefit your organization? GROW YOUR ORGANIZATION As a member, you’ll access the resources and tools you need to help innovate and inspire your organization. AGT also promotes the interests of geneticists and genetic technologists on a National scale, with representation on organizations such as NAACLS, BOC, CCCLW, CAP/ACMG and others.

MAXIMIZE EFFICIENCY Realize efficiencies and implement improved methodologies at your organization by seeing how others have met similar challenges.

BE PREPARED All organizations look different, but the challenges they face are often the same. We can help you prepare to meet any organizational challenge with the right knowledge and solutions by providing you an easy way to meet your CE needs.

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THREE EASY WAYS TO JOIN: Online: http://www.agtinfo.org/Pages/joinagt.aspx Mail: Return the application to the address listed With an Event: Sign up for membership at the same time you register for one of our events. It’s that easy!

Annual Meeting American Society for Mass Spectrometry (ASMS) Annual Conference

Indianapolis, IN

June 4-8, 2017

www.asms.org

Cambridge Healthtech Business Institute (CHI) World Association Preclinical Congress

Boston, MA

June 13-15, 2017

www.healthtech.com

International Aids Society (IAS) Conference

Paris France

July 2017

www.iasociety.org

European Society of Human Reproduction & Embryology (ESHRE) Annual Meeting

Geneva, Switzerland

July 2-5, 2017

www.eshre.com

2017-2018 Scientific Meetings July 30 – Schedule www.aacc.org

American Association for Clinical Chemistry (AACC)’s San Diego, CA Annual Meeting

August 3, 2017

IfAmerican you know Society of a relevant meeting, please send information toSan Ephrem Chin, Directorwww.ascls.org at nzelfman@gmail.com. July Relations 30 – of Clinical Laboratory Diego, CA Public August 4, 2017

Science (ASCLS) Annual Meeting

Society for Inherited Metabolic Disorders (SIMD) Meeting Annual Meeting

Rio de Janeiro, Location Brazil

September 5-8, 2017 Dates

www.simd.org

International Congress of Pediatric Laboratory American Society of Clinical Oncology (ASCO) Medicine (ICPLM) Annual Meeting

Durban, Chicago, IL South Africa

October June 2-6, 20-22, 2017 2017

www.icplm2017.org www.asco.org

American Society for Mass Spectrometry (ASMS) 2018

Indianapolis, IN

June 4-8, 2017

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American Chemical Society (ACS) National Cambridge Healthtech Institute (CHI) World Meeting & Exposition Preclinical Congress

New Orleans, Boston, MA LA

March 18-22, 2018 www.healthtech.com portal.acs.org June 13-15, 2017

American Association for Cancer Research (AACR) International Aids Society (IAS) Conference Annual Meeting

Chicago, IL Paris France

April2017 14-18, 2018 July

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European Society Reproduction & International UnionofofHuman Basic and Clinical Embryology (ESHRE) Annual Meeting Pharmacology (IUPHAR) World Congress of Basic and Clinical Pharmacology American Association for Clinical Chemistry (AACC)’s Annual Meeting American Chemical Society (ACS) National Meeting & Exposition American Society of Clinical Laboratory

Geneva, Kyoto, Japan Switzerland

July 2-5, TBD,2017 2018

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San Diego, CA Boston, MA

July 30 – August 3, 2017 19-23, 201830 – July

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Science (ASCLS) Annual American Association of Meeting Blood Banks (AABB) Annual Meeting & CTTXPO Society for Inherited Metabolic Disorders (SIMD) Annual Meeting American Society of Human Genetics (ASHG) Annual Meeting International Congress of Pediatric Laboratory Medicine (ICPLM)

San Diego, CA Boston, MA Rio de Janeiro, Brazil San Diego, CA Durban, South Africa

August 4,13-16, 2017 October 2018 September 5-8, 2017 October 16-20, 2018 October 20-22, 2017

www.ascls.org www.aabb.org www.simd.org www.ashg.org www.icplm2017.org

Job placement ads are online at http://www.AGT-info.org

2018 American Chemical Society (ACS) National Meeting & Exposition

New Orleans, LA

March 18-22, 2018 portal.acs.org

American Association for Cancer Research (AACR) Annual Meeting

Chicago, IL

April 14-18, 2018

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International Union of Basic and Clinical Kyoto, Japan Pharmacology (IUPHAR) World Congress of Basic and Clinical Pharmacology

July TBD, 2018

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American Chemical Society (ACS) National Meeting & Exposition

Boston, MA

August 19-23, 2018

portal.acs.org

American Association of Blood Banks (AABB) Annual Meeting & CTTXPO

Boston, MA

October 13-16, 2018

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American Society of Human Genetics (ASHG) Annual Meeting

San Diego, CA

October 16-20, 2018

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Job placement ads are online at http://www.AGT-info.org

The Journal of the Association of Genetic Technologists 43 (3) 2017

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The Journal of the Association of Genetic Technologists The Journal of the Association of Genetic Technologists is a peer-reviewed journal, and scientific materials for publication containing original research will be reviewed by independent referees. Manuscripts that require revision or that contain major editorial changes will be returned to the senior author of the article. Materials submitted will not be retained following publication nor will photographs, disks, or hard copies of manuscripts be returned to authors. Rejected manuscripts will not normally be returned, although an effort will be made to return original photographs and prints. Manuscript content is the responsibility of the author(s). All articles published, including editorials, letters, book reviews, invited articles, Brain Ticklers, columns, and reviews, represent the opinions of the authors and do not reflect the official policy of AGT or the institution with which the author is affiliated unless specified by the author. AGT, its members, and the editor of The Journal of the Association of Genetic Technologists make no warranty and assume no liability with respect to the information contained herein.

Information For Authors The Journal of the Association of Genetic Technologists is pleased to consider manuscripts that describe experience with cytogenetics, molecular genetics, or biochemical genetics and the application of these disciplines. Submitted manuscripts must be typed, preferably double-spaced, using a 12 point font and 1” margins. In addition to the original, three copies of the manuscript and camera-ready illustrations must be submitted to the editor-in-chief. Items to be italicized or enhanced (bold, underlined) should be clearly indicated. The conversion factor for print equivalency is as follows: two double-spaced typed pages equal approximately a one-half typeset page. Authors may supply the material on a 3½” disk, preferably in Microsoft Word, WordPerfect, or ASCII format, along with the hard copy. Macintosh disks are also acceptable, but conversion costs will be assessed accordingly to AGT and a delay in processing may occur. Materials may alternatively be supplied to the editor via email at the address shown on inside front cover. Email submission is preferred. Illustrations must be original photographs, computer-generated digitized files (preferably saved as a .tif, .eps, or .bmp file), or black and white line drawings, professionally prepared. The cost of separating and printing color photographs or illustrations will be charged to the author. Photographs must be properly identified on the back, including the author’s name, title of article, and top direction. A ball point pen should not be used for labeling. The affixing of a typewritten label to the illustration or table will prevent damage.

Notation & References Authors’ titles must be accompanied by a position description of less than 15 words, which will be printed with the article. Textual citations to the referenced literature should be parenthetically noted by author’s surname followed by year of publication, and arranged chronologically and then alphabetically, as demonstrated in the following example: (Lese and Ledbetter, 1998; Reilly, 1998a; Morgan et al., 1999). In situations with more than two authors, the first author’s surname should be followed with et al. When references are made to more than one paper published in the same year by the same author, a lower case a, b, etc. should be appended to the date of publication and should be included in both textual citations and the reference list. References should be listed completely at the end of the paper in alphabetical order by surname of first author, and then by year of publication. When more than one publication appears with the same first author, listings will be alphabetized by the first varying co-author. Irrespective of the number of authors, et al. should not be used in the reference list. Journal titles should be abbreviated according to Index Medicus and book titles should be italicized. Use the following format for references: Journal Article Brothman AR, Zhu XL, Maxell T, Cui J, Derbler DA. Advances in the cytogenetics of prostate cancer. J Assoc Genet Technol. 1999;25(1):1-6. Book Chapter Barch MJ and Lawce HJ. The cell and cell division. In: Barch MJ, Knutsen T, Spurbeck JL (eds). The AGT Cytogenetics Laboratory Manual, 3rd ed. Philadelphia: Lippincott-Raven; 1997:1-18. Book Mark HFL. Medical Cytogenetics. New York: Marcel Dekker; 2000. All references should be complete. Accuracy is the responsibility of the authors. Only published articles and those in press may be included in the reference list. If necessary, unpublished data and submitted manuscripts should be cited parenthetically within the text.

Reprint Orders Reprints of articles can be purchased by authors at cost within two years after publication. On the order request, specify the journal’s volume and issue numbers, year of publication, page numbers, article title, author(s), and quantity requested. Include the contact name(s), address(es) and phone number(s) to be used for either shipping purposes or related questions. Payment should accompany the order. Checks must be made payable to AGT. Minimum order is 50 copies. Reprints are produced on 60# white offset paper, saddle-stitched (unless under four pages), and will appear exactly as they do in the journal. Price is based on article length, quantity ordered, and color requirements. Orders are not processed until payment is received. Once payment is received, allow four weeks for printing and shipping. Prices quoted include shipping by UPS ground; expedited shipping is available at an additional charge. Journal copies can be purchased by AGT members for $25/each, if copies are available. Please forward reprint orders or questions regarding price quotations to the AGT Executive Office (see inside front cover for address).

ISSN 1523-7834