JAGT - Issue 3 2016

The Journal

of the Association of Genetic Technologists

Volume 42 • Number 3 • Third Quarter 2016

Brain Tickler

Column Editor: Helen Lawce

Brain Tickler

Submitted by:

A peripheral blood sample was received from a 17-year-old male presenting with short stature and hypogonadism.

Rikki Harrison Cytogenetics Laboratory Oregon Health & Sciences University Portland, Oregon

The answer to this Brain Tickler appears on page 118.

The Journal of the Association of Genetic Technologists Third Quarter 2016 Volume 42, Number 3

Table of Contents

The official journal of the AGT

Brain Tickler. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inside Front Cover Column Editors and Review Board . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 A Note from the Editor. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

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) Copyright © 2016 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 $5 per issue and for non-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.

JAGT Editor Mark Terry 1264 Keble Lane Oxford, MI 48371 586-805-9407 (cell) 248-628-3025 (phone/fax) Email: markterry@charter.net 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.

Review Article JAK2 in the Diagnosis and Treatment of Lymphoid Malignancies: A Review of the Literature Lakshan N. Fonseka, Beatriz Germán, Francisco Exposito, Enrique Conde, Sergio Bárcena and Carlos A. Tirado. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 Review Article Telomerase in Acute Myeloid Leukemia: A Molecular Update on Diagnosis, Prognosis, and Treatment Lakshan N. Fonseka and Carlos A. Tirado . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 Molecular Diagnostics Training to Strive for Continuous Improvement Michelle Mah. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112 Profiles and Perspectives Dr. Sheila Dobin Interviewed by Hon Fong L. Mark. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114 Brain Tickler Summary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118 Continuing Education Opportunities Test Yourself #3, 2016 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 AGT 2015-2016 Education Committee Regional Representatives. . . . . . . . . . . . . . . 123 AGT Journal Clubs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124 Association Bu siness Message from the President. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 41st Annual Business Meeting Minutes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132 AGT 2016 Sponsors and Exhibitors, Annual Meeting Photos. . . . . . . . . . . . . . . . . . . . 133 AGT and FGT Award Winners. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136 Platform, Poster and Student Poster Presentations from the 2016 Annual Meeting. . . 138 Association of Genetic Technologists BOD Contacts. . . . . . . . . . . . . . . . . . . . . . . . . 162 AGT 2017 Call for Abstracts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163 Student Research Award. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164 FGT Letter. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165 2017 FGT Grants and Awards Deadlines. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166 FGT Board of Trustees. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167 Product Order Form. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169 Job Placements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170 New Membership Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171

ISSN 1523-7834

2017-2018 Scientific Meetings Schedule. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172 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 42 (3) 2016

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The Journal of the Association of Genetic Technologists Staff

Column Editors Abstract Reviews/Genetics in the News Jaime Garcia-Heras, MD, PhD Director of Cytogenetics The Center for Medical Genetics 7400 Fannin, Suite 700 Houston, TX 77054 713-432-1991 713-432-1661 FAX jgarcia@geneticstesting.com Brain Tickler/Book Review Editor Helen Lawce, BSc, CLSp(CG) Clinical Cytogenetics Laboratory Oregon Health Sciences University 3181 SW Sam Jackson Parkway MP-350 Portland, OR 97201 503-494-2790 503-494-6104 FAX lawceh@ohsu.edu

Genetics, Government & Regulation Helen Bixenman, MBA, CLSup, CLSp(CG) San Diego Blood Bank 3636 Gateway Center Avenue, Suite 100 San Diego, CA 92102 619-400-8254 hbixenman@sandiegobloodbank.org Jennifer Crawford-Alvares Cytogenetic Technologist II Section of Hematology/Oncology The University of Chicago Medicine 5841 S. Maryland Ave., Rm. I-304 Chicago, IL jen.crawford34@gmail.com Office: 773-702-9153

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. Toronto, Ontario Canada M5G 2M9 416-946-4501 ext.5036 michelle.j.mah@gmail.com

Letters to the Editor Mark Terry, JAGT Editor 1264 Keble Lane Oxford, MI 48371 586-805-9407 (cell) 248-628-3025 (phone/FAX) markterry@charter.net

Special Interests Turid Knutsen, MT(ASCP), CLSp(CG) 17836 Shotley Bridge Place Olney, MD 20832 301-570-4965 knutsent@earthlink.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 Michelle M. Hess, MS, CLSp(CG) (Clinical cytogenetics, radiation biology, Thomas Wan, PhD (Cytogenetics, Cancer cytogenetics) toxicology; clinical molecular genetics) (Cytogenetics, Molecular genetics, Lynn Hoyt, BSc, CLSp(CG), CLSup Cancer genetics) Rodman Morgan, MS, CLSp(CG) (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 Heather E. Williams, MS, CG(ASCP) CM (Cytogenetics, Molecular, Education) Jonathan P. Park, PhD (Cytogenetics, Molecular Genetics) (Cytogenetics, Molecular genetics, Julia Kawecki, BSc, CLSp(CG) Cell biology) Su Yang, BSc, CLSP(CG) (Cytogenetics, Molecular genetics) (Education, Traditional Cytogenetics) David Peakman, AIMLT, CLSp(CG) Turid Knutsen, MT(ASCP), CLSp(CG) (Prenatal diagnosis) Jason A. Yuhas, BS, CG(ASCP) CM (Cancer cytogenetics, CGH, SKY) (Cytogenetics, Molecular cytogenetics) Carol Reifsteck, BA Brandon Kubala, BSc, CLSp(CG) (Breakage syndromes, Fanconi’s James Zabawski, MS, CLSp(CG) (Traditional Cytogenetics) 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)

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A Note from the Editor

This and That My Point

This issue of The Journal of the Association of Genetic Technologists has plenty of content. Much of it is related to this year’s Annual Meeting, which was held in Anaheim, California.

Yes, I did visit Disneyland. Yes, the new Soaring Around The World is pretty cool. And yes, they’re just getting going on the Star Wars stuff, but its presence is already pretty big.

If I have a point, it’s that being part of AGT and attending the Annual Meeting is about more than getting up-to-date about changes in your field. It’s also about networking and meeting up with friends and making new friends. Although I’m not a huge fan of the word “networking” in this context, it’s a significant aspect of being in a professional organization and attending meetings. These are your colleagues. And you never know what those relationships might become or where they might go. There are people at the AGT meetings that have been friends for over thirty years!

And before I go, I wanted to share this photograph of Gene Gnome, submitted by Julie Sanford Biggerstaff, the director of t he Ida ho Cy togenetic s Diagnostic Laboratory. I believe Gene was made by Sally Hunter.

The person holding Gene is Brittney Stone, a technologist at the laboratory.

I Digress From the meeting point of view, there were two primary highlights when I grab my first thoughts. First, the keynote speaker on the first day of the meeting was Randy Schekman, professor of Cell and Developmental Biology in the Department of Molecular and Cell Biology at the Howard Hughes Medical Institute and the University of California at Berkeley. He’s also a Nobel Laureate. It’s not often you get to hear a Nobel Laureate in person. I was struck by how fundamental his work was to our understanding of how cells work. It was an enjoyable talk.

The second was more personal. I worked in the cytogenetics laboratory at Henry Ford Hospital from 1987 to 2004 before leaving to write full time. The director of the laboratory during my years there was Daniel Van Dyke, who is currently a professor of Laboratory Medicine and Medical Genetics at Mayo Clinic.

Dan gave the Gordon W. Dewald Lecture at the AGT Annual Meeting on Saturday. I had a few minutes to talk to him and catch up a little bit before he gave his talk, which I also found very interesting. Dan left Henry Ford Hospital a few months before I did, but he was an excellent boss (and I was probably a fairly average employee), and for all my conflicting feelings over my 18 years there, I think I was generally blessed with very good bosses.

Cheers, Mark Terry, Editor

AGT Website: www.AGT-info.org Member’s Only area – User Name: First initial, Last initial and AGT ID# (Ex. CR12345) Password: genetics

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Review Article

JAK2 in the Diagnosis and Treatment of Lymphoid Malignancies: A Review of the Literature Lakshan N. Fonseka1, Beatriz Germán1*, Francisco Expósito1*, Enrique Conde1*, Sergio Bárcena1 and Carlos A. Tirado2 1. University

of Navarra, Pamplona, Spain of Pathology & Laboratory Medicine—David Geffen School of Medicine at UCLA These authors contributed equally to this work.

2. Department *

Abstract In 2016, there will be an estimated 6,590 new cases of acute lymphocytic leukemia and 18,960 new cases of chronic lymphocytic leukemia in the United States. These and other lymphoid malignancies have a key player in common, JAK2, an enzyme from the Janus kinase (JAK) family. Deviations from the normal functioning of JAK2, particularly in the JAK-signal transducer and activator of transcription (STAT) pathway, can disrupt homeostasis and drive the accumulation of intermediate progenitors, contributing to the development of myeloid and lymphoid malignancies. In this review, the recent literature on JAK2 mutations in lymphoid malignancies is summarized, concluding with a discussion of the treatment of lymphoid malignancies. New directions for future research have been underlined to advance the clinical management of lymphoid malignancies.

Keywords: JAK2, JAK-STAT, Lymphoid, Cytogenetics, FISH, Diagnosis, Treatment

Introduction

acute lymphoblastic leukemia (ALL), B-cell chronic lymphocytic leukemia (B-CLL), and Hodgkin’s lymphoma (HL). Most mutations involving the JAK2 gene in lymphoid malignancies are translocations between JAK2 and other partner genes (Salmoiraghi et al., 2013). See Figure 1 for an ideogram of chromosome 9 illustrating the JAK2 locus.

In 2016, there will be an estimated 6,590 new cases of acute lymphocytic leukemia and 18,960 new cases of chronic lymphocytic leukemia in the United States (American Cancer Society, 2016).

These and other lymphoid malignancies have a key player in common, JAK2, an enzyme from the Janus kinase (JAK) family, which also includes JAK1, JAK3, and TYK2 (Bain et al., 2014). The principal function of the enzyme is the regulation of cytokines and growth factors such as erythropoietin, IL-3, IL-5, granulocyte/macrophage colony-stimulating factor and growth hormone (Hricik et al., 2014).

Deviations from the normal functioning of JAK2, specifically in the JAK-signal transducer and activator of transcription (STAT) pathway, can disrupt homeostasis and drive the accumulation of intermediate progenitors, paving the way for lymphoid malignancies such as multiple myeloma, acute lymphocytic leukemia, and chronic lymphocytic leukemia (Springuel et al., 2015).

Previous research led to the discovery of the JAK-STAT pathway, a common signaling pathway used by many cytokines.

Once a cytokine binds to its cell-surface receptor it causes receptor dimerization and activates JAK tyrosine kinases, which lead to the phosphorylation of specific tyrosine residues on the receptor.

The phosphorylated residues recruit transcriptional factors called STATs that undergo phosphorylation by JAKs, then dimerize and exit the receptor site to initiate gene transcription in the nucleus (Darnell et al., 1998; Levy and Darnell, 2002). However, all JAK2chimeric proteins are capable of dimerization, which can lead to the self-activation of JAK2 independent of the presence of the growth factor involved in its activation, causing uncontrolled cell proliferation (Salmoiraghi et al., 2013).

Figure 1. The region outlined in blue is 9p24, the JAK2 locus.

Mutations in JAK2 can result in constitutive activation of the JAK-STAT pathway, the mechanism underlying various lymphoid malignancies (Maglott et al., 2005; National Library of Medicine, 2016).

JAK2: An Update Recent reports indicate that mutations at the JAK2 locus, 9p24, are involved in several lymphoid malignancies including

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Review Article JAK2 in the Diagnosis and Treatment of Lymphoid Malignancies: A Review of the Literature and in this way activate transcription via the JAK-STAT pathway, but simultaneously demonstrate that JAK2 inhibitors can block this autophosphorylation (Maglott et al., 2011; Schinnerl et al., 2015).

The final novel fusion discussed is SPAG9-JAK2, found in a 14-year-old male diagnosed with pediatric ALL. Cytogenetic analysis revealed t(9;17)(p24;q21), and SNP array analysis detected deletions of CDKN2A/2B, PAX5, BTG1, CDK6, ADARB2, and IKZF1. This patient faced aggressive ALL and a poor outcome. SPAG9, or sperm associated antigen 9, has been implicated in other forms of cancer, but due to its rarity it has not been thoroughly studied. It encodes a protein involved in the JUN-NH2-kinase signaling pathway, and may be important in understanding tumor progression (Maglott et al., 2011; Kawamura et al., 2015). The results of a Mitelman case search for JAK2involved fusion proteins in ALL, AML, and HL are illustrated in Figure 2. These abnormalities are potential therapeutic targets for the clinical treatment of lymphoid malignancies.

Recent studies have explored a subtype of B-cell acute lymphoblastic leukemia (B-ALL) known as Philadelphia chromosome-like ALL (Ph-like ALL), in which gene expression is remarkably analogous to cases of ALL positive for the BCRABL1 fusion protein. These patients have a poor prognosis and typically show deletion of IKZF1, a gene encoding the lymphoid transcription factor IKAROS. In 187 cases of Ph-like ALL, Mullighan et al. found that 87.5% of JAK2-involved cases involved IKZF1, and 68.9% involved CDKN2A/B (Mullighan et al., 2009).

One study analyzed the genomes of 1,725 standard-risk B-ALL patients and compared them to 154 Ph-like ALL patients; 91% of patients of the Ph-like ALL group showed genomic alterations that activated kinases, including JAK2 and ABL1 rearrangements.

JAK2 fusions resulted in phosphorylation of STAT5, independent of the cytokines that regulate the JAK-STAT pathway (Roberts et al., 2014). Though Ph-like ALL is a high-risk subtype, new treatments involving ruxolitinib and rapamycin, which target JAK1/2 and mTOR signaling, respectively, have shown promise in murine xenograft models (Maude et al., 2012).

In a study of T-cell lymphoblastic lymphoma (T-ALL), two JAK2 mutations were revealed that lead to constitutive JAK-STAT pathway activation. These mutations are H574R and R683G, with the latter most likely due to RNA editing. Further research suggests that one mechanism of this constitutive activation is silencing of SOCS3 by hypermethylation, which indicates the potential usefulness of epigenetic drug use in concert with JAKinhibitors (Roncero et al., 2016).

JAK2 Fusion Proteins

Various mutations in JAK2 have been reported in lymphoid malignancies, the most recent of which are summarized in Table 1 (see table on next page). It has long been known that an ETV6JAK2 fusion can occur in pediatric T-cell acute lymphoblastic leukemia (T-ALL), resulting in constitutive activation of tyrosine kinases (Berger et al., 1997; Lacronique et al., 1997). In three different cell lines of cutaneous T-cell lymphoma, Ehrentraut et al. examined the PCM1-JAK2 fusion derived from t(8;9)(p22;p24).

The authors used lentiviral vectors to knockdown the fusion gene, and found that suppressor of cytokine signaling 2 and 3 (SOCS2 and SOCS3) were inhibited, whereas one gene involved in T-cell development, GATA3, was partially restored. These changes have been attributed to changes in STAT5 activation due to mutated JAK2 (Ehrentraut et al., 2013).

Recently, there have been two additional fusion proteins reported in acute leukemia cases revolving around two genes: SSBP2 and PAX5. One patient, diagnosed with pre-B-ALL, was found to have a SSBP2-JAK2 fusion that again resulted in constitutive activation in the pathway (Poitras et al., 2008).

Single stranded DNA binding protein 2 (SSBP2), is involved in maintaining the stability of the genome and is located at 5q14.1.

On the other hand, the PAX5-JAK2 fusion has been found in four pediatric B-ALL cases to date with an abnormal 9p arm (Nebral et al., 2009; Coyaud et al., 2010; Roberts et al., 2012).

Located at 9p13, PAX5 (Paired Box 5) codes for an activator protein expressed only in the early stages B-cell differentiation. In 2015, Schinnerl et al. aimed to classify the role of PAX5-JAK2. It is suggested that the fusion protein is able to phosphorylate itself

Figure 2. Number of cases containing one of the six discussed JAK2 fusion proteins in acute lymphoblastic leukemia and lymphoblastic leukemia (ALL/LL), acute myeloid leukemia (AML), and Hodgkin’s Lymphoma (HL) reported in the Mitelman database.

Treatment Conventional therapies such as chemotherapy have long been considered the first line of treatment against lymphocytic leukemia. Drugs such as fludarabine, cyclophosphamide and rituximab have led to survival improvements in young CLL patients. Likewise, other therapies involving androgens, interferon, or immunomodulatory agents such as thalidomide are becoming the standard of care. Glucocorticoids have been administered for their immunosuppressive activity, and hydroxylamine is being used for its capacity to inhibit T-cell growth. However, these are only palliative drugs, and are thereby not always effective because they do not target the disease origin (LaFave et al., 2012).

The necessity of reliable and specific treatments is a fact.

Deeper knowledge of hematological disease pathogenesis has contributed to a better characterization of the mutations and molecular mechanisms implicated in the development of lymphoid diseases. This progress has highlighted a number of therapeutic

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Review Article JAK2 in the Diagnosis and Treatment of Lymphoid Malignancies: A Review of the Literature Table 1. JAK2 (9p24) mutations in acute lymphoblastic leukemia reported since 2010 in the Mitelman database.

Karyotype

Diagnosis

Reference

46,XX,t(1;19)(q23;p13),der(9)t(9;9)(p24;q22)

B-ALL

Andersen et al., 2011

46,XY,del(9)(p13p24)

B-ALL

Baughn et al., 2015

46,Y,t(X;10)(p11;p12),add(1)(p36),del(9)(p11p24)

T-ALL

Brandimarte et al., 2014

47,XX,+X,der(9)t(8;9)(q24;p24),der(12)hsr(12)(p13)t(12;22)(p13;q11),der(14)t(8;14) (q24;q32),r(22)(p13q13)

B-ALL

Chae et al., 2010

46,XY,add(7),t(9;14)(p22;q12),del(9)(p13p24)

B-ALL

Coyaud et al., 2010

46,XY,der(9)t(7;9)(q35;p24)

B-ALL

Coyaud et al., 2010

49,XY,inv(1)(p21q43),del(2)(p16),del(6)(q13q23),t(9;13)(p24;q11),add(12)(p13), +add(16),+21,+mar

B-ALL

Coyaud et al., 2010

46,XX,ins(2;9)(p23;p21p24),der(9)del(9)(p12)t(9;22)(q34;q11),der(22)t(9;22)

B-ALL

Coyaud et al., 2010

49,XY,+X,+2,+4,-9,t(9;22)(p24;q11),-11,+add(19)(q13),+20,-22,+mar

ALL

Cuesta-Dominguez et al., 2012

47,Y,der(X)t(X;22)(p22;q11),-7,+8,add(9)(p24)x2,+der(22)t(9;22)(q34;q11)/51, idem,+Y,+15,+16,+der(22)

ALL

De Braekeleer et al., 2010

46-49,XX,add(1)(p13),+3,add(6)(q13),del(7)(p15),add(9)(p24),+11,-12,t(12;21) (p13;q22),-15,+18,+mar

ALL

Gardiner et al., 2012

45-49,XX,del(6)(q2?),add(9)(p24),add(11)(p15),-15,dup(21)(q?),1dmin

B-ALL

Harrison et al., 2014

46,XY,del(7)(q?),der(9)t(9;13)(p24;q14),ider(21)(q10)dup(21)(q22q22)

B-ALL

Harrison et al., 2014

46,XY,t(9;17)(p24;q21)

B-ALL

Kawamura et al., 2015

46,XX,add(9)(p24),t(9;22)(q34;q11)/40-45,idem,-14,-16

B-ALL

Kim et al., 2011

47,XX,+8,del(9)(p21p24)

T-ALL

Le Noir et al., 2012

46,XY,der(7)t(7;9)(q11;p13)del(9)(p21p24),der(9)t(7;9)(q11;p13),del(19)(q13)

ALL

Lindqvist et al., 2015

67,XXX,-4,-5,+6,-7,-8,-9,-9,der(9)del(9)(p11)del(9)(q12),+10,+der(12)t(8;12) (q22;q12)t(8;8)(p11;q12)t(8;12)(p22;q21)t(8;12)(q22;q22),+13,+14,-15,-16,-17,der(18) t(9;18)(p24;p11)t(9;18)(q13;q11)x2,-20,-21,i(21)(q10)x2,+22

ALL

Mkrtchyan et al., 2010

46,XY,der(8)t(8;9)(p22;p24)t(8;22)(q24;q11),der(9)t(8;9)(p21;p24), der(21)t(1;21)(q21;p11),der(22)t(8;22)(q24;q11)

ALL

Patterer et al., 2013

54,XY,+X,dup(1)(q12q44),+4,+6,dup(9)(p12p24),+10,+14,+17,+18,+21, dup(21)(q21q22)

B-ALL

Paulsson et al., 2015

47,XY,+2,del(2)(p23),t(3;22;9)(p12;q11;p24)

B-ALL

Roberts et al., 2012

46,XX,i(6)(p10),add(9)(p24),inv(14)(q11q32)

ALL

Russell et al., 2014

46,XX,-4,del(6)(q25),del(7)(q32),add(9)(p24),-12,-16,+3mar

ALL

Simons et al., 2011

46,XY,t(9;22)(p24;q11)

B-ALL

Tirado et al., 2010

46,XY,ins(6;11)(q27;q23q23),t(9;12),(p24;p11.2)[5]/46,XY[15]

B-ALL

Tirado et al., 2013

46,XY,-17,+22/47,idem,+8,t(9;12)(p24;p13)

ALL

Zhou et al., 2012

46,XX,t(9;12)(p24;p13)

ALL

Zhou et al., 2012

46,XY,t(9;12)(p24;p13)

ALL

Zhou et al., 2012

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Review Article JAK2 in the Diagnosis and Treatment of Lymphoid Malignancies: A Review of the Literature lymphoid branch, justifying investigation of its potential role in dual pathogenesis (Delhommeau et al., 2007).

Despite improvements to the WHO diagnostic criteria for lymphoid malignancies, there are areas that need to be studied in depth. In cases involving JAK2 fusion proteins, Bain et al. suggests that cases involving the PCM1-JAK2 fusion protein could be identified as a distinct category. On the other hand, ETV6-JAK2 and BCR-JAK2 positive cases might deserve separate classification, and further studies can determine its grouping (Bain et al., 2014). These proposed changes should be considered carefully, as better classifications may lead to the most appropriate use of JAK2 inhibitors.

Ruxolitinib (FDA-approved drug only for myeloid fibrosis treatment) could be useful in the clinical management of various lymphoid malignancies. Preliminary studies of cell lines treated with ruxolitinib are promising, but future research in advanced animal models is necessary to grasp the complete clinical utility of ruxolitinib. If this compound succeeds, the outlook for the diagnosis and treatment of lymphoid diseases will take a step forward and may also hold the key for improving clinical outcomes in other hematological malignancies.

targets central to the most prominent pharmacological strategies, based on JAK2 cascade.

Recent efforts have led to the development and improvement of type I and II JAK inhibitors (Samanta et al., 2006; LaFave et al., 2012). Type I inhibitors act in competition with ATP, attempting to bind to the kinase when it is in its active conformation. One of the drugs in this category, ruxolitinib, has demonstrated success in clinical trials. On the other hand, type II inhibitors, such as imatinib and sorafenib, maintain the same ATP-binding site as type I inhibitors, but they also target an adjacent hydrophobic binding pocket available only when the kinase is inactive (LaFave et al., 2012). In this group, the JAK2 inhibitor NVP-BBT594 has shown strong effect against proliferation in a B-ALL cell line expressing the mutation JAK2 I682F with rearrangements of CRLF2 expression (Andraos et al., 2012).

Type I inhibitor ruxolitinib is based on the results of two international phase III studies, COMFORT I and COMFORT II. Ruxolitinib is a selective inhibitor of both JAK1 and JAK2 (Koppikar et al., 2010). Ruxolitinib has shown therapeutic promise in the subset of Ph-like ALL, though further research in this particular subtype is necessary (Maude et al., 2012). Other studies of JAK inhibitors are being carried out, but are less extensive.

Fedratinib has demonstrated similar reductions in symptom severity. However, gastrointestinal side effects have been shown, limiting its dosage (Vainchenker et al., 2013).

Some patients have JAK-mutant cells with the ability to survive after JAK inhibitor exposure. This could be due to alterations in the JH1 kinase domain of JAK2 that confer resistance. The mutation M929I in JAK2, for example, can confer resistance in cells previously treated with ruxolitinib, illustrating the necessity of developing other strategies in order to avoid disease perpetuation (LaFave et al., 2012). One such strategy involves the aforementioned fedratinib, which can be useful in cases of ruxolitinib resistance. Kesarwani et al. identified over 200 amino acid substitutions that showed relevance in ruxolitinib-resistance development, and found that none of these were insensitive to fedratinib (Kesarwani et al., 2015). However, it has to be considered that JAK2 is necessary for normal functions, so its complete inhibition is not a feasible goal. Yet there is a promising therapy that may help to resolve this: HSP90 inhibitors. As cancerous cells increase heat shock protein 90 (HSP90) chaperone folding, HSP90 inhibitors have high selective efficacy. In vitro and in vivo studies have shown JAK2 degradation when HSP90 was tested (Marubayashi et al., 2010; Jhaveri et al., 2012).

Competing Interests The authors have no competing interests.

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Corresponding Author: Carlos A. Tirado, ctirado@mednet.ucla.edu

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Review Article

Telomerase in Acute Myeloid Leukemia: A Molecular Update on Diagnosis, Prognosis, and Treatment Lakshan N. Fonseka1 and Carlos A. Tirado1 1. Department

of Pathology & Laboratory Medicine—David Geffen School of Medicine at UCLA

Abstract It is expected that 10,460 patients will die from acute myeloid leukemia (AML) in the United States in 2016. Despite progress in clinical management, AML patients still have a 25.9% survival rate in the U.S. Researchers have sought to further understand this hematological malignancy and a number of studies have focused on unveiling the role of telomerase in disease initiation, progression, and maintenance. Though the role of telomerase in diagnosis has remained relatively static, its role in prognosis and treatment has become much clearer. While variants in TERT and TERC have been associated with worse clinical outcomes, telomerase and survivin co-expression can predict improved clinical outcomes. In regards to treatment, novel therapies such as mesoindigo and sodium metaarsenite provide new insights in clinical management. The use of leukemic stem cells in mouse models has shown promising results as well. Herein, we provide an update on the role of telomerase in AML through a survey of recent literature, focusing on the diagnosis, prognosis, and treatment of AML. Keywords: Telomerase, AML, Cytogenetics, FISH, SNP, Microarray, Prognosis, Treatment

Introduction

Treatment of AML is composed of fairly limited options that either aim to cause widespread elimination of leukemic cells (remission induction therapy) or to eradicate leukemic cells that remain after widespread elimination has taken place (postremission therapy). The primary route of attack in remission induction therapy is chemotherapy, though anti-cancer drugs such as Trisenox (arsenic trioxide) can be used in conjunction with chemotherapy. Post-remission therapy may consist of an autologous stem cell transplant, in which the patient’s own stem cells that were extracted during a period of remission are re-implanted. An allogenic stem cell transplant is also a possibility, depending on the availability of a compatible donor to provide the stem cell infusions that assist in reestablishing a healthy bone marrow (Mayo Clinic, 2015). These transplants may lead to death or other serious risks, and research regarding their appropriate application is still inconclusive (American Cancer Society, 2014).

Yet these diagnostic and treatment techniques may yield results that are not specific to AML; thus, it is necessary to develop more accurate methods in order to achieve a higher level of care. With this in mind, we provide an update on the role of telomerase in AML through a search of the recent literature and organize the resulting summary into three sections: diagnosis, prognosis, and treatment.

Acute myeloid leukemia (AML), a rapidly-progressing blood and bone marrow neoplasia, is expected to cause around 10,460 deaths in the United States in 2016 (SEER Cancer Statistics Factsheets, 2015). In early stages, patients may present with a variety of symptoms including fever, fatigue, and soreness originating in bones (Mayo Clinic, 2015). Patients diagnosed with AML between 2005 and 2011 had a 25.9% five-year survival rate (SEER Cancer Statistics Factsheets, 2015). Due to the symptom variety and low survival rate, it is imperative that research focuses on improving both the diagnosis and treatment of this disease.

An understanding of telomerase can guide us towards reaching this goal. An enzyme often found to be expressed in malignant tumors, telomerase may be involved in tumorigenesis.

Likewise, because this enzyme regulates telomere shortening and can contribute to the development of cell immortality, studies investigating its role may improve diagnoses by enabling earlier recognition of the transition from normal cell apoptosis to cancerous cell immortality (Deville et al., 2009).

Currently, AML is diagnosed primarily through blood and bone marrow tests, and spinal taps. AML blood samples typically show insufficient red blood cells and platelets, while white blood cell counts are significantly above the normal range. These white blood cells are mostly myeloblasts—immature cells that do not function exactly like their matured versions. However, confirmation of leukemia usually occurs via bone marrow biopsy or spinal tap. If patients are found with greater than 20% of myeloblasts, it suggests an AML diagnosis, as a normal sample contains fewer than 5% of myeloblasts. In addition, if a patient presents with blurred vision, headaches, or balance issues, it can suggest that AML cells have spread to the peripheral nervous system, in which case a spinal tap is performed (American Cancer Society, 2014; Mayo Clinic, 2015).

Telomerase and the Telomere Before delving into the review, it is essential to have an understanding of telomerase and the telomere. Human telomeres consist of TTAGGG repeats at the ends of each chromosome.

While they prevent chromosomes from fusing to each other, they have an additional role in cellular aging and chromosome integrity. DNA polymerase in standard DNA replication creates strands that are 50-200 base pairs shorter than the original strand, leading to continually shortened telomeres with repeated cell division. When these telomeric ends are reduced sufficiently,

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Review Article Telomerase in Acute Myeloid Leukemia: A Molecular Update on Diagnosis, Prognosis, and Treatment was unassociated with abnormal cytogenetics. This research team continued to conclude that despite finding variants in both TERT and TERC in pediatric AML patients, these variants appear to be irrelevant in developing the disorder (Aalbers et al., 2013).

Yet when Capraro et al. examined correlations between TERT expression and telomerase activity, both ALL and AML illustrated a positive correlation that reached significance (RAML = 0.401, RALL = 0.372). By calculating the ratio of telomerase activity to TERT expression, all groups were found to be significantly different (p = 0.006). Of all these groups, AML had the highest ratio of 1059 and the second highest value was 100 in T-ALL. Between AML subtypes, AML0 and AML3 were found to have the least levels of TERT expression (Capraro et al., 2011). Differences between subtypes of AML are well-categorized by the World Health Organization in four primary groups. These groups are summarized in Table 1 and are composed of recurrent cytogenetic abnormalities, multilineage deregulation, secondary AML, and lastly the morphology-based classification for patients who cannot be confidently placed in the other three categories (Flandrin, 2002).

it triggers cellular senescence, which is typically followed by cellular death.

Nevertheless, there exists an enzyme complex that can increase telomere length. This complex is telomerase, which contains both an RNA component and a reverse transcriptase component that enables telomerase to restore TTAGGG repeats to the shortened strand (Lingner et al., 1995; Mu et al., 2002; Hornsby, 2007). At the start of cellular division, chromosomes are replicated, but in this process the DNA transitions from its compact form to an unwound strand so that replication can take place. Thus, the primers can bind to their complementary sequence and signal DNA polymerase to begin replicating the sequence. Because the primer is composed of RNA and does not stay attached, the newly created strand lacks a short sequence at this point (see Figure 1 for an illustration of this strand). This loss is repeated with each cellular division, and each normally dividing cell undergoes this loss fifty to seventy times in its lifetime.

During this process, telomerase continually adds repeats to the telomeres, lengthening the strand and allowing the cell to continue functioning as it normally would. While telomerase frequently lengthens the telomeres of young cells, it is produced at low levels in older cells. The telomeres in these older cells shorten in greater increments and will soon reach senescence (University of Utah, 2016).

Prognosis Despite the limited number of studies of telomerase in uncovering new diagnostic information, there have been many more experiments that aim to develop more accurate prognoses.

One key example is evident in SNP rs2853669, a proposed risk factor for AML development. Through genotype analysis, Mosrati et al. found that patients with this variant in TERT, displayed significantly shorter survival time than the other patients. This is especially important in patients that present with a normal karyotype, as they can now be classified as high risk and monitored more closely if this SNP is detected (Mosrati et al., 2015). Variants in TERT and TERC were investigated by Yan et al. in 72 AML patients, of which 61 cases were de novo. Through this analysis of variants, these researchers identified three variants in TERT. Patients who carried these mutations had a poorer prognosis.

In addition, telomere 3â&#x20AC;&#x2122; overhangs, which act as substrates for the telomerase complex, were analyzed in this study. Although overhangs in AML were confirmed to be shorter than those in controls (p < 0.001), AML patients with shorter overhangs also tended to have a more abnormal karyotype and a poorer prognosis than the other patients (p = 0.001). In addition to overhang length, the researchers found that age and certain unfavorable chromosomal abnormalities were prognostic markers (Yan et al., 2013). Favorable and unfavorable abnormalities found in AML are summarized in Table 2.

Also investigating TERT, Aref et al. examined a specific mutation in exon 15. This mutation is TERT A1062T, and in the sample population, the mutation was present in 18 out of 153 AML patients, as well as in one out of the 197 cases used as control; 53.3% of patients in the wild-type AML group entered complete remission, as compared to 16.7% of those with the TERT A1062T mutation. On a similar note, 62.5% of the subjects with the mutation relapsed whereas the wild-type AML group had a reduced relapse rate of 28.2%. Patients with this mutation displayed significantly lower survival than wild-type AML patients, but the prognostic capabilities of this potential marker are not as robust when controlled for components such

Figure 1. During DNA replication in typical somatic cell division, the primer attracts the DNA polymerase, which then adds bases after the primerâ&#x20AC;&#x2122;s position in the telomeric region.

Thus, less bases will be present in the telomere of the newly synthesized strand, as illustrated in the figure. Telomerase will lengthen these telomeres, and in this way avoid cellular senescence triggered by short telomeres (University of Utah, 2016).

Diagnosis An enhanced understanding of telomerase has the potential to generate better methods of diagnosing AML with earlier recognition of disease initiation. This includes developing more efficient methods of distinguishing AML from other diseases with similar characteristics, such as B-cell acute lymphoblastic leukemia (B-ALL). Capraro et al. investigated the role of not only telomerase activity, but also TERT and telomerase gene expression in adult leukemias, specifically 37 AML patients, 13 B-ALL patients, and seven patients with T-cell acute lymphoblastic leukemia (T-ALL). From these patients, samples of leukemic cells were extracted from the bone marrow, all of which contained greater than 85% blasts. In comparison to control cells with normal karyotypes, both the AML and ALL cells with abnormal cytogenetics showed shorter telomeres (Capraro et al., 2011).

However, this was only statistically significant in ALL (p < 0.05), which is similar to another research teamâ&#x20AC;&#x2122;s confirmation that telomere length, measured by quantitative PCR, in pediatric AML

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Review Article Telomerase in Acute Myeloid Leukemia: A Molecular Update on Diagnosis, Prognosis, and Treatment Table 1. World Health Organization system of AML subtype classification. Here we focus on the common cytogenetic abnormalities associated with each (Flandrin, 2002).

AML With Recurrent Cytogenetic Translocations

Multilineage AML

Secondary AML

Morphological Classification of AML

t(8;21)(q22;q22)

-5

Similar to Not based on cytogenetic Multilineage AML abnormalities

Inv/del(16)(p13q22)/del(16)(q22)/t(16;16)(p13;q22)

-7/del(7q)

Abnormalities of 5q, -7, del(7q)

t(15;17)(q22;q21)

+8

t(9;11)

t(11;17)(q23;q21)

+9

t(8;21)

t(9;11)(p21;q23)

+11

t(15;17)

t(11;19)(q23;p13.1)

del(11q) del(12p) del(17p) -18 +19 del(20q) +21

Table 2. Favorable and unfavorable cytogenetic abnormalities in AML (American Cancer Society, 2014).

Favorable

Unfavorable

Translocation between Chr.8 and Chr. 21

Deletions in Chr. 5 or Chr. 7

Inversion of Chr. 16

Translocation or inversion of Chr. 3

Translocation between Chr. 16 and Chr. 16

Translocation between Chr. 6 and Chr. 9

Translocation between Chr. 15 and Chr. 17

Translocation between Chr. 9 and Chr. 11q23 abnormalities Complex changes involving multiple chromosomes Analyzing the expression levels of TERT, Kim et al. also sought to investigate the prognostic relevance of telomerase.

Samples from this study came from 151 adult AML patients.

The authors found increased expression of not only TERT, but also WT1 and survivin proteins. Patients with more peripheral blood leukemic blasts at diagnosis demonstrated increased TERT expression. TERT co-expressed with survivin were associated with better survival (p = 0.0115). Additionally, there were a handful of factors associated with event-free survival, such as the combination of lower WT1, survivin, and TERT (p = 0.0454), and lower levels of survivin co-expression with WT1 (p = 0.0031) (Kim et al., 2013).

as cytogenetics (Aref et al., 2014). Even so, Calado et al., found that this variant was present in 60% of AML patients (n = 594), significantly higher than those in the control group (n = 1,110) (Calado et al., 2009).

In 2013, Eid et al. used fluorescence in situ hybridization (FISH) in a study population of 20 adult patients with AML. They investigated the role of TERC and TERT genes, and examined gene amplification to determine if these two genes had prognostic relevance. In 19 out of the 21 cases, TERC amplification was detected, with each interphase cell having two to five copies.

Similarly, TERT amplification was present in a different set of 19 cases. In the TERT gene, each interphase cell showed two to nine copies. De novo cases consistently showed greater amplification than relapse cases. In addition, amplification of TERT was found to be associated with patients of poor clinical outcome, while no correlation between these factors was found in regards to TERC (Eid et al., 2013).

Treatment The bulk of recent research on telomerase in AML has been in the development of potential therapies. These therapies have a wide array of targets, but a large number of studies revolve around

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Review Article Telomerase in Acute Myeloid Leukemia: A Molecular Update on Diagnosis, Prognosis, and Treatment Another study investigated TERT and P-gp (P-glycoprotein), both of which are involved in signaling in STAT genes and FLT3 during AML pathogenesis. One study has shown that STAT5 expression (present in all 18 examined cases of de novo AML) may be a transcription factor for P-gp and TERT (Katsumi et al., 2013).

Thus, future therapies may be able to target STAT5 in order to regulate its expression. Recently, the first study examining the role of sodium metaarsenite has shown that TERT can be significantly reduced in HL-60 and HL-60R cells. By targeting telomeres, this compound can induce the shortening of telomeric terminal restriction fragments. Moreover, it inhibits the JAK/STAT pathway, in which the aforementioned STAT5 would also be affected, while activating the MAPK pathway and inducing apoptosis (Yoon et al., 2015).

TERT expression may be in part controlled by AML1/MTG8, a leukemic fusion gene that is common in AML. Researchers knocked down this fusion gene in two t(8;21)-positive cell lines and found reduced levels of TERT expression and shortened telomeres, suggesting that this gene is involved in maintaining telomere length. In fact, when AML1/MTG8 depletion lasted seven to twelve days, levels of CDKN1B and beta-galactosidase increased. Interestingly, the increase in beta-galactosidase may indicate an induced-dysregulation of leukemic self-renewal, as beta-galactosidase is associated with cellular senescence. Moreover, both cell lines showed reduced TERT transcript protein levels (Gessner et al., 2010).

Using the DEK oncoprotein, researchers were able to halt the transcription of TERT in AML cells. DEK, which is involved in a significant portion of transcriptional modifications, was isolated from the TERT promoter in AML cells, as well as from Taxexpressing cells. Tax acts as a TERT repressor. The outcomes of chromatin immunoprecipitation assays and reporter assays suggest that DEK’s effect is likely due to its interaction with the promoter, not solely through its expression (Karam et al., 2014).

Though not studied as frequently, mesoindigo may have therapeutic applications in AML. It is already utilized in the treatment of CML in China; thus, Lee et al. investigated its role in AML through primary AML cells and animal models, as well as NB4, HL-60, and U937 cell lines. In the NB4 and HL-60 cell lines, TERT promoter activity was significantly downregulated, and qRT-PCR revealed reduced TERT mRNA levels. Mesoindigo was able to work efficiently in conjunction with chemotherapy drugs cytarabine and idarubicin by increasing their cytotoxity (Lee et al., 2010). Both cytarabine and idarubicin are usually included in AML treatment protocol for induction therapy—the first phase of AML treatment—and cytarabine is also used in second phase post-remission therapy (Seiter, 2013). Future research is needed to understand the mechanism by which mesoindigo interacts with TERT, but the authors have suggested the involvement of STAT3 (Lee et al., 2010).

Lastly, there is a promising line of work from Bruedigam et al. using leukemic stem cells (LSCs), which play a role in maintaining AML. The authors examined LSCs in mouse models, targeting telomerase to induce LSC apoptosis. Mice in which telomerase deficiency was induced (using a retroviral construct targeting TERC) showed delayed latency compared to the wildtype group. However, despite delaying the onset of the disease,

TERT and TERC (see Figure 2 for ideograms of these genes). One study attempted to take advantage of the possibility that TERT expression may be sensitive to methylation, a possibility built upon the fact that the TERT promoter is located in a CpG island.

To examine this, the authors used 5-Aza-2’-deoxyctidine, a derivative of 5-azacytidine, also known as decitabine (DAC). DAC is currently a therapy for myelodysplastic syndrome that operates through the inhibition of DNA methylation to ultimately decrease the silencing of tumor suppressors, which can otherwise occur by promoter hypermethylation. Because AML cases can commonly display promoter hypermethylation, the authors aimed to test this drug in three AML cell lines: HL-60, OCI/AML3, and U937.

The results varied widely. For instance, while the latter two cell lines showed no significant DAC-induced telomerase shortening, the telomeres of HL-60 cells was significantly decreased, as compared to control. In regards to telomerase activity, each cell line displayed different results. The activity of telomerase showed a DAC-independent decrease in HL-60 cell lines. In OCI/AML3 cells there was a significant DAC-induced decrease in telomerase activity, but the opposite occurred in U397 cells, in which DAC significantly increased telomerase activity. Likewise, results varied when analyzing TERT expression as well. Though DAC significantly diminished the expression of TERT in both OCI/AML3 and HL-60 cells, it had no such effect on the U937 cells (Pettigrew et al., 2012).

DAC may also have therapeutic potential when introduced in conjunction with busulfan, a drug that alkylates DNA and is used in myeloablative pretransplant conditioning.

One study suggests that DAC, together with busulfan, can result in synergistic cytotoxicity and may induce apoptosis in myeloid leukemia cells, but this result is dependent on p53 (Valdez et al., 2010). A related study showed that 5-azacytidine—from which DAC is derived—downregulated TERT in AML patient samples and induced telomere dysfunction (Zhang et al., 2015). Although future research is needed, these results indicate the potential of both DAC and 5-azacytidine in a telomerase-targeted treatment.

Figure 2. Ideograms illustrating the location of TERC (Telomerase RNA Component) and TERT (Telomerase Reverse Transcriptase) (Huret et al., 2013; Maglott et al., 2011).

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Review Article Telomerase in Acute Myeloid Leukemia: A Molecular Update on Diagnosis, Prognosis, and Treatment

Figure 3. The four primary components of the human telomerase complex (Cong et al., 2002; Cohen et al., 2007; Maglott et al., 2011; Montanero et al., 2011)

Despite the therapeutic potential involving telomerase, such potential has yet to be fully realized in diagnoses and prognoses.

Particularly in diagnosis, the aforementioned studies show no concrete manner in which telomerase can aid clinicians either in more accurately diagnosing or in providing earlier diagnoses of AML. Though various correlations suggest that telomerase possesses diagnostic utility, future research is necessary to determine if these correlations truly illustrate that telomerase has diagnostic value.

The research by Capraro et al., for example, has illustrated that AML is significantly different from ALL, B-ALL, and T-ALL when the ratio of telomerase activity to TERT expression is calculated (Capraro et al., 2011). Through this ratio, the researchers translated the data gathered about telomerase into a value that may lead to more specific, and possibly earlier, recognition of AML. The promise of clinical potential is clear, but future research is necessary to fully develop these into utilizable diagnostic values.

Greater prognostic utility of telomerase is evident as compared to diagnostic utility. Specific forms of telomerase involvement have been associated with worse clinical outcomes. These forms include amplification of TERT and a number of TERT variants, such as SNP rs2853669. Yet despite the fact that telomerase has been more frequently implicated in unfavorable clinical outcomes, one study showed that if co-expressed with survivin, patients demonstrated improved survival. Future research is essential in understanding whether telomerase signals improved or diminished clinical outcomes in various situations. Research of the different contexts of telomerase holds the key to revealing its prognostic potential in AML. In conclusion, telomerase is clearly essential to our understanding

both groups of mice showed no differences in disease burden 30 days later. More interestingly, the authors found that inducing telomerase deficiency could decrease the amount of LSCs in the mice and thereby increase survival. This decrease in LSCs is dependent on p53, as p53 knockdown was able to decrease the amount of apoptotic LSCs present in the telomerase-deficient mice. These LSC mice models have revealed prognosticallyrelevant information, specifically that the genes SMC4, CPD, MRC1, SLC2A1, and SLC44A2 predicted favorable outcomes in human AML (Bruedigam et al., 2014).

Discussion The future of AML treatment appears bright, with novel therapies such as mesoindigo and sodium metaarsenite showing promising results. Moreover, research on LSCs has shown the benefits of LSC therapeutic strategies in mouse models, moving LSCs to the forefront of new treatments as compared to the others conducted using AML cell lines. Future research must be conducted to better understand these treatments, not only on the mechanism by which sodium metaarsenite and mesoindigo operate, but also on developing LSCs for larger mouse model studies with the ultimate goal of introducing a refined therapy suitable for clinical trials. Though most of the studies target TERC or TERT, there are two other genes involved in the human telomerase complex: TEP1 and DKC1 (Cohen et al., 2007). These key components of the human telomerase complex are detailed in Figure 3. Future research directed at understanding the role of these less frequently studied genes may reveal new therapeutic options, as well as new diagnostic and prognostic information.

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Review Article Telomerase in Acute Myeloid Leukemia: A Molecular Update on Diagnosis, Prognosis, and Treatment of and capacity to treat AML. Though the diagnostic and prognostic utility requires further investigation to actualize the potential of telomerase, numerous studies have illustrated the potential of a variety of new therapeutic strategies. It may be some time before these strategies are validated for patient use, but the future appears bright for the development of AML treatments with enhanced accuracy, efficacy, and above all, improved clinical outcomes.

Karam M, Thenoz M, Capraro V, Robin J.-P, Pinatel C, Lancon A, Galia P, Sibon D, Thomas X, Ducastelle-Lepretre S, Nicolini F, El-Hamri M, Chelghoun Y, Wattel E, Mortreux F. Chromatin redistribution of the DEK oncoprotein represses TERT transcription in leukemias. Neoplasia.

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Kim H-J, Choi E-J, Sohn H-J, Park S-H, Min W-S, Kim T-G. Combinatorial molecular marker assays of WT1, survivin, and TERT at initial diagnosis of adult acute myeloid leukemia. Eur J Haematol. 2013;91(5): 411–422.

Lee C-C, Lin C-P, Lee Y-L, Wang G-C, Cheng Y-C, Liu HE. (2010).

Meisoindigo is a promising agent with in vitro and in vivo activity against human acute myeloid leukemia. Leuk Lymphoma. 2010;51(5): 897–905.

Lingner J, Cooper JP, Cech TR. Telomerase and DNA end replication: no longer a lagging strand problem? Science. 1995;269: 1533–4.

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Montanaro L, Brigotti M, Clohessy J, Barbieri S, Ceccarelli C, Santini D, Taffurelli M, Calienni M, Teruya-Feldstein J, Trerè D, Pandolfi PP, Derenzini M. Dyskerin expression influences the level of ribosomal RNA pseudo-uridylation and telomerase RNA component in human breast cancer. J Pathol. 2006 Sep;210(1): 10-8.

Mosrati MA, Willander K, Falk IJ, Hermanson M, Höglund M, Stockelberg D, Wei Y, Lotfi K, Söderkvist P. Association between TERT promoter polymorphisms and acute myeloid leukemia risk and prognosis.

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Pettigrew KA, Armstrong RN, Colyer HA, Zhang SD, Rea IM, Jones RE, Baird DM, Mills KI. Differential TERT promoter methylation and response to 5-aza-2’-deoxycytidine in acute myeloid leukemia cell lines: TERT expression, telomerase activity, telomere length, and cell death.

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Yoon JS, Kim ES, Park BB, Choi JH, Won YW, Kim S, Lee YY. Anti-leukemic effect of sodium metaarsenite (KML001) in acute myeloid leukemia with breaking-down the resistance of cytosine arabinoside. Int J Oncol.

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Zhang X, Li B, de Jonge N, Björkholm M, Xu D. The DNA methylation inhibitor induces telomere dysfunction and apoptosis of leukemia cells that is attenuated by telomerase over-expression. Oncotarget. 2015;6(7): 4888–4900.

Competing Interests The authors have no competing interests.

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Aref S, El-Ghonemy MS, Abouzeid TE, El-Sabbagh AM, El-Baiom MA. Telomerase reverse transcriptase (TERT) A1062T mutation as a prognostic factor in Egyptian patients with acute myeloid leukemia (AML). Med Oncol. 2014;31(9): 158.

Bruedigam C, Bagger FO, Heidel FH, Paine Kuhn C, Guignes S, Song A, Austin R, Vu T, Lee E, Riyat S, Moore AS, Lock RB, Bullinger L, Hill GR, Armstrong SA, Williams DA, Lane SW. Telomerase inhibition effectively targets mouse and human AML stem cells and delays relapse following chemotherapy. Cell Stem Cell. 2014;15(6): 775–790.

Calado RT, Regal JA, Hills M, Yewdell WT, Dalmazzo LF, Zago MA, Lansdorp PM, Hogge D, Chanock SJ, Estey EH, Falcao RP, Young NS. Constitutional hypomorphic telomerase mutations in patients with acute myeloid leukemia. Proc Natl Acad Sci U S A. 2009;106(4): 1187–1192.

Capraro V, Zane L, Poncet D, Perol D, Galia P, Preudhomme C, BonnefoyBerard N, Gilson E, Thomas X, El-Hamri M, Chelghoun Y, Michallet M, Wattel E, Mortreux F, Sibon D. Telomere deregulations possess cytogenetic, phenotype, and prognostic specificities in acute leukemias.

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Deville L, Hillion J, Ségal-Bendirdjian E. Telomerase regulation in hematological cancers: A matter of stemness? Biochim Biophys Acta.

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Flandrin G. Classification of acute myeloid leukemias. Atlas Genet Cytogenet Oncol Haematol. 2002;6(3), 215-219.

Gessner A, Thomas M, Garrido Castro P, Büchler L, Scholz A, Brümmendorf TH, Soria NM, Vormoor J, Greil J, Heidenreich O. Leukemic fusion genes MLL/AF4 and AML1/MTG8 support leukemic self-renewal by controlling expression of the telomerase subunit TERT. Leukemia.

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Huret JL, Ahmad M, Arsaban M, Bernheim A, Cigna J, Desangles F, Guignard JC, Jacquemot-Perbal MC, Labarussias M, Leberre V, Malo A, Morel-Pair C, Mossafa H, Potier JC, Texier G, Viguié F, Yau Chun WanSenon S, Zasadzinski A, Dessen P. Atlas of genetics and cytogenetics in oncology and haematology in 2013. Nucleic Acids Res. 2013 Jan;41(Database issue):D920-4.

Corresponding Author: Carlos A. Tirado, ctirado@mednet.ucla.edu

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For In Vitro Diagnostic Use

Molecular Diagnostics

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

Training to Strive for Continuous Improvement

By Michelle Mah & Anna Haasen Genetic diagnostic services are becoming more widespread in todayâ&#x20AC;&#x2122;s evolving laboratory landscape, with molecular techniques expanding into different laboratory medicine disciplines. For example, virology and microbiology laboratories are turning to molecular assays to more efficiently detect or quantify particular species of pathogens. With the increased demand to provide accreditation to laboratories providing genetic services, the Institute for Quality Management in Healthcare (IQMH) invited me and my colleague in the field to attend a three-day assessor training course in downtown Toronto. This training not only addressed specific requirements that genetics laboratories must adhere to in order to provide clinicians with reliable and accurate results, but also examined the role of a quality management system (QMS) in a laboratory as a whole. Upon completion of the training, we gained a newfound appreciation for implementing quality assurance programs in the laboratory environment we each work in. Can you define QMS in one sentence?

laboratory demonstrates with respect to each IQMH requirement.

The requirements include all-inclusive guidelines categorized into eleven tenets of quality management: I. organizational structure; II. quality management system; III. physical facilities; IV. equipment, reagents, and supplies; V. pre-analytical process; VI. analytical process; VII. quality assurance; VIII. post-analytical process (reporting); IX. laboratory information system; X. safety and XI. point-of-care testing. IQMH also has discipline-specific requirements for different specialties. For example, a laboratory whose practice falls under Molecular Diagnostics (MD) has 62 additional unique requirements to conform to in order to achieve accreditation.

Accreditation in Molecular Diagnostics The discipline specific checklists are quite comprehensive and even include the design of workflow, as special consideration is needed for certain testing like PCR assays that may pose a risk of contaminating future reactions leading to false positive or false negative results. Consequently, at least six requirements in the IQMH accreditation framework relate to mitigating this risk including having physically separated spaces for pre-PCR and postPCR manipulations (IQMH requirement III.10 MD002) and using aerosol barrier tips while handling reagents and specimens prior to amplification (IQMH requirement VI.1 MD017).

The most recent updates of the accreditation requirements in December 2015 include systematically addressing the validation and use of next-generation sequencing (NGS) testing. For example, requirements for the analytical process state that NGS validations shall determine performance characteristics of the entire sequencing process including instruments and bioinformatics algorithms (IQMH requirement VI.2 MD082). Examples for the quality assurance of laboratory examination include descriptive requirements to determine a variety of NGS quality metrics like proper alignment of sequences, appropriate depth of coverage, and passing quality filters (IQMH requirement VII.1 MD101). And given the surplus of genetic information, requirements for the post-examination process include a policy regarding the disclosure of incidental or secondary findings (IQMH requirement VIII.1 MD104). The requirements are not designed to be prescriptive so the focus of the assessment is on whether policies and procedures set by individual laboratories not only reflect the work that is being performed, but that there is evidence for follow-up and improvement.

The general and discipline-specific accreditation checklists can be purchased in the IQMH online store and freely available for all enrolled accreditation participants.

The History of IQMH IQMH evolved from the Quality Management ProgramLaboratory Services (QMP-LS) in 2014. QMP-LS was responsible for providing proficiency testing in Ontario since the 1970s, and accreditation in the last decade in several Canadian provinces.

Rebranding to IQMH has allowed the organization to extend the number of services it provides to their clients and gain a broader international presence. The Institute is a not-for-profit corporation and currently manages three centres: Centre for Accreditation, Centre for Proficiency Testing and Centre for Education.

The Centre for Accreditation is responsible for administering laboratory assessments that evaluate a facilityâ&#x20AC;&#x2122;s QMS which includes the processes and procedures used in medical laboratory testing. Simply put, accreditation is the evaluation of laboratory processes to ensure quality and competence through conformance to an acknowledged standard (IQMH, 2015). When a lab is awarded a Certificate of Accreditation from IQMH, it means that international standards have been achieved. IQMH accreditation requirements strictly adhere to International Organization for Standardization (ISO) standards, specifically ISO 15189 (quality and competence in medical laboratories), but also integrate guidelines from the Clinical and Laboratory Standards Institute (CLSI) among other reputable sources. That being said, the requirements do not prescribe exactly how a laboratory should do certain practices, but rather the onus is on the laboratory to demonstrate compliance. After all, the goal of implementing a QMS is not to simply meet an accreditation standard but to strive for continuous quality improvement.

Perhaps unique to IQMH is the pool of knowledgeable, trained peer assessors that are deployed to evaluate laboratories participating in the Accreditation program. The peer assessors work in the field and have specific technical expertise in the discipline that they are evaluating. As such, the assessors are able to readily identify and classify the degree of conformance a

IQMH and its International Presence Perhaps more commonly, laboratorians have worked with the laboratory accreditation program offered by the College of American Pathologists (CAP). CAP accreditation also supports compliance with the clinical laboratory improvement amendments (CLIA)

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Molecular Diagnostics Training to Strive for Continuous Improvement

Sources

regulatory requirements to meet U.S. Federal regulations. The standard CAP accreditation is not equivalent to gaining accreditation from IQMH because the underlying foundations are not structured the same. The striking difference is that by implementing ISO 15189, the focus of IQMH is on continual improvement of operational systems. This operational system is based on four conceptual pillars: management responsibility, resource management, service realization, and measurement, analysis, and improvement. These four pillars all work in unison to support a functional QMS (IQMH, 2015). Since its inception, IQMH has worked with numerous laboratories worldwide to strengthen technical standards, with the goal of supporting medical laboratories in their endeavour to achieve continual excellence. Information about IQMH can be found on their website https://iqmh.org.

Accreditation Requirements and Guidance Information Molecular Diagnostics. Institute for Quality Management in Healthcare (IQMH), Centre for Accreditation. Version 6.1. 2015.

By Michelle Mah, MLT, MB(ASCP) CM & Anna Haasen, BSc, MLT (Genetics) Hamilton Regional Laboratory Medicine Program McMaster University Medical Centre

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Profiles and Perspectives

Column Editor: Hon Fong L. Mark, PhD, MBA, FACMG

Dr. Sheila Dobin Interviewed by Dr. Hon Fong L. Mark Sheila M. Dobin is a Joint Associate Professor in the Department of Molecular and Cellular Medicine/Pathology/Internal Medicine/ Pediatrics and Ob/Gyn for Texas A&M University, Health Science Center Medical School. She is also the Section Chief of the Cytogenetics Laboratory in the Department of Pathology at Scott and White Memorial Hospital in Temple, Texas, part of Baylor Scott & White Health. She has served as the founding Division Director of Genetics in the Department of Internal Medicine from 2008-2010 and is now a PhD Medical Geneticist within that department. Dr. Dobin was educated at the University of Texas at Austin in 1975 and subsequently received a PhD from the University of Texas Graduate School of Biomedical Sciences in 1981. She was board certified by the American Board of Medical Genetic (ABMG). Due to her interest in ethics, she serves on the IRB and ethics committee for Scott & White Memorial Hospital, and also the Baylor Scott & White System Ethics Council. She is also the current chair for the Pastoral Advisory Committee. Dr. Dobin’s research interests are multidisciplinary. They include family history, chromosomal abnormalities in leukemias, lymphomas and solid tumors.

Techniques practiced in her lab included classical cytogenetic banding, FISH (Fluorescence or Fluorescent in Situ Hybridization) techniques and more recently chromosomal microarray analysis.

Dr. Sheila Dobin

After initial coursework and qualifying exams, I started my project which looked at the effect of drug selfadministration in primates on brain wave activity. Due to sexual harassment and unethical practices of my graduate advisor (which was not talked about back in those days), I transferred to one of my other rotation laboratories.

The laboratory was the biochemical genetics laboratory in pediatrics under Dr. Rodney Howell.

I never really told him the details of why I needed to transfer, but just that there was a conflict with my advisor and I needed to leave the current lab. He was extremely kind during a difficult time in my life. I knew I wanted to finish my degree but was totally disillusioned and felt marginalized. The previous advisor told me that I would never make it as a professional in science due to me turning down his attempts. I was not the only graduate student that he did this to. Two of us turned him into HR of the institution he worked for. Nothing was done.

He continually lied about the number of animals in the experiment and the outcomes and if you did not go along you were chastised.

I credit Dr. Howell with saving my future career. At the time, I only had one undergraduate course in general genetics that was not taught very well. So, I started with coursework, again. I found a warm and welcoming environment in the pediatric department and a love of genetics. I graduated with my PhD just a year later than the four years I had originally planned in 1981.

My dissertation was “Zinc Concentration of Hair as an indication of Trace Metal Imbalances.” Dr. Scott introduced me to his work with Little People of America and the struggles and successes of individuals with skeletal dysplasias. Dr Charlene Moore introduced me to cytogenetics. Dr. Margery Shaw’s course is where my interest in genetics, law and ethics started and would grow into a future passion.

HFLM: Where were you in the 1960s?

SD:

In the 1960s I was in elementary and junior high school.

I lived in Houston, Texas. Back in those days I had a chemistry set and when someone asked what I wanted to do, I would answer be a teacher or mad scientist. We played a lot of school and I mixed up a lot of things that I probably should not have mixed.

I am the first generation to be born in the U.S. in my family. My parents and grandparents were Holocaust survivors who escaped Austria and Germany and lived in Shanghai for 10 years before they were allowed into the U.S. due to quota systems.

HFLM: Where were you in the 1970s?

SD:

I went to Jones High School for one year and then my family moved across town and I did the last two years at Westbury High School in Houston. I then attended the University of Texas at Austin. I graduated with a B.A. with honors and special honors in Plan II (Interdisciplinary Honors Program) in December rather than in May as I had planned. My concentration was in Psychology. My senior thesis was pheromones and thermoregulation.

Since I finished earlier than I had planned, I took some graduate coursework in psychology until I was accepted to graduate school. That fall, I began as a PhD student at the University of Texas Graduate School of Biomedical Sciences in Houston. After rotating through three labs, I decided that my interests were best aligned with psychopharmacology.

HFLM: As a bright young woman, obviously there were many options open for you. Why did you choose to study genetics?

SD:

It was totally an accident. I thought I would be doing research in psychology and in pharmacology. Sometimes the road you plan is not the road you end up on but it is a much better road for you. By the time I graduated, I was married to Loui. I knew that there were fewer biochemical labs than cytogenetics labs and I needed to have a job

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where a Jewish summer camp director and a geneticist could live. Dr Moore had introduced me to cytogenetics so I wanted to do a post-doc in cytogenetics. I met Dr Patricia Howard—Peebles at a Texas Genetic Society meeting and contacted her. Dallas would be a place that both of us could continue our careers. I worked on submitting a grant, and found a job teaching at a private high school until there was a spot in her lab. A week after I started teaching there was a position, but I had signed a contract and did not want to break the contract. So she allowed me to start the following year while I worked on the grant. I again lucked into an incredible mentor and friend. The experiences I was given were incredible. Dr Jan Friedman introduced me to the clinical world of genetics and Dr. Mary Jo Harrod proved to me that a PhD could do clinical work and see patients. Both Dr. Harrod and Dr Peebles were boarded as PhD Medical Geneticists. All three individuals encouraged me to be successful in multiple areas to make myself more marketable.

on life and what he had learned. He loved talking about his trials and told me about medical care in the country we were visiting. He talked to me about their insurance and the quality of their physicians and how many of his physicians he considered friends. The following day he was back fishing with us. The tumor turned out to be a Merkel cell carcinoma. I learned from him the real respect that patients can have with their physicians, if the physicians work to build those relationships.

HFLM: You have been a board certified clinical cytogeneticist for many years. Please tell us about your most interesting clinical case(s).

HFLM: As our readers probably noticed, behind every successful student scientist there is usually at least one patient teacher. Who are your mentors?

SD:

During my postdoctoral fellowship at UT Southwestern, the first patient I saw with Dr. Jan Friedman was a girl who was pregnant. She was in the hospital and I was to interview her and do a physical exam. The whole time I tried to engage her; she watched the cartoons on the TV. I could not get her interested in what I was doing or trying to tell her. When I told Dr. Friedman of my problem, he said what do you expect of a 12-year- old? I learned probably the most important lesson—always read a chart before walking into a room or signing out a report. If I had known her age, I would have engaged her in a different manner.

There have been multiple patients that taught me things. The two most memorable had babies with severe chromosomal problems. One taught me how resilient families can be in the face of adversity and how important a family support system is. That individual still comes with her child to visit me and talk to the techs in the lab.

The second was an individual whose baby would die. At the time I told them the abnormality should not occur in a future pregnancy. They said they would never become pregnant again. A few years later, she called and they were pregnant. An amnio and U/S was performed and everything looked good. A healthy baby was born and they were thrilled and remembered what I had told them and said it was a ray of hope. So I learned to never accept or say the word never.

I also learned that he was not always compliant with his physician’s orders. For him it was a choice and control.

He needed to control some things because others were completely out of his control. Patient compliance is multifactorial. We can make gains in some areas but not all. Patients have autonomy and it is not always up to us.

They should have some control and take charge, even when it may not be what we think is best. His spirit and resilience was amazing. I now tell his story to the students.

SD:

I am not sure that I have a most interesting clinical case. I find in running a clinical lab that I come across something interesting almost weekly. When I write a report, it has to have enough information for a clinician to be able to interpret that information for the patient. Reports should not be too long but they also can’t just give a result.

Physicians have little time to gain the background and look up unusual results. We need to help them help the patient. Therefore, I need to look up stuff constantly. So I am learning all of the time. I am never bored signing out cases. Even routine cases can teach you something new because every patient is different and things change over time. I keep a file of all the interesting cases, just in case I can ever have time to write them up. Unfortunately, you can no longer do some of the things we would kind of do on the side to further investigate these cases because of costs.

HFLM: What do you think are the most urgent scientific questions that need to be answered in our field in the coming years?

SD:

One of the lectures I give to medical students is on polycystic kidney disease. This past year, half a world away from where I live, on a fishing trip, I met a man who has lived with polycystic kidney disease. He had a strong family history. The next day he was having a tumor removed (which he showed me) and showed me a number of scars from having skin cancers removed. He had been married and had a family. He had a very positive outlook

I think trying to understand the meaning of gains that we see on chromosomal microarrays will be extremely important. We know so little about gain of function of genes. We can learn more about losses as well, but we really know very little about gains. Having this information will allow interpretation to become easier. I am excited about the possibility of having national databases with clinical information and lab information to help answer these types of questions.

HFLM: What challenges do you see clinical cytogenetics laboratories face in the near future?

SD:

There are multiple challenges. We have never seen a time like this before. I know that many have predicted the downfall of the cytogenetics lab. I don’t believe that

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will happen for the reasons that others state, which are advances in technology. I don’t believe that there will be one technology that will give us all of the answers.

I think the real threats have to do with non-scientific ones. There are problems with reimbursement and coding.

There are decreases in reimbursement coming down the pike. There will be DRGs for the clinical areas. There will be pay for performance for physicians in the clinic which will put pressure on them to deliver cost-effective care for better outcomes. We will need to prove that our results change outcome. That is a very difficult thing to do. Most of what we do involves laboratory developed tests. In addition, multiple groups have different coding schemes for reimbursement. There are the CPT codes that we are familiar with, but then some insurances want Z codes and LOINC codes. We still have the uninsured and I feel we have a duty to serve that population. So far, the duty has fallen to the hospital and academic labs. Some commercial labs have patient assistant programs, but having more labs with these types of programs could be beneficial. All of these pressures require more people to be involved which then costs more money and drives up the cost of testing while reimbursement is declining.

predict what will happen in the future, others have tried and failed. Instead, I try and learn from history and try and see opportunities that challenges present. I do believe there is room and a need for both types of laboratories.

While the labs may have different missions, both types of labs should work together for a common goal and work to try and agree on areas that neither is hurt. That is a vision that I hope we can strive toward. There is enough business for all.

We also hear that there is a limited pot of funds. That is true, but it depends on where you place your priorities for that pot of funds. How much does healthcare receive vs infrastructure vs defense etc. As many in business have said, “no margin, no mission.” Cytogenetic labs need to become lean and most of us have become as lean as possible. So the threat is one of reimbursement and policy choices. Then on top of that you have changes in technology and commercial companies driving the conversations and having the influence while smaller labs and academic labs have less influence. Commercial labs have stockholders. Those individuals want to make money on their investments either in the long-term or short-term.

Our retirement funds are made up of those stocks as well.

So we want them to make money.

HFLM: Do you see the study of chromosomes being completely replaced by Microarrays? Next Generation Sequencing?

Digital Molecular Pathology?

SD:

However, they are facing some of the same pressures and will have to make choices about research and how they charge. The small labs, not-for-profit labs and the academic labs are needed and serve a purpose. There is room for both. To my way of thinking those are the threats. We have to become advocates in the political and reimbursement arena for our patients. We have to always remember that there is a patient that is behind the lab test. We must be able to explain what we do in an elevator speech because all people now have short attention spans.

We must show our passion for what we do for our patients.

I do not. I don’t think any one technology can do everything well. What we have to remember is that there is a patient at the end of that laboratory test. We should aim to do the best possible test to answer a specific question. It may require more than one type of test. For example, in radiology you would not say you can either do a CT scan or an MRI or a PET, but you can never do all three. In some cases CT may be the best and the only test you need, but at other times you may need two or all three of them. I do think we need to be flexible and nimble and find new ways that we add value to a diagnosis, treatment or prognosis for a patient. We should be the ones counseling the complex chromosomal or molecular cases and we should be the ones helping physicians understand the complexity of the testing options.

HFLM: What advice would you have given a young and talented person today who is contemplating going into the field of human genetics?

SD:

HFLM: Where do you think laboratory genetics is heading? Do you see genetic testing being performed mainly in large commercial laboratories?

SD:

Competition provides an opportunity to learn and improve, but some of the things that I see today are harmful. They are not just harmful to the small and academic labs but to the patients and the physicians that care for them. I could give you multiple examples of false claims and pressures that sales individuals from some labs use to get physicians to use their tests that may or may not have been fully validated and tested. It is now similar to pharmaceutical sales. We are now having to teach physicians tactics to watch out for and to be able to call the experts before instituting a test. Pathologists and geneticists (for genetic tests) should be involved in helping individual physicians choose the right lab test for the patients question based on clinical information. It is a team effort.

I come from a fairly small laboratory. In a way, small makes you very nimble to adjust to changes. But in another way, small labs have greater financial pressure. It’s hard to

It is a great field. If you like to continually learn, it is the field for you. If you get bored easily, this is the field for you because it is constantly changing. You need to have an inquisitive mind, have lots of areas of interest which includes business, management, ethics, science, and research. You should be able to work in a team environment and be good at building relationships. Learn from every experience. Even the bad experiences have something to teach you. The tough or changing times also have something to teach you. I have learned from bad and

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tough experiences, including my time spent in graduate school in pharmacology. It is amazing how often I draw on something that I learned during my coursework and how I have learned to stand up for what is ethical and right.

References Mark HFL. Cytogenetics in the 1960s. J Assoc Genet Tech.

1999;26: 72-73.

Hon Fong L. Mark, PhD, MBA, FACMG, Editor of the cytogenetics textbook, â&#x20AC;&#x153;Medical Cytogenetics,â&#x20AC;? is President of KRAM Corporation, a small consulting firm specializing in medical genetics, grant review and scientific review administration. Dr. Mark is a Clinical Cytogeneticist board-certified by the ABMG (1993), and was formerly Director of Cytogenetics and Clinical Professor at the Lifespan Academic Medical Center/Brown University in Providence, RI, Director of Human Genetics, RIDOH, also in Providence, RI, and Director of the Cytogenetics Department at Presbyterian Laboratory Services/Novant Health in Charlotte, N.C. She was recruited to the Boston University School of Medicine as Director of the Cytogenetics Laboratories (and Clinical Professor of Pathology and Laboratory Medicine) in 2004, a position from which she resigned in 2007. This column is dedicated to the technologists and laboratory directors in all the cytogenetics laboratories in the U.S. and throughout the world whom she had the good fortune of meeting through the years.

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Brain Tickler

Brain Tickler Summary (see inside front cover)

KARYOTYPE RESULTS: 46,XX.ish der(X)t(X;Y)(p22.3;p11.3)(DXZ1+,SRY+) Twenty cells were 46,XX in a phenotypically male patient. FISH using a probe for the centromere of X (green) and the SRY locus (red) confirmed that the SRY was located on the end of one X short arm. The patientâ&#x20AC;&#x2122;s features of short stature and hypogonadism are consistent with the XX male syndrome. There is some overlap in XX male and Klinefelter phenotypes, but the short stature usually distinguishes between the two syndromes.

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Continuing Education Opportunities

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

Test Yourself #3, 2016 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 42, Number 2, Second Quarter 2016 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 is on page 123 of this issue. 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 9, 2016.

Passing score is 87% or 21 out of 24 questions answered correctly. Compiled by Doina Ciobanu and Sally Kochmar.

The following questions are from Hiral P et al. CML in Chronic Phase Abnormalities: A Case Report. J Assoc Genet Technol. 2016;42(2): 6-8.

The following questions are from Leel P et al. An adult male presenting with concurrent plasma cell myeloma involving a CCND1-IGH translocation and chronic myelogenous leukemia with a variant (9;22) translocation. J Assoc Genet Technol.

2016;42(2): 9-12.

1. All of the following are considered major-route chromosomal aberrations, except: a. b. c. d.

5. Plasma cell myeloma:

Isochromosome 17 Trisomy 7 Additional Philadelphia chromosome Deletion of the Y chromosome

I. is characterized by complex chromosomal aberrations.

II. is characterized by an uncontrolled proliferation of a single plasma cell clone.

III. has clones with low proliferative activity in culture.

IV. is associated with a Philadelphia chromosome.

2. Deletion of 13q14 has been strongly linked to: I. CLL II. AML III. Myeloproliferative disorders IV. Myelodysplastic syndromes a. b. c. d.

a. b. c. d.

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

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

6. The most frequently observed translocation in plasma cell myeloma is t(11;14)(q13;q32). a. true b. false

3. Clonal evolution is defined as occurrence of additional chromosome abnormalities besides the Ph- chromosome.

7. All of the following are true except:

a. true b. false

a. t(11;14) is found in about 15% of plasma cell myeloma patients. b. CML is associated with the presence of the Philadelphia chromosome. c. The Ph- chromosome is detected in about 40% of patients with CML. d. Variant deletions on der(9) are present in 9-15% of CML cases.

4. Choose the incorrect statement: a. The degree of genomic instability is proportional to the level of BCR-ABL kinase activity. b. CE can occur in any phase of CML. c. CE frequency increases from 30% in accelerated phase to 60% in blast crisis. d. This article presents a novel secondary change with t(9;13)(q34;q12-13).

8. The case presented in this article: I. was an 87-year-old male with anemia and monocytosis.

II. was positive for CCND1 and IGH fusion in 92% of nuclei.

III. revealed a t(9;22)(q34;q11.2) in 40% of metaphases.

IV. had a mildly hypercellular marrow. a. b. c. d.

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

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Continuing Education Opportunities

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

9. The concurrence of plasma cell myeloma and chronic myelogenous leukemia is very rare with less than 20 cases reported.

15. All of the following are true, except: a. The FDA will be required to review about 60,000 LTDs. b. Harmonization of lab testing is an old topic. c. Patient Access to Medicare Act was a topic reviewed at the symposium. d. Four main topics were discussed this year.

a. true b. false The following questions are from Mark HLF. Dr. Rachel Burnside. J Assoc Genet Technol. 2016;42(2): 14-16.

The following questions are from Ma M. A Journey of Continuous Learning Through Teaching. J Assoc Genet Technol. 2016;42(2): 19.

10. According to this article, it took Dr. Burnside four years to complete her undergraduate degree.

16. According to this article, Sanger sequencing has a limit of detection of:

a. true b. false

a. 20% b. 25% c. 2% d. 15%

11. All of the following are true except: a. Dr. Burnside began her undergraduate studies at Texas A&M. b. She graduated from University of Houston. c. She was a serious student at the beginning of her undergrad studies. d. She decided to specialize in cytogenetics as a result of working in a cytogenetics Lab.

17. What happens once results are forwarded to annotation specialists?

12. Where did Dr. Burnside complete her postdoc fellowship? a. University of Kentucky b. LabCorp c. University of Houston d. UAB in Birmingham, Alabama

I. II. III. IV.

Benign polymorphisms are determined Functional consequences are established Variant interpretation is being performed Variants of unknown significance are being determined

a. b. c. d.

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

The following questions are from Crawford-Alvares J. 2016 ASCLS Legislative Symposium Overview. J Assoc Genet Technol. 2016;42(2): 17.

The following questions are from Garcia-Heras J. The Molecular Revolution of Genomic Editing with Programmable Nucleases. J Assoc Genet Technol. 2016;42(2): 22-27.

13. This year’s ASCLS symposium:

18. Conventional gene targeting has been used to inactivate genes via homologous recombination.

I. was held in Alexandria, VA. II. discussed four main topics.

III. included speakers from ASCLS, CLMA, ASCP and AMP. IV. was held in March. a. b. c. d.

a. true b. false 19. RNA-guided nucleases adapted from a microbial adaptive immune defense system from bacteria first became available in:

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

a. 1994 b. 2016 c. 2012 d. 2013

14. According to this article, the employment outlook for clinical laboratory personnel shows a high percentage of retiring workers and a small percentage of graduates entering the work field. a. true b. false

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Continuing Education Opportunities

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

22. The first example of genome editing restoring normal function in human genetic disease was:

20. What are some of the advantages of hESCs and iPSCs cells?

I. II. III. IV.

They grow in culture for long periods of time.

They retain a normal karyotype.

They retain pluripotency.

They allow molecular manipulation of their DNA in the lab.

a. b. c. d.

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

a. the correction of the Duchenne Muscular Dystrophy gene. b. factor IX cDNA for hemophilia B. c. the correction of the L2R receptor gene that is mutated in X-linked immunodeficienc.y d. the correction of the CFTR locus.

23. The first human genome editing in embryos was reported in: a. 2013 b. 2014 c. 2015 d. 2016

21. Genome editing with programmable nucleases: I. II. III. IV.

can inactivate deleterious mutations. corrects defective genes. adds therapeutic genes into specific sites. supersedes limitations of standard gene therapy.

a. b. c. d.

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

24. The most noteworthy therapeutic success with genome editing has been its application to patients affected with: a. Muscular dystrophy b. Cystic fibrosis c. AIDS d. Hemophilia B

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

Answer Sheet 1.____

13.____ 14.____ 15.____ 16.____ 17.____ 18.____

7.____ 8.____ 9.____ 10.____ 11.____ 12.____

2.____ 3.____ 4.____ 5.____ 6.____

Answers to Test Yourself #2, 2016

Please Print Clearly 19.____ 20.____ 21.____ 22.____ 23.____ 24.____

1.

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4.

5.

6.

Passing Score: (passing score is 21/24 or 87%) 7. d 13. a 8. a 14. b 9. b 15. d 10. c 16. a 11. a 17. d 12. c 18. c

c d a c d c

The Association of Genetic Technologists Name:

19.

20.

21.

22.

23.

24.

a a d d a d

CEU REPORTING FORM

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(check appropriate box) Address

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T ES T YOURSE L F , 3 r d QUAR T ER 2015 6 J P544 Qtr

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___________________ Date

Continuing Education Opportunities 2015-2016 AGT Education Committee Regional Representatives If you have questions or experience difficulty locating your representative, please contact the AGT Education Director (see page 162 for address).

Great Lakes

Mountain States

Northern Pacific

(Illinois, Indiana, Michigan, Minnesota, Ohio, Wisconsin)

(Arizona, Colorado, Idaho, Montana, New Mexico, Utah, Wyoming)

(Alaska, Northern California, Oregon, Washington)

Audra Birri Cincinnati Children's Hospital Cytogenetics Laboratory TCHRF Room 1003 3333 Burnet Ave. Cincinnati, Ohio 45229 513-636-4474 513-636-4373 FAX Audra.Birri@cchmc.org

Great Plains (Arkansas, Iowa, Kansas, Missouri, Nebraska, North Dakota, Oklahoma, South Dakota) Julie Carstens Cytogenetics Laboratory Munroe-Meyer Institute 985440 Nebraska Medical Center Omaha, NE 68198-5440 402-559-4965 402-559-7248 FAX jcarsten@unmc.edu

Mid-Atlantic (Delaware, District of Columbia, Maryland, New Jersey, Pennsylvania, Virginia, West Virginia) Deborah Ketterer Pittsburgh Cytogenetics Laboratory Magee-Womens Hospital of UPMC 300 Halket St., Room 1229 Pittsburgh, PA 15213 412-641-6688 412-641-2255 FAX dmketterer@verizon.net

Uma Van Roosenbeek 574 W. Vekol Ct. Casa Grande, AZ 85122 520-509-1130 umaswati@gmail.com

New England (Connecticut, Maine, Massachusetts, New Hampshire, Rhode Island, Vermont) Gail Bromage DIANON Systems, Inc. Cytogenetics Laboratory 1 Forest Parkway Shelton, CT 06484 800-328-2666 203-380-4554 FAX gbromage@sbcglobal.net

New York State (New York) Laura Benz 4756 Black Warrior Road Truxton, NY 13158 607-842-6009 benzl@upstate.edu

Christine Donovan University of Washington Medical Center Cytogenetics Laboratory Box 356100 1959 NE Pacific Seattle, WA 98195 206-598-4489 206-598-2610 Fax chrisd19@u.washington.edu

Southeast (Alabama, Florida, Georgia, Kentucky, Louisiana, Mississippi, North Carolina, South Carolina, Tennessee) Joan Bishop Greenwood Genetics Center 106 Gregor Mendel Circle Greenwood, SC 29646 864-388-1719 864-941-8133 FAX jtbishop@ggc.org

Southern Pacific (Hawaii, Nevada, Southern California) Shree Merchant 26381 Silver Creek Dr. San Juan Capistrano, CA 92675 949-231-0327 shreegoradia@gmail.com

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Texas (Texas) Su Yang Cytogenetics Laboratory U.T. M.D. Anderson Cancer Center 1515 Holcombe Blvd.

Unit 350 Houston, TX 77030 713-792-6330 713-745-3215 FAX suyang@mdanderson.org

Canada Michelle Mah Advanced Diagnostics Lab Princess Margaret Cancer Centre University Health Network 610 University Ave. Toronto, Ontario Canada M5G 2M9 416-946-4501 ext.5036 michelle.j.mah@gmail.com

Non-U.S./Canada Sally Kochmar Pittsburgh Cytogenetics Laboratory Magee-Womens Hospital of UPMC 300 Halket St., Suite 1233 Pittsburgh, PA 15213 412-641-4882 412-641-2255 FAX skochmar@upmc.edu

At-Large Doina Ciobanu 487 Burns Rd. Fair Haven, VT 05743 cdoini@gmail.com

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 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

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 58 – General Content Area: Solid Tumor and FISH–2007 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 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 AutismAssociated Segmental Maternal Heterodisomy of the Chromosome 15q1113 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

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Continuing Education Opportunities Glycaemic Control in the Majority of Insulin-Treated Patients

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 miR-127 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.

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 85 – General Content Area: Esophageal Cancer–2010

READING LIST 91 – General Content Area: Hutchinson-Gilford Progeria Syndrome–2011

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

2. Combining Next-Generation Sequencing

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.

READING LIST 95 – General Content Area: Cell Death–2011 1. Hypoxia induces autophagic cell death in apoptosis-competent cells through a mechanism involving BNIP3.

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Continuing Education Opportunities 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.

3. Evidence for Three Loci Modifying Ageat-Onset of Alzheimer’s Disease in EarlyOnset PSEN2 Families.

READING LIST 96 – General Content Area: Genetic Associations of Cerebral Palsy– 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.

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 1. Genetics of Alzheimer Disease.

2. New mutation in the PSEN1 (E120G) gene associated with early onset Alzheimer’s disease.

READING LIST 101 – General Content Area: Multiplex PCR and Emerging Technologies for the Detection of Respiratory Pathogens–2011

neuronal trigger for inflammation and Alzheimer’s pathology.

3. The inflammasome: a caspase-1activation platform that regulates immune responses and disease pathogenesis.

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 102 – General Content Area: Single Nucleotide Polymorphism (SNP) Array Analysis–2011

READING LIST 107 – General Content Area: HERV-K and Its Correlation With Melanoma Cells–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.

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 103 – General Content Area: Research of BRAF Gene Related to Cancer–2011

READING LIST 108 – General Content Area: Refractory Myeloma–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 105 – General Content Area: Inflammasome Activation by Proteins–2011 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

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".

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.

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Continuing Education Opportunities 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 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

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

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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Continuing Education Opportunities

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Life Sciences

scientists enabling scientists.

Association Business

Message from the President Greetings all you genetic technology professionals!

Every time I return home from an AGT annual meeting I say, “Was that great, or what?” I said that every year, except for maybe two or so, for 35 years. But, I think this year, in Anaheim, California, was the greatest. Not because I was the president. Certainly not. It was because of the people who were there. The speakers, including a Nobel laureate, were all stellar. The Gordon Dewald Lecturer was spot on. The student presenters were very impressive. All the speakers were great. The audience was even greater! I felt an excitement and enthusiasm that I had rarely felt before.

Most of the attendees were AGT members already, so my spiel about joining was probably not necessary. Sorry…

Except that many people join just to get the meeting discount, so to those of you who are thinking about not renewing your membership next year because it is not your turn to go to the annual meeting, think again! Your continued support for AGT enables the organization to stay strong and relevant. More educational opportunities can be developed, better annual meetings can be planned, communication with your peers can be enjoyed, and the latest in genetic technology can be absorbed from AGT publications. Belonging to professional organizations is what professionals are expected to do.

Planning for next year’s annual meeting began soon after this year’s meeting ended, with an enthusiastic bunch of planners hot on the trail of new and better ideas for structuring the meeting. Your responses to the meeting evaluation have been heeded. There will be more opportunities for face-to-face interaction with experts and peers, so make a list of things you need help with, and don’t be shy about sharing what you do well with your fellow technologists. The number of talks with a molecular genetics theme will be increased to be about equal that of cytogenetics topics. I encourage the people who work mostly or entirely in cytogenetics to partake of the molecular genetics content, and the molecular genetics folks to partake of the cytogenetics topics. Both methodologies are extremely useful for making a correct diagnosis for the patient. A comprehensive understanding of all diagnostic strategies makes for a well-rounded and inspired genomics professional. The Trivia Game will also make a repeat appearance, so bone up on your genetics facts.

I don’t want to give away too much, but the keynote speaker for the St. Louis meeting will be dynamite! Mark your calendars, and RENEW YOUR MEMBERSHIP! Communication about the meeting will be all over social media, including Facebook and Twitter, so stay tuned! Cheers!

Pat Dowling, AGT President

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Association Business

Association of Genetic Technologists 41st Annual Business Meeting Minutes Saturday, June 25, 2016 Hyatt Regency Orange County Orange County, California 7:05 a.m. – 7:55 a.m.

AGT President Patricia Dowling called the 41st Annual Business Meeting to order at 7:05 a.m. on Saturday, June 25.

President Patricia Dowling introduced the newly elected and appointed members of the Board of Directors and Council of Representatives for 2016-2017:

• President Patricia Dowling thanked members for their participation and continued support. She also thanked the exhibitors and sponsors of the 41st Annual Meeting.

• Annual Meeting Co-Director – Christina Mendiola The outgoing members of the Board of Directors and Council of Representatives were recognized for their service and presented with plaques.

• The current, 2015-2016, AGT Board of Directors were introduced: President-Elect – Jason Yuhas, Secretary-Treasurer – Denise Juroske-Short, Education Director – Sally Kochmar, Public Relations Director – Ephrem Chin, Annual Meeting Director – Adam Sbeiti and Annual Meeting Co-Director – Jennifer Sanmann.

• Adam Sbeiti – 2016 Annual Meeting Director and 2014-2015 Co-Director • Jennifer Sanmann – 2017 Annual Meeting Director was presented with the Annual Meeting traveling plaque.

• Members present at the business meeting RESOLVED to approve the 40th Annual Business Meeting Minutes.

President Patricia Dowling addressed the membership with final remarks.

• President Patricia Dowling reported to the membership on the items completed in the past year and the initiatives taken at the Board of Directors Meeting.

There being no further business to come before the AGT membership, President Patricia Dowling adjourned the 41st Annual Business Meeting at 7:55 a.m. Pacific Standard Time.

• Secretary/Treasurer, Denise Juroske-Short, presented a brief overview of the current financial condition of the association and of the approved 2016-2017 Operations budget.

Respectfully submitted,

• Members were invited to ask questions about the Board initiatives, budget or any related organizations. No questions were asked.

Denise Juroske-Short Secretary/Treasurer

President Patricia Dowling introduced the following Council of Representatives members and asked them to provide brief reports: • BOC – Representatives: Helen Bixenman • FGT – Representative: Robin Vandergon • NAACLS – Representative: Peter Hu • CAP – Representative: Jun Gu • CCCLW – Representative: Hilary Blair

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Association Business

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 41st 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.

ADS Biotec, Inc. Affymetrix, Inc. Agilent Technologies, Inc. Applied Spectral Imaging, Inc. BioDot Inc. BioView USA Inc. Chroma Technology Enzo Life Sciences Foundation for Genetic Technology Genial Genetics/Rainbow Scientific, Inc. Innopsys, Inc. Irvine Scientific Leica Biosystems MetaSystems Oxford Gene Technology Percival Scientific Personal Genome Diagnostics Promega Psyche Systems SciGene Staff Icons, LLC STEMCELL Technologies, Inc. Thermotron WaveSense

Gold Sponsors

Silver Sponsors

AGT would like to express a special thanks to the following individuals who spent countless hours of planning and hard work making this 41st Annual Meeting a great success.

Pat Dowling, AGT President Adam Sbeiti, Annual Meeting Director Jennifer Sanmann, Annual Meeting Co-Director

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Association Business

AGT 41st Annual Meeting

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Association Business

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Association Business

Awards Presented at the AGT 41st Annual Meeting

Kristina Regmi, winner of the AGT Student Abstract Award, presented her submission, Prognostic Impact of MYC Rearrangement in Plasma Cell Myeloma, as a platform presentation during the meeting.

She is pictured with AGT President, Patricia Dowling.

The AGT Margaret Barch Memorial Workshop Award was presented to Diane Pickering, University of Nebraska Medical Center, for her presentation, Clinical Microarray Testing – Current Applications and Methodology.

Daniel Van Dyke, Mayo Clinic, this year’s Gordon W. Dewald Lecturer, pictured with Patricia Dowling, AGT President

Pamela Althof, University of Nebraska Medical Center, receives the Florence Dowling Genome Award from Patricia Dowling, AGT President.

Dr. Dowling sponsors this award in honor of her mother.

Adriel Kim receives the EXCEL Award from award sponsor representative, Aysel Heckel of Oxford Gene Technology (OGT).

Eduardo Zavala receives the Barbara J. Kaplan Scholarship from Helen Bixenman, who was a close friend of Barbara Kaplan.

Carlos Alonso Muñoz receives the Outstanding Technologist Grant from FGT Secretary, Denesha Criswell. This award is sponsored by Leica Biosystems.

The Best Poster Award for the poster, Issues Relevant to Fish Semi-Automated Spot Counting System was presented to Ken Sterns, Angela Teng, Jiaqi Chen, Cristina Garcia, Roberto Guajardo, Crystal Lee, Dominique Cline, Victoria Nettles, Sylvia Wong, Ming Zhao and Jun Gu from the University of Texas, M.D. Anderson Cancer Center. Several authors are pictured here with Irvine Scientific representative, Lan Truong. The best poster award is sponsored by Irvine Scientific.

Not pictured: Charlie Smith was the recipient of the New Horizons Award sponsored by Rainbow Scientific.

Not pictured: Amy Groszbach, Mayo Clinic, winner of the Joseph Waurin Excellence in Education Award, was unable to attend the meeting. Jason Yuhas accepted the award on Amy’s behalf. Jim Waurin sponsors this award each year in honor of his father.

Not pictured: Sarah Ahlong Kim was the winner of the Best Platform Presentation Award for her presentation, Efficacy of Plasma Cell Enrichment Technique in Multiple Myeloma: An Institutional Experience. Co-authors were Rosemarie Schmidt, Haisu Yang and Renu Bajaj.

OGT won Best Exhibit Booth as chosen by AGT Annual Meeting attendees.

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Association Business

2016 AGT Award Winners Outstanding Achievement Award

Sponsored by Martha Keagle Because this year’s Outstanding Achievement Award winner was unable to attend the Annual Meeting, AGT will honor two winners in 2017.

Student Research Award

Kristina Regmi, University of Texas, Houston, TX

AGT Margaret Barch Memorial Workshop Presentation

Diane Pickering, Human Genetics Laboratory, University of Nebraska Medical Center, Omaha, NE

Dr. Gordon W. Dewald Lecture

Daniel Van Dyke, Mayo Clinic, Rochester, MN

2016 FGT Award Winners Florence Dowling Genome Award

Sponsored by Pat Dowling Pamela Althof, Human Genetics Laboratory, University of Nebraska Medical Center, Omaha, NE

Excel Award

Sponsored by Oxford Gene Technology Adriel Kim, University of Texas, M.D. Anderson Cancer Center, Houston, TX

New Horizons Award

Sponsored by Rainbow Scientific Charlie Smith, NeoGenomics, Irvine, CA

Joseph Waurin Excellence in Education Award Sponsored by Jim Waurin Amy Groszbach, Mayo Clinic, Rochester, MN

Barbara J. Kaplan Scholarship

Eduardo Zavala, University of Texas, M.D. Anderson Cancer Center, Houston, TX

Outstanding Technologist Grant

Sponsored by Leica Biosystems Carlos Alonso Muñoz, Laboratorios Mendel, Morelia Michoacan, Mexico

Best Poster Award

Sponsored by Irvine Scientific ISSUES RELEVANT TO FISH SEMI-AUTOMATED SPOT COUNTING SYSTEM Ken Sterns, Angela Teng, Jiaqi Chen, Cristina Garcia, Roberto Guajardo, Crystal Lee, Dominique Cline, Victoria Nettles, Sylvia Wong, Ming Zhao, Jun Gu, University of Texas, M.D. Anderson Cancer Center, Houston, TX

Best Platform Presentation Award

EFFICACY OF PLASMA CELL ENRICHMENT TECHNIQUE IN MULTIPLE MYELOMA: AN INSTITUTIONAL EXPERIENCE Sarah Ahlong Kim, Rosemarie Schmidt, Haisu Yang, Renu Bajaj, LabCorp Specialty Testing Group, New York, NY

Best Exhibitor Booth Award Oxford Gene Technology

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Platform Abstracts, AGT 2016

AGT 41st Annual Meeting

June 23–25, 2016 Hyatt Regency Orange County — Orange County, California

Platform, Poster and Student Poster Presentations from the 41st Annual Meeting The Journal of the Association of Genetic Technologists proudly publishes the platform, poster and student poster abstracts presented at the 41st AGT Annual Meeting in Orange County, California. 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 41st Annual Meeting Final Program and has not been edited further.

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

MORPHOLOGICALLY DIAGNOSED SECONDARY ACUTE LYMPHOBLASTIC LEUKEMIA WITH MYELOID NEOPLASIA ASSOCIATED CYTOGENETIC ABNORMALITIES FOLLOWING LENALIDOMIDE TREATMENT FOR MULTIPLE MYELOMA Melinda A. Claydon, BA, CG(ASCP); Annemarie W. Block, PhD; Sheila N. J. Sait, PhD, Roswell Park Cancer Institute

Therapy related acute leukemia is a heterogeneous disease. Recent reports based on small numbers have consistently shown an increased incidence of hematologic malignancies in patients treated with the immunomodulatory drug Lenalidomide vs placebo in patients with multiple myeloma (MM) and chronic lymphocytic leukemia (CLL). Myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) have been reported to be the more common types of secondary leukemias with chromosome 5 and 7 abnormalities. Secondary acute lymphoblastic leukemia (ALL) is reported much less frequently than therapy-related AML. Most cases develop a secondary MDS or AML, with a predominance of cases showing deletions of 5q and 7q. When present, secondary ALL is associated with 11q23 abnormalities, 9p rearrangements, and t(9;22). Secondary MDS/AML with deletions of chromosomes 5q and 7q have been reported in patients post Lenalidomide therapy. Here we report 3 patients with a diagnosis of MM and CLL who developed a secondary leukemia post Lenalidomide therapy. All three had a pathology diagnosis of ALL, but cytogenetically had myeloid associated abnormalities including del(20q) in 2/3 patients and 7q abnormalities in 2/3 patients. The patients include 1 male and 2 females with a median age of 66 years. The incidence of myeloid associated chromosome abnormalities in patients with morphologically diagnosed secondary ALL raises interesting questions about the lineage of these cells. Identification of more of these cases would help to elucidate the underlying mechanism of transformation to secondary acute leukemia.

QUANTIFICATION OF LYSOSOMAL STORAGE DISEASE SPECIFIC URINARY OLIGOSACCHARIDES FOR POTENTIAL TREATMENT MONITORING

Teresa Thompson, BS, MB(ACSP)CM; Rongrong Huang, PhD; Allison Cason, BS; Laura Pollard, PhD, FACMG; Tim Wood, PhD, FACMG, Greenwood Genetic Center Our laboratory has developed a UPLC-MS/MS assay utilizing reducing-end labeling of urinary free oligosaccharides (FOS) to screen for various lysosomal storage diseases (LSDs), predominantly the glycoproteinoses, which result from impaired degradation of glycoproteins in the lysosome. Urine samples are derivatized by butyl-4-aminobenzoate (BAB) using a modification of a previously reported method. An amount of urine corresponding to 30ug of creatinine is used for each sample, to which 1ug of ΔHexA-GlcNAc as the internal standard (IS) is added prior to BAB derivatization. After derivatization, a Sep-Pak Aminopropyl (NH2) cartridge was used for sample cleanup. Eight FOS compositions were selected and detected in SRM mode, and the semi-quantification was conducted by calculating the peak area ratio of each FOS versus IS. Total ion chromatogram (TIC) analysis of the eight FOS is used to create a fingerprint for the detection of at least eight LSDs, including aspartylglucosaminuria (AGU) using the native oligosaccharide, which is not amenable to end-labeling. We analyzed 39 urine samples from patients with one of eight LSDs: aspartylglucosaminuria (n=9), fucosidosis (n=2), alpha-mannosidosis (n=16), beta-mannosidosis (n=1), beta-galactosidase deficiency (n=4), Sandhoff disease (n=3), sialidosis (n=2) and galactosialidosis (n=2), which were collected either as part of the Glycoproteinoses Natural History Study or through routine diagnostic testing. Age-specific normal ranges were developed for each FOS using 50 samples from unaffected controls. The increased abundance of the disease-specific FOS in affected individuals compared to age-matched controls ranged from 3 – 150 fold, with aspartylglucosaminuria (average 33-fold), fucosidosis (average 142-fold), betagalactosidase deficiency (average 50-fold) and galactosialidosis (average 107-fold) showing the widest dynamic range. The reproducibility of the semi-quantitative analysis was demonstrated using two samples, which each displayed approximately 6% relative standard deviation (%CV) for IS peak area (n=8) and <20% CV for each FOS/IS peak area ratio, with the exception of Hex1HexNAc1 for beta-mannosidosis (35% CV), which has the earliest retention time (0.42 minutes). Treatments are not currently available for the glycoproteinoses, but research is currently underway. Preliminary evidence that this semi-quantitative analysis of urine FOS can be used for biomarkers in treatment monitoring was obtained using patients who have received bone marrow transplants. The Hex3HexNAc1 level in a treated alpha-mannosidosis patient was normal and the Hex3HexNAc2Fuc1 level in a treated Fucosidosis patient was only 8-fold elevated compared with >100 fold elevated in untreated patients. Finally, a beta-mannosidosis patient showed a 50% decrease in his Hex1HexNAc1 level after receiving a bone marrow transplant. Future work will involve developing true quantitative assays for individual FOS to improve the accuracy and reproducibility of the results for use in clinical trials. Overall, our data demonstrates that the use of LCMS/MS for urine FOS analysis allows for both diagnostic screening and quantitative analysis for treatment monitoring.

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Platform Abstracts, AGT 2016 MEDIA AND HARVEST METHODOLOGY COMPARISONS FOR CHROMOSOME ANALYSIS OF ACUTE LYMPHOCYTIC LEUKEMIA Angelique Jermac, CG(ASCP)CM; Lane Wilcox; Daniel L. Van Dyke, PhD, Mayo Clinic

Acute lymphocytic leukemia (ALL) is a neoplastic disease that is typified by colonization and expansion of leukemic cells throughout the bone marrow. This disease represents approximately 80% of childhood leukemias in the United States. Genetic aberrations contribute to increased proliferation, prolonged survival and/or lack of differentiation in hematopoietic progenitor cells. Many of these genetic aberrations can be observed microscopically through cytogenetic testing. One significant challenge facing many cytogenetics labs is finding well-spread, high-quality metaphase chromosomes that show the genetic abnormalities in pediatric ALL. We compared two different media and two different culture and harvest methodologies in an effort to produce a higher mitotic index while maintaining chromosome length in abnormal cells. We tested our method, using commercially available CHANG Medium BMC, with a new harvest method that utilized media made with RPMI 1640 with Glutamax, supplemented with supernatant from a urinary bladder carcinoma cell line. Data were collected through blinded analysis for mitotic index, chromosome length and presence of abnormal cells. The first phase of the study revealed that chromosome length was increased with the new method using the supplemented media. Mitotic index and metaphase quality were slightly better. Mitotic index for our clinical method was 0.07 and 0.4 for the new method. Metaphase quality comparison was assessed using a quality rating scale, with 1 being poor, 2 being below average, 3 being average, 4 being good and 5 being excellent. Our standard protocol rated at 2.1 and the new method rated at 2.7. However, in the second phase of the study, an abnormal clone was identified in 7 of 20 patients grown using CHANG BMC, but in only 2 of these 7 patients with the supplemented media. One possible explanation for the less favorable performance of the supplemented media may be a generally lower ability to stimulate the abnormal cells to proliferate. We were not able to show improved ability of the new media to support growth of cytogenetically abnormal ALL cells. A third phase of the study compared our standard harvest method with the new harvest method, both cultured in CHANG Medium BMC. An abnormal clone was identified in 3 of 6 patients using our standard protocol, but in only 1 patient with the proposed method. While we cannot justify a method change, we will continue investigations to find an approach that produces higher mitotic indices, better metaphase quality and longer chromosomes in the chromosomally abnormal ALL cells.

EFFICACY OF PLASMA CELL ENRICHMENT TECHNIQUE IN MULTIPLE MYELOMA: AN INSTITUTIONAL EXPERIENCE

Sarah Ahlong Kim, CG(ASCP)CM; Rosemarie Schmidt CG(ASCP)CM, DLM CM; Haisu Yang, PhD; Renu Bajaj, PhD, CG(ASCP)CM, FACMG, Integrated Oncology, LabCorp Specialty Testing Group Description of the Experiment: Plasma Cell Neoplasm/ Multiple Myeloma (PCN/MM) is a bone marrow based disease associated with an overproduction of M protein by abnormal plasma cells in serum or urine. Disease stratification for the outcome, targeted therapy, and risk for drug resistance has been dependent upon molecular cytogenetics techniques including cytogenetics and FISH (Fluorescence in situ hybridization). However, this has always been a challenge to target the specific cell type (plasma cells) in PCN/ MM. Therefore ‘The International Myeloma Workshop Consensus Panel’ recommends performing FISH for the multiple myeloma prognostic markers on the CD138+ enriched samples. In this study, we would like to share our experience with this enrichment technique using the autoMACS™ cell separator. The autoMACS™ Separator from Miltenyi Biotec is a benchtop instrument for highspeed magnetic cell sorting of multiple samples. The compatibility of MACS technology with CD138 MicroBeads allows enriching CD138 positive plasma cells. Utilizing this design to enrich plasma cells for testing has enhanced our ability to detect genetic abnormalities at a concentrated level. Statement of Result: We present here data for enriched multiple myeloma (eMM) and non enriched multiple myeloma (MM) from 2015 at our institution. For the purpose of this presentation, we compared SRD/CKS1B (1p/1q), DLEU1/TFDP1 (13q), FGFR3/IGH (4;14), IGH/MAF (14;16), CCND1/IGH (11;14), and TP53 (17p) tests between eMM and MM, although there are more tests included in our complete multiple myeloma panel. A total of 320 enriched samples and 822 nonenriched samples were available for the study from the year 2015. The detection rate in eMM vs MM for normal cases decreased from 70% to 14% and the abnormality detection rate increased from 30% in MM to 86% in eMM. The comparison is statistically significant with a ‘p’ value of <.0001. A significant increase in the number of events (total abnormalities) with 846 in eMM vs 944 in MM was also observed when individual test results were compared for both categories with a ‘p’ value of <.0001. An increase in the total abnormality rate as well as individual prognostic markers including SRD/CKS1B(1p/1q), DLEU1/TFDP1 (13q), FGFR3/IGH (4;14), IGH/MAF (14;16), CCND1/IGH (11;14), and TP53 (17p) was observed. Informative Conclusion: We evaluated the plasma cell enrichment technique in detecting cytogenetic abnormality of plasma cell neoplasm and demonstrated that the technique increased the detection and frequency of abnormalities in the disease, therefore providing more accurate and reliable diagnosis for risk stratification and patient management.

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Platform Abstracts, AGT 2016 PROCESS IMPROVEMENT AND QUALITY MEASUREMENT OF HEMATOLOGIC CONVENTIONAL CHROMOSOME ANALYSIS CASES BY USE OF DATA DERIVED FROM THE LEICA CYTOVISION GSL-120 James Fangel, CG(ASCP)CM; Robert Jenkins, MD, PhD, Mayo Clinic

Introduction: Advanced quality metrics and process improvements are a continuing focus for our Cytogenetics laboratory. We use an automated GSL-120 system to capture metaphase images for conventional chromosome analysis. Following a standard imaging SOP, a full 20 cell analysis was not possible for some cases. We hypothesized that by using the GSL-120 quality measures, we would be able to predict which cases had an insufficient quantity of quality metaphases. If these cases could be accurately predicted, then we might be able to preemptively scan additional slides to generate extra images for analysis. Methods: We use the GSL-120 system to capture metaphases for routine chromosome analysis. The recorded GSL-120 quality data and outcome from 200 hematologic cases were combined to generate a cutoff to select cases that need additional images. When a case fell below the cutoff, extra slides were scanned before analysis to provide additional images. The process was monitored to determine if the cutoff ensured maximum efficiency. Results: By comparing the GSL-120 quality data and the final outcome of 200 retrospective cases, we generated a cutoff of 15 high quality images per slide. Cases with less than this number of high quality images were predicted to require additional scanning to complete analysis. Of 3,816 cases prospectively evaluated, 547 (14.3%) fell below the 15 image cutoff. Of 506 cases that had additional slides scanned, 483 (95.5%) were completed using these extra slides. The remaining 23 cases (4.5%) did not use the additional scanned slides. Of the 506 cases that had additional slides scanned, 23 (4.5%) required additional microscope work in combination with the additional scanned slides. Of the 3,269 (85.7%) cases that fell above the 15 image cutoff, only 2.5% required additional microscope work. With this new process 17.1 hours of technologist time was saved per week. Conclusions: Recording GSL-120 quality data from each case allows us to predict which cases will have an insufficient quantity of images for analysis. The quality data is now recorded for every hematologic case and is used to generate additional images before chromosome analysis. Overall, the new process has provided the laboratory with pre-analytic quality measurements, increased productivity, better turn-around-time, higher quality analysis and, most importantly, better patient care.

INNOVATIVE STRATEGY FOR ACHIEVING OPTIMAL CARE AND SIGNIFICANT COST SAVINGS FOR CRITICALLY ILL NEWBORNS Veronica V. Ortega, CG(ASCP); Gopalrao Velagaleti, PhD, FACMG, University of Texas Health Science Center-San Antonio

According to the American Medical Association, 4 million babies are born annually in the US. Approximately 15% of those babies are premature and account for up to 75% of NICU admissions. Term newborns with a variety of pathological conditions account for the remaining 25%. Recent studies have also shown that 1 in 33 babies is born with congenital anomalies. Frequently, developmental delay and/or intellectual disability (DD/ID) is present with one or more congenital anomalies or dysmorphic features.

Establishing underlying genetic etiology early can help physicians and families with diagnosis, prognosis and comorbidity information, all of which have significant implications for the childâ&#x20AC;&#x2122;s long-term prognosis. American Academy of Pediatrics practice guidelines recommend that chromosome analysis (CA) is required in the evaluation of children with DD/ID. Conventional cytogenetic methods such as CA and FISH, while effective to some extent, are shown to be sub-optimal in diagnosing the underlying etiology of DD/ID. American Academy of Neurology, the Child Neurology Society, and the American College of Medical Genetics, all recommend that chromosomal microarray analysis (CMA) should be considered the first-line genetic test to aid in the diagnostic evaluation of DD/ID. A CMA can detect chromosomal variations (gain and loss of genomic material) at higher resolution than a routine karyotype. However, the average turnaround time (TAT) for CMA is currently about 21 days and the daily NICU cost exceeds $3,500 per infant, easily reaching $1 million overall for extended stays. With test utilization and cost becoming an important issue among hospitals and other healthcare institutions, we developed a new approach to perform a chromosome analysis study with reflex testing to SNP microarray on all pediatric blood samples. A CA preliminary result (prelim) is reported within 24 hours to clinicians. If the prelim is positive for major abnormalities like the common trisomies, then final result is provided within 48-72 hours.

If the prelim is normal, then the testing is reflexed to a SNP microarray study based on practice guidelines mentioned above. Clients are only charged for either CA or CMA. Since inception in January 2015, we carried out reflex testing on 43 patients. Of the 43 cases, 6 have resulted in a positive prelim requiring no further testing. With our approach, we estimate a cost savings of ~$63,000 per infant since the NICU stay for these infants is reduced by about 16-18 days (72 hours with our approach vs 21 days waiting for CMA). By employing the 24 hour prelim method we were able to reduce the parental anxiety and prolonged NICU stay for these critically ill newborns. Apart from an estimated cost savings of $378,000, our approach also helped parents and physicians make informed and timely decisions regarding optimal care of these children. Given the intense debate whether the invasive and heroic measures for repairing major anomalies inflict more pain and suffering and hence only comfort care is optimal for these neonates with poor prognosis, an early diagnosis is immensely helpful for both physicians and families to decide what is optimal in each case. Reflex testing to SNP microarray after performing a 24 hour CA prelim study shows potential for extensive cost savings while maintaining optimal care for NICU patients.

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Student Poster Abstracts, AGT 2016

Student Research Award Winner PROGNOSTIC IMPACT OF MYC REARRANGEMENT IN PLASMA CELL MYELOMA

Kristina Regmi; Shimin Hu, MD, PhD; Peter C. Hu, PhD; Jun Gu, MD, PhD; Ming Zhao, MD; Zhenya Tang, University of Texas, M.D. Anderson Cancer Center Plasma cell myeloma (PCM) is a neoplasm of plasma cells and is often associated with complex chromosome abnormalities. MYC is a transcription factor that regulates cell proliferation, differentiation and apoptosis. In PCM, MYC gene is rearranged in approximately 15% of cases and is correlated with disease progression and poor outcome. Currently, the prognostic value of MYC rearrangement in PCM patients is not well understood. Here, we study the prognostic significance of MYC gene rearrangement in a cohort of 66 PCM patients with 8q24.1/ MYC gene rearrangements confirmed by conventional karyotyping and fluorescence in situ hybridization.

There were 38 men and 28 women with a median age of 58 years (range, 20 - 81). The G-banded chromosomal studies revealed complex MYC rearrangement involving translocations with multiple partner chromosomes. Most cases (37/66, 56%) had MYCimmunoglobulin fusions: t(8;14) (n=16), (8;22) (n=14), and t(2;8) (n=7). The frequently observed non-immunoglobulin fusion partners were t(1;8) (n=8) and t(6;8)(n=4). MYC rearrangement was detected at initial diagnosis in 15 patients and occurred subsequently during disease course in 51 patients. The median interval between diagnosis of PCM and emergence of MYC rearrangement was 6.8 months. The median overall survival (OS) from initial diagnosis of PCM was 33.7 months whereas the median OS from detection of MYC rearrangement was only 10.3 months. Plasma cells had high/intermediate grade morphology in approximately 58 cases (88%) and low grade in 8 cases. The median OS for the group with high/intermediate grade morphology was 25.9 months vs. 44.3 months for the low-grade morphology group. Similarly, the median OS from detection of MYC for the high/intermediate group was 9.4 months vs. 35.8 months for the low-grade group. In conclusion, MYC rearrangement in plasma cell myeloma is associated with aggressive clinical features, complex karyotype, intermediate or high grade morphology and unfavorable survival outcome.

S1

ISSUES RELEVANT TO FISH SEMI-AUTOMATED SPOT COUNTING SYSTEM

Ken Sterns; Angela Teng; Jiaqi Chen; Cristina Garcia; Roberto Guajardo; Crystal Lee; Dominique Cline; Victoria Nettles; Sylvia Wong; Ming Zhao; Jun Gu, University of Texas, M.D. Anderson Cancer Center FISH semi-automated and automated spot counting system became available and has gained some popularity during recent years.

Fully-automated FISH spot counting system is suitable for high-volume clinical diagnostic laboratories once validated but could be very expensive. Semi-automated spot counting system might be a cost-effective alternative. However, many clinical cytogenetic laboratories hesitated to adapt this approach due to the difficulty of determining efficiency and accuracy. In order to explore issues relevant to the efficiency and accuracy of the semi-automation, we conducted a quantitative FISH analysis using X/Y numerical and bcr-abl fusion FISH probes. We hypothesized that semi-automated spot counting system could improve the efficiency and accuracy of quantitative FISH analysis with the solutions to the relevant issues identified by this study. Such issues include FISH sample preparation (sample types, cell density, background, and signal intensity), image acquisition setup using semi-automated system, software configuration/calibration using either positive or negative controls, software functionality improvement, variation between semi-automate/automate counting and manual counting, misclassification review and reclassification, digital images storage for future review and documentation. Samples were scored manually and semi-automatically using the CytoVision platform. The time for setup and scoring was also recorded for analysis of efficiency. Our results demonstrated that semi-automated method could differentiate samples quantitatively. An average disagreement between manual and semi-automated FISH scoring was 7.9%. An average disagreement between manual and semi-automated FISH scoring with manual review and reclassification was only 0.39%.

Semi-automated method failed to show significant time saving when scoring less than 200 interphase cells. Enumeration probes could be more efficiently scored by semi-automated method than fusion probes. Our study identified issues relevant to FISH semiautomated counting for improvement.

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Student Poster Abstracts, AGT 2016

S2

CHROMOSOME 8P12-21 ALTERATION IN LUNG CANCER

Mohamed Baity; Xiaoshan Zhang; Hui Yi Yon; Maha El Naofal; Adriel Kim; Ming Zhao; Jun Gu; Peter Hu; Bingliang Fang, University of Texas, M.D. Anderson Cancer Center Introduction: Chromosome 8p12-21 region is frequently altered in human cancers, including lung cancers. The most frequent alterations in this regions are deletions and loss of heterozygosity, although gene amplifications have been reported. Several genes in this region have been investigated as potential tumor suppressor genes or oncogenes, including EXTL3, NRG1 and FGFR1. One of the genes located in this region is GSR, which plays a critical role in redox homeostasis and cellular defense against oxidative stress. Genetic alterations in this gene may affect treatment responses to many anticancer treatments that induce oxidative stress, such as radiotherapy, and chemotherapy with cisplatin and paclitaxel. Purpose: The goal of this is to develop a FISH method that can quickly identify lung cancers with GSR gene (8p12p21) deletion or amplification and to evaluate the feasibility of using GSR gene as a marker for assessment of treatment responses in lung cancer. Methods: Touch prepared tissues were obtained from patients with non-small cell lung cancer. A bacterial artificial chromosome (BAC) FISH probe covering the entire GSR gene at 8p12p21 was prepared with spectrum orange color. CEP8 (p11.1q11.1) labeled with spectrum green was used together with GSR probe as an internal control. Results: Preliminary probe validation confirmed the sensitivity and the specificity of the home-brewed probe. Deletion of GSR gene has been observed in H1703 lung cancer cell line and hale of the lung cancer cases screened.

Conclusion: Our study indicated that FISH using probe against GSR gene region could be an effective quick screening for lung cancer patients.

S3

SIMILARITIES BETWEEN A PATHOGENICITY ISLAND BETWEEN GROUP-A STREPTOCOCCI AND GROUP-C AND GROUP-G STREPTOCOCCI Eduardo Zavala; Randy Chu; Caitlin Evers; Varduhi Petrosyanm; Khushalia Patel; Vahid Bahranbeigi; Haowei Du; Xiqi LI; Ana Bolivar; Xuemei Shi; Awdhesh Kalia, University of Texas, M.D. Anderson Cancer Center

Background: Streptococcus dysgalactiae subsp. equisimilis bearing group carbohydrate type-C or –G are emerging pathogens that are increasingly associated with invasive infections. Group-C and –G streptococci (GCS and GGS, respectively) are the closest genetic relatives of group-A streptococcus, the human pathogen responsible for the ‘flesh-eating’ disease. Both species can co-exist in human throat or skin epithelium, and frequently exchange genes via horizontal gene transfer (HGT). No vaccine exists that confers immunity to GAS or GCS-GGS. Here we investigated the genomic architecture of the streptococcal ‘FCT’-region, which is a prime target for vaccine development. Methods: 21 SDSE genomes where investigated using genome assembly and alignment tools to investigate the genetic structural components of the FCT region. For those loci whose annotation remained in “hypothetical protein” status, the protein sequence was searched using BlastP in order to access relevant protein domains and sequence Identity.

Results: Comparison between the FCT regions among the available GCS-GGS genomes reveals the presence of genes encoding Collagen-binding protein (Cpa), and ferrous iron transporter (FeoB). Interestingly, FeoB shows between 64-81% sequence identity to fibronectin-binding protein (PrtF1). Additionally, rofGC (a positive regulator of both prtF1 and cpa), and srtC genes were also present in the majority of the strains analyzed Conclusion: Overall there is a clear similarity between the FCT regions of GAS and GCSGGS. Further work will be necessary in order to determine the degree to which the FCT region contributes to GCS-GGS virulence.

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Student Poster Abstracts, AGT 2016

S4

DESIGN AND VALIDATION OF A PYROSEQUENCING ASSAY FOR MYOFASCIAL TRIGGER POINT SUSCEPTIBILITY GENES Taylor A. Eaves; Ashley M. Rivera; Brad S. Allen, ScD, PT, COMT; Troy L. Hooper, PhD, PT, ATC, LAT; Phillip S. Sizer, PhD, PT, MOMT, FAAOMPT; Ericka C. Hendrix, PhD, MB (ASCP); Katie M. Bennett, PhD, MB(ASCP), NRCC-CC, Texas Tech University Health Sciences Center

A myofascial trigger point is generally described as a region in a taut muscle band that is hypersensitive. This hypersensitivity usually results in referred pain throughout the body. The purpose of our study was to design and validate a pyrosequencing assay to detect three human single nucleotide polymorphisms (SNPs) that are hypothesized to be associated with myofascial trigger point formation. The three SNPs are located in the calcitonin gene-related peptide (CGRP) promoter (rs3781719), serotonin receptor (rs6313), and osteopontin (rs28357094) genes. With a validated assay, clinical studies can be conducted to determine whether or not these genetic variations are linked to trigger point formation. PCR and sequencing primers were designed for the three SNPs using the Qiagen PyroMark Assay Design software and Primer Blast (NCBI). Control DNA samples were amplified by PCR and analyzed by pyrosequencing using the Qiagen PyroMark Q24 instrument. DNA samples were also confirmed by Sanger sequencing to establish genotyped controls for the assays. The pyrosequencing assays were further assessed for accuracy and precision, including inter- and intra-run variability. All parameters met validation criteria (>95% concordance). This new assay shows promise in identification of polymorphisms in the genes of interest and could be expanded to include other candidate genes. Future studies will include determining sample type requirements and examining the relationship of the polymorphisms to myofascial trigger points in a clinical study.

S5

DELETION OF CDKN2C AND CDKN2D AS NOVEL PROGNOSTIC MARKERS IN MEDULLARY THYROID CARCINOMA PATIENTS Maha El Naofal; Adriel Kim; Hui Yi Yon; Mohamed Baity; Zhao Ming; Jun Gu; Peter Hu; Jacquelin Bui-Griffith; Elizabeth Gardner Grubbs; Gilbert Cote, University of Texas, M.D. Anderson Cancer Center

Introduction: Medullary thyroid carcinoma (MTC), an aggressive form of thyroid cancer, accounts for approximately 1200 new cancer cases annually. Sporadic cases account for 85% of MTCs and clinical outcomes vary depending on tumor stage. Because RET and RAS mutations play a role in about 40% and 15%, respectively, of sporadic MTCs and are predominant drivers in MTC pathways, these mutations are some of the most comprehensively described and screened for in MTC patients. However, other aberrations are implicated in the tumorigenesis of MTC. Recent studies suggest that mutations in CDKN2C and CDKN2D also contribute to MTC tumorigenesis. Preliminary Data: Comparative genomic hybridization analysis revealed that approximately 40% of sporadic MTC samples have loss of the p18 gene (CDKN2C) at chromosome 1p32. In addition, the p19 gene (CDKN2D) at chromosome 19p13 is frequently lost in MTC patients. Purpose: To determine the extent to which p18 and p19 copy number loss contributes to MTC progression and to assess the feasibility of using fluorescence in situ hybridization (FISH) to screen MTC patients for p18 and p19 deletions. Methods: Thirty Formalin-fixed, paraffin-embedded MTC samples with defined RET/RAS mutations were subjected to dual-color FISH assays to detect the loss of the p18 and/or p19 genes. The spectrum orange probes were prepared using the bacterial artificial chromosomes RP11-779F9 for p18 and RP11-177J4 for p1 as well as spectrum green control probes to the 1q25.2 and 19q11 regions (RP11- 1146A3 and RP11-942P7, respectively). Nine Formalin-fixed, paraffinembedded normal thyroid tissue samples were used to establish the cutoffs for the signal patterns observed. Results: The bacterial artificial chromosome probes and the control probes showed 100% specificity for the p18 and p19 regions. We anticipate that a significant percentage of MTC samples will have loss of heterozygosity of the p18 and p19 genes and show 1R2G and other signal patterns. Prospective Utilization/Implication of Results: The FISH data will be combined with RET/RAS mutation status, molecular analysis of the p18 and p19 gene copy number, other biomarkers identified with protein studies, and patientsâ&#x20AC;&#x2122; clinical outcomes (disease aggressiveness and survival).

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

143

Poster Abstracts, AGT 2016

1

EVALUATION OF THREE CASES WITH DISCORDANT RESULTS BETWEEN NON-INVASIVE PRENATAL SCREENING AND FOLLOW-UP CYTOGENETIC TESTING

Monica Carrillo, BS, CG(ASCP) CM, Cytogenomics and Molecular Pathology, Department of Pathology and Immunology; Amie Stanley, MS, CGC, Department of Obstetrics and Gynecology; Diana Gray, MD, FACMG, Departments of Obstetrics and Gynecology and Radiology, Division of Maternal-Fetal Medicine and Ultrasound; Yoshiko Mito, PhD, FACMG, Cytogenomics and Molecular Pathology, Department of Pathology and Immunology, Washington University School of Medicine Non-invasive prenatal screening (NIPS) is an aneuploidy screening test in which cell-free fetal DNA circulating in maternal blood is analyzed by next generation sequencing or microarray. Based on recent large-scale studies in a high risk population, high sensitivities and specificities have been reported for detection of common autosomal trisomies such as trisomy 21, 18 and 13.

These studies suggested that NIPS is a much more efficient screening method than conventional maternal serum screening methods. However, NIPS is a screening method, and the degree of discordance against cytogenetics follow-up testing particularly in sex chromosome abnormalities has yet to be elucidated. Our laboratory performs chromosome analysis and Fluorescence in situ hybridization (FISH) on prenatal specimens including amniotic fluid (AF) and chorionic villus sampling (CVS). In 2015, approximately 20% of the prenatal cases received were ordered as confirmatory testing following abnormal results or increased risk by the evaluation of NIPS. Here, we report three cases in which sex chromosome abnormalities detected by NIPS were not confirmed by follow-up cytogenetic testing. Two cases were AF specimens with positive NIPS results for monosomy X, and the third case was a CVS specimen with a finding of XXY. In all three cases, both chromosome analysis in 20 metaphase cells and FISH analysis with sex chromosome probes in 100 nuclei showed no evidence of cytogenetic abnormality. The discordance in results may be due to technical difficulties of NIPS in detecting correct sex chromosome complement, or reflect previously unknown high incidence of sex chromosome mosaicism. No such discordance between NIPS and cytogenetic testing had been observed for trisomy 21 and 18 in our laboratory. This study illustrates a higher incidence of cases with positive NIPS and normal cytogenetics for sex chromosome abnormalities, and further investigation is warranted to elucidate the etiology of this discordance. Positive results of NIPS should be regarded with caution and follow-up cytogenetic testing should be considered.

2

AMPLIFICATION OF 5’ DDT3 GENE IN A LOW GRADE SARCOMA

Jodie Williams, BS; Maricar Fernando; James Tepperberg, Laboratory Corporation of America A 74-year-old patient diagnosed with low grade sarcoma, favoring myxoid liposarcoma (MLS) was referred for fluorescence in situ hybridization analysis (FISH) utilizing a DDIT3 (CHOP) gene specific probe. The FISH analysis showed two intact DDIT3 genes with an additional 5’ DDTI3 amplification observed in 71% of cells consistent with a diagnosis of liposarcoma. Liposarcomas are tumors that arise in the body’s fat tissues with well differentiated (WDLS), dedifferentiated, (DDLS) and myxoid/round cell liposarcomas (MLS/ RCLS) are among the more common types. These liposarcomas are characterized by a rearrangement of DDIT3. A translocation of DDIT3 at 12q14 with the FUS gene at 16p11, t(12;16)(q13;p11) was reported to be present in >95% of myxoid/round cell liposarcomas and were virtually a diagnostic marker for MLS and round cell (RC) liposarcomas. In addition to the 12;16 translocation, amplification of the 5’ segment of DDIT3 has been reported to be associated with WDLS/DDLS. Data concerning the fine structure of the 12q1315 amplicon, which contain CDK4, MDM2 and HMGA2, in well-differentiated and dedifferentiated liposarcomas (WDLPS/DDLPS) is limited. The questions in this study are: 1) Does the 5’ DDIT3 amplification extend to CDK4, MDM2 and HGMA2 genes, and 2) Does the inclusion of various amplification gene(s) help identify the liposarcoma subtype? FISH targeting HMGA2, DDIT3, and CDK4, qPCR and chromosome microarray CMA (if enough tissue is available) can help further identify and characterize the amplified genes/ segments. Amplification of 5’ DDIT3, CDK4, MDM2 and HGMA2 DNA would provide further support that multiple genes in the 12q1315 amplicon are associated with well differentiated liposarcoma/dedifferentiated liposarcoma.

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

144

Poster Abstracts, AGT 2016

3

INCREASED PRODUCTIVITY IN CYTOGENETIC LABORATORY DUE TO IMPLEMENTATION OF THE ASI 81 SLIDE LOADER AND METAPHASE FINDER

Tishome Persaud, CG(ASCP); Randi Bambach, CG(ASCP); Rosemarie Schmidt, CG(ASCP); Pauline Brenholz, MD, FAMCG, Integrated Oncology, LabCorp Specialty Testing Group In the current medical environment, productivity coupled with accuracy is paramount in providing good patient care. The ASI 81 Slide Loader and Metaphase Finder runs overnight and provides a gallery of digital metaphase images ready for analysis, eliminating the time to ascertain analyzable metaphases and reducing the analysis time, ultimately increasing technologist productivity, compared to traditional methods based on microscopy. Eleven technologists were subdivided into categories based on number of years of experience: 3 technologists had 1-4 years of experience, 5 had 5-9 years, and 3 had 10+ years of experience.

Productivity data, in the form of completed bone marrow/leukemic blood half-cases (10 cells) per day, was averaged for 3 months using conventional microscopic analysis. The ASI Metaphase Finder was then introduced to the same technologists and their productivity followed for another 3 months. Technologists with 1-4 years of experience increased their productivity from 3.9 to 6.4 cases/day (62.2%). Those with 5-9 years of experience increased from 4.0 to 5.4 cases/day (33.8%), and technologists with 10+ years of experience increased from 4.8 to 6.4 cases/day (26.7%). An inverse relationship was present between the technologistsâ&#x20AC;&#x2122; number of years of experience and the percentage of productivity increase. This could be in part due to less experienced technologists being slower in analyzing cells under the microscope, compared to more experienced technologists who are at their peak productivity using the microscope. Also, more experienced technologists spend less time searching for metaphases compared to less experienced ones, therefore their productivity was increased by a lesser percentage when the metaphase finder eliminated this part of analysis. The smaller increase in productivity of more experienced technologists could further be explained by the fact that they analyzed more complex cases, and therefore spent less time looking for metaphases and more time analyzing and preparing complex karyotypes for supervisor review. The average productivity of all 11 technologists was increased by 39.6%, and therefore the ASI Metaphase Finder clearly contributed to the efficiency of our technologists at each level of experience.

Additionally, the generated gallery of metaphases helped our supervisors and directors to review cases more efficiently, improving their productivity as well.

4

NOVEL FOUR COLOR FISH PROBE SET FOR SIMULTANEOUS DETECTION OF RET BREAK-APART AND PDGFRÎą AMPLIFICATION Aditi Khurana, CG, CGMBS; Suman Verma; Phil D Cotter; Matthew Moore, ResearchDx

Identifying the genomic abnormalities in cancer is critical to improve the efficacy of cancer therapy by matching targeted drugs to patients that will respond favorably to treatment and also identify non-responders. A number of critical genomic rearrangements are associated with therapeutics in non-small cell lung carcinoma (NSCLC). There is frequently limited FFPE material available and in some cases assays must be prioritized. In the interests of efficiently utilizing scarce clinical samples, we developed a four-color RET/ PDGFR/KIT/CC4 FISH probe set that provides coverage of the PDGFR gene (4q12), the KIT gene (4q12), detection of RET gene rearrangements and a copy control for the centromeric region of chromosome 4. All the genes targeted in this probe are clinically actionable and associated with either a current therapeutic or with on-going Phase II trials. The benefits of screening for three genes at the same time are not only a cost effective strategy for labs but also added benefit to the patient by providing additional information from limited FFPE material.

5

UTILIZING GALAXY FOR CLINICAL NGS SEQUENCE INTERPRETATION

Aditi Khurana, CG, CGMBS; Micheal Ta; Phil D. Cotter; Matthew Moore, ResearchDx Galaxy is an open source, customizable, scalable and extensible, web based platform commonly used for data intensive biomedical research. Galaxy has many positive features for clinical use, including secure access, a simple web based interface, automated (versioned) pipeline processing, load balancing, and an inbuilt set of display and analysis tools for NGS data. We have generated and validated a robust bioinformatics pipeline using Galaxy for the analysis of NGS panel and exome datasets. The unified pipeline encompasses four analysis modules: input, pre-processing of FASTQ files, alignment and post alignment process with two sub modules consisting of variant calling tools and copy number identification. The Galaxy workflow performs the following tasks: pre-processing raw FASTQ files using the FASTQ parallel groomer and Trimmomatic, alignment to the genome, post-alignment processing using Picard tools (such as MarkDuplicates) and intersection of the aligned reads with the target region of the panel using Intersect BAM alignments tool found in the Galaxy toolshed. Variant calling is accomplished using the following software components: Freebayes (snps, short indels), Pindel (indels), HardSearch (Structural variants), and Varscan2 (copy number).

Output from each variant caller is collated and compiled into a report for review and analysis. The pipeline demonstrated analytical specificity and sensitivity of >95% and a 100% precision and concordance between different reproducibility runs for variant calling on the same datasets. The pipeline is able to detect <10% allele frequency for each variation type tested by the assay with a positive predictive value of >95%. The currently reported variants include SNVs, insertions, deletions, translocations and copy number variations. With changes in command line tools and new version of Galaxy tools the pipeline can be revalidated, version controlled and duplicated at other sites if needed.

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

145

Poster Abstracts, AGT 2016

6

A RARE CASE OF MYC GENE AMPLIFICATION IN AML

Beth Barnett, MS, CG(ASCP) CM ; Sandi Hall, BS, CG(ASCP) CM ; Suzanne Hurley, MS, CG(ASCP) CM ; Brandice Nowell, BS, CG(ASCP)CM; Hong Huang, MD, CG(ASCP)CM; Jade Sikes, BS, CG(ASCP)CM; Shanorria Greer, BS; Siraj El Jamal, MD; Holly H. Hobart, PhD, University of Mississippi Medical Center Here we report a case of AML with monocytic differentiation and double minute chromosomes containing the c-MYC locus. While not an absolutely unique case, this finding is sufficiently rare that none of the personnel in the Cytogenetics lab had seen the phenomenon in careers extending back as far as 20 years and thus is worth calling attention to. Gene amplification in neoplasia is one mechanism for generating overproduction of a gene product that leads to dysregulation and loss of control of the cell cycle. The balanced translocation rearrangement of genes found in many forms of leukemia and lymphoma is another mode of dysregulation.

Gene amplification is not uncommon in solid tumors, found at relatively high frequency of cases of neuroblastoma (perhaps 30%), for example. In solid tumors, the physical manifestation of gene amplification takes two primary forms, homogeneously-staining regions (HSR) and double-minute chromosomes (dmin). In leukemias, the phenomenon is much less frequent, less than 3% of cases of AML and for some FAB groups, less than one quarter of that. In AML, data suggest that dmin are most frequently found in cases of acute erythroleukemia (FAB M6), with myeloblastic leukemia (groups M1 and M2) next most frequent. The complex karyotype found during G-band analysis had dmin in addition to other structural and numerical abnormalities. FISH for PML/RARA, t(15;17), had proved to be normal. FISH was performed using probes for c-MYC and KMT2A (MLL), two loci often found to be in dmin associated with hematological disease. The c-MYC probe showed signal on the dmin, KMT2A did not.

7

A CYTOGENETIC COMPARISON OF TWO CASES INVOLVING MOSAIC TRIPLOIDY

Elizabeth Hamelberg, CG(ASCP) CM ; Elizabeth Hamelberg; Cecelia Green-Geer; Sayaka Hashimoto; Theodora Jacobson; Matthew Meleski; Donald Roman; Jeffrey Wobser; Scott Hickey; Shalini Reshmi; Caroline Astbury, Nationwide Childrenâ&#x20AC;&#x2122;s Hospital, Cytogenetics Laboratory The finding of mosaicism for a diploid (or near diploid) cell line with a triploid cell line is rare. Mosaic triploidy (or diploid/triploid mixoploidy) is thought to arise by either double fertilization of an ovum by two sperm or by delayed incorporation of the second polar body into one of the early blastomeres. We describe two cases that have a mosaic triploid karyotype, with markedly different phenotypic presentations. Case 1 is a 5 year old male who presented with global developmental delay, hypomelanosis of Ito, hemihyperplasia, and borderline microcephaly. Chromosome analysis on skin fibroblasts yielded a karyotype of mos 69,XXY[10]/46,XY[10]. Case 2 involved a comparative chromosomal study between amniotic fluid (obtained at 27w5d gestation) and peripheral blood obtained at birth (32 week gestation). The indication for study included advanced maternal age, polyhydramnios and fetal hydrops, while prenatal ultrasound showed lower extremity ectrodactyly and ascites. The final chromosome analysis on the amniotic fluid showed two abnormal cell lines and the karyotype was designated: mos 69,XXX[8]/45,X[7]. At birth, the physical findings included complex heart abnormalities. Peripheral blood was sent for chromosome analysis to elucidate further the prenatal finding of mosaic triploidy. The final G-banded result was mos 45,X[12]/69,XXX[8]. Although both cases reported here have a triploid cell line, the clinical presentation between the two cases differs in their severity for each patient. A difference in the distribution of the triploid cell line in different tissue types could possibly explain the variation in the phenotypic presentation between the two patients. The possible origin of each mosaic triploidy will be discussed.

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

146

Poster Abstracts, AGT 2016

8

DISCORDANT RESULTS IN CULTURED CELLS VS TOUCH IMPRINTS AND PARAFFIN-EMBEDDED TISSUE

Jennifer Conner; Christine M. Higgins; Pamela A. Althof; Michele L. Wiggins; Ningxia Lu, Human Genetics Laboratory, MunroeMeyer Institute for Genetics and Rehabilitation, University of Nebraska Medical Center; Hina Naushad Qureish, Department of Pathology and Microbiology, University of Nebraska Medical Center; Bryan Teruya, VA Nebraska-Western Iowa Health Care System, U.S. Department of Veterans Affairs; Stefan Costinean; John Gross, Department of Pathology and Microbiology, University of Nebraska Medical Center; Bhavana J. Dave; Jennifer N. Sanmann, Human Genetics Laboratory, Munroe-Meyer Institute for Genetics and Rehabilitation at the University of Nebraska Medical Center Lymphoma is a hematopoietic solid malignancy of the lymphoid tissue characterized by uncontrolled proliferation of clonal lymphocytes in the lymphatic system. In addition to traditional pathologic assessments, genetic markers serve as important diagnostic and prognostic indicators for lymphoma. Often, the sample is divided for pathology assessment and for cytogenetic testing. Conventional cytogenetic analysis allows for a whole-genome assessment of the sample; however, in some cases limited resolution, sub-optimal morphology, and culture failure can limit the utility of conventional cytogenetic analysis of lymphomas.

Therefore, indication-specific, adjunct fluorescence in situ hybridization (FISH) studies are often performed concurrently. We report two lymphoma cases sent to the Human Genetics Laboratory for conventional cytogenetic analysis and FISH testing. Short-term suspension cultures were established in a closed culture system from the fresh tissue submitted for both cases, and an additional long-term flask was established for Case 2 based on the indication for testing received at specimen intake. FISH studies for the BCL6 and BCL2 loci were performed on the short-term culture (24 hour overnight with colcemid) for Case 1 with negative results.

Based on previous genetic studies for this patient (positive for BCL6 and BCL2 rearrangements) and given the pathologic findings of the current specimen, FISH studies were repeated on touch imprints generated from the fresh sample upon receipt. These slides were positive in 12% and 11% of cells, respectively. FISH studies for assessment of the ALK locus was performed on cells from the established flask for Case 2 with negative results. However, the tissue received for cytogenetics was noted to be significantly necrotic at sample intake, and the sample exhibited strong positivity for ALK by immunohistochemistry. Therefore, paraffinembedded tissue from the same sample was requested for Case 2 and was determined to be positive for rearrangement of ALK in 96% of cells. The results from these two studies affirm the need for careful documentation (condition of the sample, texture, size, cells liberated) upon receipt in the laboratory. In addition, these cases highlight the importance of diligent review of genetic testing results in coordination with the pathologic assessment, as well as in the context of any previous genetic studies. Laboratories should be acutely aware of the possible pitfalls associated with cell culture alone and should consider other methodologies (e.g., assessment on touch imprints and/or paraffin-embedded tissue) when performing FISH studies on solid tissues, particularly in cases where there is marked discordance between the pathologic and cytogenetic findings, before issuing a clinical report.

9

MLL: THE GENE AND ITS TRANSLOCATION PARTNERS. 2 CASES WITH A T(11;17) INVOLVING MLL AND MIMICKING THE VARIANT T(11;17) RARA TRANSLOCATION

Erik Schulz, BS, CG(ASCP); Tiffany Chouinard, CG(ASCP)CM, MB(ASCP)CM; Felix De La Cruz, CG(ASCP)CM; Erica Elwell, CG(ASCP)CM; Christopher Mixon, MD; Robert Gasparini, MS, CG(ASCP)CM, DLM(ASCP)CM, NeoGenomics

The MLL gene or KMT2A as it is now called is located at 11q23 and was first identified in 1992 by Rowley, et. al., as a “gene involved in human leukemia”. Since then, the ‘myeloid/lymphoid leukemia’ or ‘mixed-lineage leukemia’ gene has been one of the most extensively studied and characterized genes, perhaps even more so than BCR or ABL. MLL is implicated in just over 10% of all acute leukemias including myeloid (AML), lymphoid (ALL), bi-phenotypic, childhood, and treatment-related leukemia. In virtually all cases when MLL is abnormal, the prognosis is poor. MLL abnormalities including deletion, amplification and gene rearrangements have been reported in hundreds of peer-reviewed journal articles. Of these various abnormalities, MLL gene rearrangement is perhaps the best known and reported. To-date MLL has been identified as one of the genes involved in 85 separate, recurrent translocations earning it a reputation as (one-of) the most promiscuous genes in the human genome.

We report on two cases where acute leukemia was suspected and a t(11;17) (q23;q21) was identified via cytogenetic testing. The translocation certainly looked like the “classic” (11;17) RARA variant translocation but FISH analysis for both cases utilizing the (15;17) dual-color, dual-fusion probe set (PML 15q22 red, RARA 17q21 green) and the RARA break-apart probe set were normal with two green (RARA) signals and two fusion signals respectively, in all interphase nuclei. Metaphase FISH analysis on both cases with the RARA break-apart probe set showed a RARA fusion signal on one of the number 17 chromosomes and a second fusion on one of the number 11 chromosomes as identified through a reverse banding technique to help identify the respective chromosomes involved in the translocation. Interphase FISH analysis of both cases with the MLL break-apart probe set showed the classic 1R1G1F abnormal signal pattern in the majority of interphase nuclei. Metaphase FISH analysis via reverse banding on both cases with this same MLL probe set showed one red signal on the derivative 11 chromosome and one green signal on the derivative 17 chromosome. Four of the MLL translocation partners are located at 17q21 where the hematopoietic gene RARA (retinoic acid receptor alpha) is located. The t(11;17) (q23;q21) is a well-known RARA variant translocation and is important to identify when acute promyelocytic leukemia (APL) is suspected. Per the current version of the WHO classification, this variant (11;17) RARA translocation would be diagnostic of APL regardless of blast count in the appropriate clinical setting. However, in our two cases, the t(11;17) (q23;q21) did not involve RARA at 17q21 but involved one of the other MLL gene rearrangement partners in this same band. A negative result for either the t(15;17) probe set (this probe set eliminates the 15 as a translocation partner) or the RARA break-apart probe set and a positive (abnormal) result for the MLL break-apart will help differentiate a true RARA variant translocation from one that cytogenetically looks exactly the same. Being aware of these MLL translocations with any of its partner genes located at 17q21 can help prevent a diagnosis of a variant APL translocation.

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

147

Poster Abstracts, AGT 2016

10

DNA-BASED MICROARRAY CGH AS A HIGH-RESOLUTION TECHNIQUE FOR HER2 EQUIVOCAL BREAST CANCER

Cynthe Sims, PhD, MB(ASCP), CGMBS; Shailin Govender, BS; Aditi Khurana, CG(ASCP) MB(ASCP), PacificDX; Nancy Morales, HT(ASCP)CM, Biocare Medical; Mathew W. Moore, PhD; Philip D. Cotter, PhD, FACMG, FFSc(RCPA); Shelly Gunn, MD, PhD, FCAP, ResearchDX Accurate reporting of HER2 (ERBB2) status in newly diagnosed breast cancer is critical for selecting patients predicted to respond to anti-HER2 therapy. Current CAP/ASCO guidelines recommend all newly diagnosed and metastatic breast tumors be evaluated for HER2 positivity by protein-based immunohistochemistry (IHC) and/or chromosome-based fluorescence in situ hybridization (FISH).

In the majority of cases, these testing modalities provide a clearly actionable “positive” or “negative” answer.

However, in an estimated 10 to 20% of breast cancers, both tests are reported as “equivocal” leaving the clinician with a treatment decision dilemma and no alternative testing method. In the current study 20 previously HER2 characterized breast tumors were analyzed by DNA-based microarray comparative genomic hybridization (CGH). Multiple QC steps were incorporated into the testing workflow to ensure high quality microarray results for enzymatic labeling of DNA extracted from formalin fixed paraffin embedded (FFPPE) tissue. Following hybridization and data analysis, CGH microarray results were compared to results by IHC and/or FISH. CGH results were highly sensitive and specific for detecting HER2 gene copy number alterations supporting our hypothesis that DNA-based CGH microarray testing can provide an alternative clinical testing method for determining HER2 status in breast cancer.

Further investigation through clinical trials is needed to incorporate DNA-microarray testing into the HER2 testing algorithm for all newly diagnosed breast cancers.

11

MULTIPLE ABERRANT SEGREGATION EVENTS IN AN 8-WEEK PRODUCT OF CONCEPTION FROM A TWIN PREGNANCY

Sheri Hedrick, CG(ASCP); Cecelia Green-Geer; Mollie Haughn; Theodora Jacobson; Mariana Kekis; Kelley Kneile; Aimee McKinney; Matthew Meleski; Donald Roman; Ruthann Pfau; Caroline Astbury, Nationwide Children’s Hospital, Cytogenetics Laboratory We describe the case of a product of conception sample received from a 34-year-old G10 P4 A6 woman. Documentation of one of the patient’s previous pregnancy losses included the cytogenetic finding of a ring chromosome and associated anencephaly, while maternal chromosomes were normal. Dissection of the current sample revealed a dichorionic, diamniotic twin pregnancy with connected gestational sacs. A number of morphological differences were observed between the fetuses and their placentas, including a size discrepancy between the fetuses and a dysplastic placenta for only one twin. Samples for cytogenetic tissue culture were collected from the chorionic villi of each of the twin sacs and separately from each fetus. Cytogenetic analysis of Fetus A showed a 68,XX karyotype in the chorion /villi, while the tissue sample for Fetus B tissue sample failed to grow. Microarray analysis of snap frozen tissue from Fetus B was consistent with a 68,XX karyotype. Microsatellite analysis showed that the fetuses were identical and that the triploid karyotype most likely resulted from digyny. Markers from all examined chromosomes showed one maternal allele except for chromosome 18, which showed two different maternal alleles. The absence of a third sex chromosome, combined with the unusual segregation pattern of the maternal chromosomes, suggests multiple aberrant segregation events. We will describe possible mechanisms which may have led to the observed findings.

13

A REPORT OF CYTOGENETIC ABNORMALITIES FOUND IN 246 MEXICAN PATIENTS CLINICALLY DIAGNOSED WITH MYELODYSPLASTIC SYNDROME (MDS) Carlos Alonso Muñoz, BS,CG(ASCP)CM; Victor Alfredo Perez-Contreras, MS; Elik Enrrique Alonso-Muñoz, BS; Carlos Ivan AndradeCardenas, BS; Carlos Cortes-Penagos, PhD, Laboratorios Mendel

Myelodysplastic syndrome (MDS) is a group of clonal cell disorders characterized by progressive cytopenias and dishematopoiesis.

Although the etiology is still unknown, primary and secondary MDS may be due to the use of chemotherapy, radiation and chemicals substances contained in cigarette smoke. MDS is associated with constitutional diseases in children. Biological features include impaired hematopoiesis, which may be accompanied by cytogenetic aberrations, as well as molecular and/or immunologic abnormalities. Among the cytogenetic alterations found in this study, the absence of chromosome Y (-Y) , complex karyotypes and abnormalities of chromosomes 5 and 7, were observed. Many of these abnormalities are associated with good, intermediate and poor prognosis. This paper describes the cytogenetic abnormalities found in a Mexican population of 246 people which were clinically diagnosed with MDS and referred to Laboratorios Mendel for Cytogenetic testing.

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

148

Poster Abstracts, AGT 2016

14

SHORTER HYBRIDIZATION TIMES USING CYTOCELL AQUARIUS ® PROBES

Steve Chatters BSc(Hons), DipRCPath; Alex Hobbs, BSc; Faidra Partheniou, BSc MSc; Richard Frodsham, BSc(Hons); Gothami Lakshika Fonseka, PhD; Karina Mak-Hannah, MSc; Martin Lawrie, PhD, Cytocell Ltd. Fluorescence in-situ hybridization (FISH) analysis is the ‘gold-standard’ method for the detection of balanced and unbalanced chromosomal rearrangements plus gains and deletions in neoplastic specimens. Standard FISH protocols incorporate an overnight hybridisation step; however, shorter hybridisations are sometimes desired as a result of laboratory operational requirements. The Cytocell Aquarius FISH probe range from Oxford Gene Technology (OGT) delivers bright, clear and precise signals when hybridised overnight. Data are presented in this poster to show that, for a range of probe and specimen types, hybridization time for Cytocell Aquarius probes may be shortened from overnight to just a few hours with no loss of accuracy or probe signal quality, giving laboratories the flexibility to use these probes in a variety of differing settings.

15

A TRIPLOID PRODUCTS OF CONCEPTION WITH TETRASOMY OF CHROMOSOME 2 AND 20 DETECTED BY SNP MICROARRAY: IMPLICATIONS FOR MULTIPLE MEIOTIC ERRORS Tara Ellingham, MS, CG(ASCP); Iya Znoyko, PhD; Daynna Wolff, PhD; Cynthia Schandl, MD, Medical University of South Carolina

Whole genome single nucleotide polymorphism (SNP) microarray, a technique using arrayed DNA sequences as targets for hybridization, has become increasingly useful in prenatal diagnosis over standard cytogenetic analysis and/or fluorescence in-situ hybridization due to its high resolution analysis and the ability to obtain results even when cells are nonviable. We present a case of a 22 year old female with a missed abortion at 11 weeks that had abnormalities detected using SNP microarray. The laboratory received a fresh piece of chorionic villus sample that had bulbous, grape-like clustered villi. A portion of the sample was cultured for routine cytogenetics but the cells failed to grow in culture. Genomic DNA was extracted from another portion and this was used for SNP microarray analysis (Illumina 850K Beadchip). The SNP microarray study revealed triploidy with two copies of the X chromosome and one copy of Y, and also showed four copies of chromosome 2 and four copies of chromosome 20. Analysis of the B-allele frequency revealed complex allelic composition of the two tetrasomic chromosomes, with balanced gain of whole chromosome 20 and an unbalanced gain of 2q and part of 2p. Due to the appearance of the villi and the results of the allelic analysis, we hypothesized that the fetus was dyandric, with fertilization of an abnormal egg with an extra chromosome 20 by one normal sperm and one sperm with a nondisjoined chromosome 2. Triploidy with tetrasomy of selected chromosomes is rare. Application of SNP microarray in this case not only allowed for analysis of non-viable tissue, but also provided insight into the genetic mechanisms underlying this rare and complex abnormality.

16

EVI1 AND C-MYC REARRANGEMENTS IN TREATED BLAST PHASE OF CHRONIC MYELOID LEUKEMIA

Parameswar Ganeshan; Anna S. Nam, MD; Patricia Massino; Ana Revelo-Sanchez; Junyu Chen; Susan Mathew, PhD, NewYorkPresbyterian Hospital MYC overexpression has been observed in patients with chronic myeloid leukemia in blast phase (CML-BP) and is associated with progression of disease. However, MYC rearrangement in CML-BP has not yet been reported. Moreover, EVI1 rearrangement is a rare finding in CML-BP. Here, we describe a patient with Philadelphia chromosome in CML-BP who acquired a t(3;8)(q26.2;q24.2) translocation involving EVI1 and MYC genes two years after the diagnosis. The patient, a 41-year-old male, initially presented in CML-BP with a white blood cell count of 224x103/mL and increased circulating blasts. At the time of diagnosis, conventional cytogenetic analysis showed a 46,XY,t(9;22)(q34;q11.2) karyotype in twenty evaluated metaphase cells. Interphase fluorescence in situ hybridization (FISH) analysis confirmed BCRABL1 rearrangement. He achieved chronic phase of disease after standard induction therapy with daunorubcin, cytarabine and dasatinib. However, upon progression of disease several months later, the patient was switched to nilotinib and subsequently to ponatinib. A bone marrow biopsy revealed clonal evolution with acquisition of trisomy 19 and an extra Philadelphia chromosome. Cytogenetic analysis of the most recent biopsy identified a 46,XY,t(9;22) (q34;q11.2)[15]/48,idem,+19,+der(22)t(9;22)[4]/46,idem,t(3;8)(q26.2;q24.2)[1] karyotype. FISH analysis using break-apart probes for MYC and EVI1 revealed rearrangements of both genes suggesting that these genes are involved in the translocation. The corresponding pathology revealed a fibrotic marrow with increased CD34+ blast cells, consistent with persistent disease. Although EVI1 rearrangement in a t(3;8) translocation has been reported in CML-BP in a single case report, to our knowledge this is the first documentation of rearrangement of both MYC and EVI1 genes in a patient treated for CML-BP.

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

149

Poster Abstracts, AGT 2016

17

DETECTION OF UNIPARENTAL HETERODISOMY THROUGH SNP-BASED TECHNOLOGY: A REPORT OF RUSSELLSILVER SYNDROME

Denae Golden, BS, CG(ASCP)CM; Diane L. Pickering, MS, CG(ASCP)CM; Julie M. Carstens, MS, MB(ASCP)CMCG; Shelly D. Nielsen, MS, MT, LCGC, Genetic Medicine; Bhavana J. Dave, PhD, FACMG; Lois J. Starr, MD, FAAP, FACMG, Genetic Medicine; Jennifer N. Sanmann, PhD, FACMG, Human Genetics Laboratory, Munroe-Meyer Institute for Genetics and Rehabilitation at the University of Nebraska Medical Center Russell-Silver syndrome (RSS) is characterized by intrauterine growth restriction and poor postnatal growth with normal head circumference, broad forehead, blue sclerae, narrow chin commonly with micrognathia, fifth-finger clinodactyly, hemihypotrophy, and an increased risk for developmental delay. The underlying genetic etiology for the majority of patients with RSS involve the imprinting center at 11p15.5; however, approximately 10% of patients with RSS have maternal uniparental disomy (UPD) of chromosome 7. We present a 3 year and 8 month old female patient with short stature, fine motor delay, speech and language impairment, autism spectrum disorder and poor weight gain with an oral aversion to food. Initial genetics evaluation resulted in a normal karyotype. Following a subsequent clinical genetics evaluation, sequencing for 86 genes associated with autism, intellectual disability, and multiple anomalies and a high-density SNP array (HDSA; Affymetrix CytoScan HD Array, Santa Clara, CA) were performed. Studies were negative for pathogenic sequencing variants and clinically-significant copy number changes. However, the HDSA revealed several regions of homozygosity (ROH) ranging from 3.9 Mb – 49.4 Mb in size isolated to chromosome 7 indicative of uniparental heterodisomy. Subsequent methylation studies confirmed UPD(7)mat. Current guidelines recommend microarray as a first-tier test for the evaluation of patients with intellectual disability, autism, and multiple congenital anomalies. Clinically-significant copy number changes are most commonly identified in patients with the aforementioned clinical indications for testing. However, as microarray platforms have evolved from BAC and oligonucleotide to single-nucleotide polymorphism (SNP) designs, identified regions of homozygosity may also provide valuable and unexpected diagnostic information. We present this patient to emphasize the clinical utility of ROH identified by SNP microarray platforms.

18

CASE REPORT: DETECTION OF UNIQUE AND UNUSUAL DELETION OF RUNX1 IN AN AML PATIENT BY FISH TESTING FOR T(8;21), IN LINE WITH STUDIES IN MOUSE AND IN VITRO CELL MODELS Suying Xu, MS; Patricia J. Mouchrani; Tina Clark; Ju-Hsien Chao; Joseph Hersh, University of Louisville

A patient diagnosed as acute myeloid leukemia (AML) presented 95% of deletion of RUNX1(AML) gene, in both interphases and metaphases, by using FISH probe to detect translocation of t(8;21). The abnormality could not be clearly recognized by routine chromosome analysis, although one of two chromosome 21 appeared to be a bit shorter. No other chromosome abnormalities were found. RUNX1, involved in the development of normal hematopoiesis, encodes the DNA-binding subunit of the Core-Binding Factor transcription factor complexis. It is a frequent target of human leukaemia-associated gene aberrations in form of translocation between chromosome 8q and 21q. Loss of RUNX1/AML1 arginine-methylation impairing peripheral T cell homeostasis has been recently reported by a knock-out study in mice. Another study demonstrated successful in vitro modeling of familial platelet disorder (FPD) with predisposition to acute myeloid leukemia with patient-specific iPSCs and confirmed that RUNX1 mutations are responsible for megakaryopoietic defects in FPD patients. Our very interesting clinical report of this case is the 1st RUNX1 deletion pattern as presented in AML patient in human. It will shed new insight into the mechanism of AML, as well as its diagnosis and treatment.

19

WASTE TREE – SIMPLE, VISUAL, IMPROVEMENT TOOL

Chris Andreshak, CG(ASCP)CM; Kate Kroeger, MS, CG(ASCP)CM, Seattle Cancer Care Alliance; Min Fang, MD, PhD, FACMG, Fred Hutchinson Cancer Research Center Purpose: Our institution has committed to several Values and Beliefs that center around Continuous Performance Improvement. The “elimination of all forms of waste” is one commitment. To facilitate and organize the work of eliminating waste we created a simple, visual tool; a Waste Tree. Hypothesis: Eliminating waste (processing, correction, inventory, wait time, search time, transportation, space, complexity) allows for time to be spent on those activities that add value to the patient experience and reduces or eliminates tasks that don’t. The theory is that the people who best know how to improve the work are those people doing the work. There is a necessity then to have everyone participate in bringing forward ideas for eliminating waste, tracking the ideas, and measuring the outcomes. Method: We adopted a Waste Tree approach. People write their improvement ideas on small paper forms and place them on the Tree. Once a week the ideas are discussed as a group to initially evaluate them and have the group think through them, and then to give status updates as projects progress. Whenever possible measurements are used to guide decision making and prove successful project completion. Results: To date 57% of the team members have submitted an idea. Nine ideas have gone to completion; 8 are currently in the process of being worked on; and 2 have been tabled. The projects have been of different scopes and durations and have been worked on solo or with a group. They have eliminated wastes of processing, correction, inventory, transportation, and search time. Over the course of a year we will save 23.8 hours of time, 1.6 miles of walking, $660, and 89700 hand written characters. Conclusions: These results suggest that the Waste Tree is working. It has provided an ongoing forum for bringing forward, vetting, and tracking ideas and project statuses from many engaged individuals. We believe this is a quality improvement tool that can easily be implemented in any organization and therefore would like to share this methodology.

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

150

Poster Abstracts, AGT 2016

21

A PATIENT WITH MOSAIC 45,X/47,XY,+21 DOUBLE ANEUPLOIDY

Alyssa Pollard, CG(ASCP); Sheryl Senior; Amro Mahmoud; Violeta Villani; Alma Ganezer; Daniel Di Bartolo; Carol Teplis; Julie Cox; Loretta Mahon; Philip Mowrey; Steven Gersen, AmeriPath Northeast Individuals who are mosaic for two numerical chromosome abnormalities are extremely rare. We present one such case of double aneuploidy. The proband, a seven-year-old phenotypic female, was referred for genetic testing based on a clinical indication of short stature (growth along the 5th percentile), delayed bone age, speech delay, and sensory processing disorder. She has broad shoulders and external female genitalia. Pelvic ultrasound identified a uterus with a small focus of gonadal tissue in the left adnexa, while the right gonad could not be visualized; however, she manifests signs of gender dysphoria, preferring to wear boyâ&#x20AC;&#x2122;s clothing and explicitly expressing the desire to be a boy. Hormone studies are pending, and serological screening for Celiac disease, based on family history, is also underway. Constitutional chromosome analysis identified a 45,X[12]/47,XY,+21[8] karyotype. Fluorescence in situ hybridization analysis with probes for the sex chromosomes and for chromosome 21 confirmed the karyotype but demonstrated approximately 80% monosomy X cells; FISH also confirmed the presence of SRY. There are two possible scenarios by which such mosaicism can arise: through the fusion of two abnormal zygotes, or through two mitotic nondisjunction events occurring (very early in development) in separate cells from the same 46,XY zygote. Oligonucleotide-SNP microarray analysis was performed; the results are consistent with the presence of both abnormal cell lines, with percentages similar to but even more disparate than those detected with FISH. The results provide no evidence of chimerism, suggesting two nondisjunction events to have occurred; however, given the low percentage of XY,+21 cells, chimerism cannot be completely ruled out.

22

IMPORTANCE OF PRENATAL MICROARRAY IN ELUCIDATING AN UNBALANCED TRANSLOCATION IN A CASE OF MOSAIC TURNER SYNDROME Annmarie Rupp, MA, CG(ASCP); Janine Rosenberg, MS, CGC; Shamsa Naqvi, BS, CG(ASCP); Debra Rita, MD; Jillene Kogan, MD, PhD, ACL Laboratories

We present a case report describing the comprehensive prenatal and postnatal cytogenetic workup on a 38-year-old woman at 23 gestational weeks following noninvasive prenatal screening indicating an increased risk for monosomy X, in order to highlight the importance of prenatal microarray in clarifying an unclear, abnormal prenatal karyotype. Prenatal cytogenetic studies were performed on an amniotic fluid specimen and included prenatal fluorescence in situ hybridization (FISH), chromosome analysis, and single nucleotide polymorphism (SNP) microarray. Direct prenatal FISH on the amniotic fluid identified a pattern consistent with mosaic monosomy X in 80% of the interphase cells examined. Subsequent in situ analysis of cultured amniocytes identified an abnormal mosaic karyotype of 45,X[10]/46,X,add(X)(p11.4)[5], indicating two abnormal cell lines with mosaic monosomy X and an unbalanced rearrangement involving one X chromosome. Prenatal SNP microarray results confirmed the karyotype but clarified the presence of an unbalanced translocation involving chromosomes X and 22. Nomenclature of the copy number changes was as follows: arr[hg19] Xp22.33(169,921-50,262,452)x1,Xp11.22q28(50,263,200-155,233,731) x1-2,22q11.

1q11.21(16,888,899-21,202.202,767)x2-3. Postnatally, neonatal blood chromosome analysis was reported as 45,X[11]/46,X,der(X) psu dic(X;22) (p11.22;q11.21)[9]. Direct neonatal blood SNP microarray identified negligible variation in breakpoints; nomenclature was as follows: arr[hg19] Xp22.33(168,546-50,278,715)x1,Xp11.22q28(50,281,651-155,233,731)x1-2,22q11. 1q11.21(16,888,89920,312,661)x2-3. The degree of mosaicism calculated in the postnatal microarray analysis correlated to the 45,X cell line being present in approximately 70% of the sample whereas the derivative X chromosome was present in approximately 30%. Maternal blood chromosome analysis revealed a normal, 46,XX karyotype. These findings indicate the importance of microarray testing in the context of an unclear but abnormal karyotype, but they also raise various challenges in both the cytogenetic and clinical settings. The difference between the mosaicism levels in the prenatal and postnatal cytogenetic studies illustrate not only the difference between the cell types found in amniotic fluid versus those present in the blood but also whether testing was completed on a cultured or a direct specimen. Additionally, these findings present a diagnostic challenge to the clinician due to the inability to predict the pattern of X-inactivation and its influence on phenotype, specifically whether or not the X-inactivation occurring in cells with an abnormal X chromosome derived from an X; autosome translocation will spread to include any or all of the autosomal chromosome material.

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

151

Poster Abstracts, AGT 2016

24

HISTOLOGIC CHARACTERISTICS OF GASTROESOPHAGEAL ADENOCARCINOMAS AND THE CHALLENGES OF HER2 FISH TESTING AND ACCURATE INTERPRETATION Meghan Kozub, CG(ASCP); Jason A. Yuhas; Kathryn E. Pearce; William R. Sukov, Mayo Clinic

Accurate HER2 testing on gastroesophageal (GE) adenocarcinomas is essential but poses a significant challenge for clinical cytogenetics laboratories. In recent years, effective therapies directed at tumor overexpression of the human epidermal growth factor receptor 2 (HER2) have become more widely available for breast carcinomas. Patients with GE adenocarcinomas testing positive for HER2 amplification have demonstrated a clinical benefit with Trastuzumab (Herceptin); which targets the HER2 protein. Anatomical location and histologic characteristics of GE tumors make fluorescence in situ hybridization (FISH) analysis for HER2 amplification difficult. Initial assessment of the GE lesion depends on obtaining a small biopsy via endoscopy. Invasive gastroesophageal adenocarcinoma often arises in a background of interstinal metaplasia with high-grade dysplasia (HGD) and it can be difficult to differentiate the invasive tumor from the dysplastic area. This is critical because it has been well documented that interstinal metaplasia with HGD has a very high frequency of HER2 amplification. Conversely, invasive adenocarcinoma has a much lower frequency of amplification, even in the presence of highly HER2 amplified HGD. As treatment is directed toward the invasive component, assessment of the incorrect area of tissue may lead to erroneous results and incorrect treatment. Technologists evaluating HER2 status in GE specimens with a large amount of high-grade dysplasia has many practical difficulties. It can be challenging for technical staff to differentiate between well differentiated adenocarcinoma and HGD on FISH specimens. Accurate identification of invasive tumor versus HGD by a pathologist both before and during FISH analysis can provide reliable detection of HER2 gene status in adenocarcinoma. Of over 1,700 clinical GE biopsies analyzed, a subset of recent cases that were negative for HER2 gene amplification was identified. Slides were reviewed by a pathologist to identify biopsies demonstrating areas of both invasive carcinoma and HGD. The HER2 status of the respective areas was then compared to determine the frequency at which the HER2 status was discordant. The frequency of discordance highlights the need for cooperation of the pathologist and the FISH technologist during HER2 analysis of GE biopsies to provide accurate interpretation.

25

INTRACHROMOSOMAL AMPLIFICATION OF CHROMOSOME 21 (IAMP21) IN PEDIATRIC ALL

Jennifer Alvares, CG(ASCP); Tracy Raoul, CG(ASCP); Olyesa Borinets, CG(ASCP), The University of Chicago Medicine We discuss the case of a 9-year old female with a past medical history of juvenile idiopathic arthritis (JIA) who was admitted to The University of Chicago Medical Center with symptoms of increased nausea, generalized pains, increased fatigue, decreased appetite, intermittent headaches and 30% circulating blasts in peripheral blood smear. Additional workup established the diagnosis of B-cell acute lymphoblastic leukemia (B-ALL). Upon receipt of the bone core sample, one unstimulated culture was set up for chromosome analysis and fluorescence in situ hybridization (FISH). An aliquot of 0.7ml of bone core suspended in media was reserved for chromosomal microarray analysis (CMA). Karyotype analysis showed one abnormal clone 47,XX,+X,dup(21) pter>q11.2::q22.3::q11.2->pter)[19]/46,XX[1]. Interphase FISH analysis revealed an amplification of the RUNX1 (21q22) locus in 98% of cells and was negative for BCR/ABL1 and ETV6/RUNX1 translocations, an MLL rearrangement, and trisomy for chromosomes 4, 10, and 17. Chromosomal microarray analysis (CMA) confirmed trisomy for chromosome X and detected numerous complex chromosomal rearrangements of chromosome 21. Multiple breakages on chromosome 21 consistent with a chromothripsis event were detected resulting in intrachromosomal amplification of the region 21q21.2q22.3 (iAMP21). In addition, CMA revealed homozygous deletions at 9p21.3 and 13q14.2 regions associated with a biallelic loss of the CDKN2A and RB1 tumor suppressor genes, respectively. RB1 deletion and the gain of chromosome X have been reported as a secondary markers associated with iAMP21 to assist in diagnosis of B-ALL (Harrison et al., 2014). The presence of iAMP21 in childhood B-cell precursor ALL is associated with a high relapse rate under standard treatment regimens for ALL, whereas an intensive therapy treatment has shown an improved outcome (Moorman et al, 2013; Heerema et al, 2013; Harrison et al, 2014). The patient tolerated induction therapy per AALL0932 with plans to begin consolidation therapy per AALL1131. FISH analysis of a post-treatment sample for residual disease was negative for a gain of RUNX1 (21q22) and the most recent hematopathology report revealed a hypocellular marrow with regenerative trilineage hematopoiesis and no evidence of residual B-ALL.

26

FISH ASSAYS FOR CRLF2 REARRANGEMENTS IN ALL - ONE LABORATORY’S EXPERIENCE

Jessica Lincoln, CG(ASCP); Billie Carstens, CG(ASCP); Lynne Meltesen, CG(ASCP); Kim Harding, CG(ASCP); Karen Swisshelm, PhD, Colorado Genetics Laboratory Overexpression of the lymphoid cytokine receptor gene, CRLF2, is a more recently described abnormality in high risk acute lymphoblastic leukemia (ALL). It’s found especially in Down syndrome, Hispanic, and adolescent ALL patients and associated with Ph-like expression pattern of JAK1, JAK2, and IKZF1 mutations. Because rearrangement of CRLF2 is cryptic by standard cytogenetic chromosomes, an interphase FISH assay can be helpful to identify potential patients. This is especially true in cytogenetic laboratories that do not offer other molecular techniques such as genomic profiling by sequencing, SNP array, gene expression profiling or targeted arrays. The CRLF2 overexpression occurs in one of three mechanisms: an interstitial deletion leading to P2RY8 in the pseudo-autosomal region of X and Y, a rearrangement with IGH (14q32), and a mutation of the gene. The first two scenarios may be investigated using dual color FISH probe, while the last would need to be studied by PCR and sequencing. Our laboratory has been performing FISH analysis for CRLF2 since 2014 on diagnostic studies of eleven pediatric ALL cases, especially those enrolled in Children’s Oncology Group (COG) clinical trials. Using Cytocell FISH probe two cases with deletion and one case with translocation have been identified.

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

152

Poster Abstracts, AGT 2016

27

VALIDATION OF TWO CANCER NEXT GENERATION SEQUENCING PANELS: HEREDITARY BREAST AND OVARIAN CANCER AND LEUKEMIA PANELS

Brittany Hennigan, MB(ASCP)CM; Renee Bend, PhD; Raymond Louie, PhD; Katharine Kubiak, MS; Stephen McGee, MS; Mike Friez, PhD, FACMG, MS, Greenwood Genetic Center; Julie Eggert, PhD, Clemson University; Alka Chaubey, PhD, FACMG, MS; Fatima Abidi, PhD, MS, Greenwood Genetic Center Breast cancer is the most common cancer in women, affecting about 1 in 8 women in their lifetime. Approximately 5 to 10% of breast cancers and 10 to 15% of ovarian cancers are believed to be hereditary. Leukemia is a group of malignant disorders that affects the blood and bone marrow. It is the leading cause of death among children and young adults under the age of 20. To find a genetic cause for patients with newly diagnosed hereditary breast and ovarian cancer (HBOC) or leukemia, we have designed and validated two Next Generation Sequencing panels. The HBOC panel consists of 16 genes, and the Leukemia panel contains 62 genes. These panels are designed to be more comprehensive because they not only target hot-spot regions as do other cancer panels, they also sequence the entire coding region of each gene. The SmartChip NGS Target Enrichment System (WaferGen Biosystems, USA) was used to enrich the coding regions of the genes of interest followed by massively parallel sequencing of 16 breast cancer samples and eight leukemia samples via Illumina’s MiSeq (Illumina, USA) chemistry. This method allows for analysis of greater than 99% of the targeted sequence using our in-house bioinformatics pipeline. The 16-gene HBOC panel and the 62-gene Leukemia panels were validated using positive blinded samples. As part of an ongoing collaborative research study, 115 newly diagnosed cases of breast cancer and 26 newly diagnosed patients with leukemia have subsequently been sequenced. For HBOC samples, this study has identified pathogenic mutations in 12% of the patients, and variants of uncertain clinical significance in 28%. The pathogenic mutations were identified in the following genes: BRCA1 (3), BRCA2 (4), PALB2 (2), PTEN (1), MSH2 (1) and MUTYH (1). For the remaining 59% of the cases, no variants deemed to be novel or potentially clinically significant were identified. The leukemia cases are only 15% completed, however, we have identified pathogenic mutations in the FLT3 and NPM1 genes. Our findings show the diagnostic performance of our custom NGS panels and are likely to have enhanced clinical utility over “hot-spot” panels, which could have a significant positive impact on patient management and care.

28

UTILIZING CELL ENRICHMENT AND FLUORESCENCE IN SITU HYBRIDIZATION (FISH) TO IDENTIFY BONE MARROW INVOLVEMENT OF MATURE B-CELL NEOPLASMS

Shannon Chan, CG(ASCP) CM; Derek Lyle, MD; Mayuko Imai, MD; Austin Mauch, CG(ASCP) CM; Kyle Lynes, CG(ASCP) CM; Erik Cancino; Rene Castillo; David Kolker, Genoptix; J. Dianne Keen-Kim, PhD, FACMG, Color Genomics; Farzad Nooraie, MD, DABGG, Genoptix Mature B-cell malignancies are a form of Non-Hodgkin Lymphoma (NHL) that often mimic normal cell differentiation and can thus be difficult to classify, yet make up 80-85% of the subtype. Once an initial diagnosis has been made, further studies are needed to determine the type of malignancy. Cytogenetic analysis is an invaluable tool that can provide diagnostic and prognostic information about mature B-cell neoplasms; however, mature B-cells do not typically grow well in cytogenetic cultures. Once patient samples are analyzed, abnormal FISH results may be missed if bone marrow or peripheral blood involvement is minimal. To increase the sensitivity of abnormal mature B-cell identification, we developed a cell enrichment method using pan B-cell antibodies. This cell enrichment separates mature B-cells from the remaining bone marrow and/or peripheral blood cells, allowing for increased detection. The assay was validated using 59 bone marrow and peripheral blood specimens. Specimens used had 0.1-10% monoclonal mature B-cells detected by flow cytometry and were enriched using cell-separating technologies. Each specimen was divided into four parts, then cultured or enriched using three different pan B-cell antibodies. Non-enriched and enriched samples were compared for results. A variety of FISH probes were hybridized (based upon initial reason for referral) and analyzed; FISH results were obtained by two independent groups of scoring technologists. Validation results demonstrate enrichment increased the sensitivity of FISH abnormality detection: 29% (17/59) of samples had abnormalities that were detected in both the enriched and non-enriched portions. However, detection was on average 15-fold more sensitive in the enriched portions. Overall, FISH abnormalities were detected at a higher incidence in the enriched samples versus non-enriched. New abnormalities were detected within the enriched portion in 42% (25/59) of samples that were not detected in the standard non-enriched portion. Of these, 64% (16/25) had a FISH abnormality that was a critical finding for the diagnosis, prognosis, and/or management of the patient. The average detection rate of FISH aberrations in the non-enriched portion was 3% (at or near the experimentally determined cut-off value for most FISH probes). In contrast, the average detection rate of FISH abnormalities in the enriched portion was 56%. In 5% (3/59) of cases, detection of FISH aberrations in enriched specimens helped to distinguish two separate neoplastic processes in the bone marrow. NHL cases with low-level bone marrow and/or peripheral blood involvement can be difficult to detect. By utilizing mature B-cell enrichment and FISH analysis, higher abnormality detection rates can be obtained and more informative patient results can be delivered.

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

153

Poster Abstracts, AGT 2016

29

IMPORTANCE OF CYTOGENETIC TESTING IN DISORDERS OF SEX DEVELOPMENT TO CLARIFY ETIOLOGY AND FUTURE CLINICAL COURSE

Kimberly Gobac, BS, CG(ASCP) CM ; Rhonda Bachman, BS, CG(ASCP) CM ; Gloria Haskell, PhD; Anna Horne, BS; Laurelin Cottingham, BS, CG(ASCP)CM; Kristen Deak, PhD, FACMG; Sarah Horn, PhD, DABMG; Catherine Rehder, PhD, FACMG, Duke University Health System The sex-determining region Y gene, SRY, located on the Y chromosome, begins the process of directing undifferentiated gonads into forming testes, while an absence of the gene defaults to form ovaries. Areas in the pseudoautosomal regions (PAR) of the X and Y chromosome recombine during meiosis I, however, when crossover events occur outside of the PAR, these exchanges can cause XX males and XY females. Disorders of sex development (DSD) can also be caused by other factors, including Congenital Adrenal Hyperplasia (CAH), which is the most common cause of ambiguous external genitalia in newborns. In 2015, our laboratory received two peripheral blood samples from phenotypic male patients with DSD. Chromosome analysis showed a 46,XX karyotype for both cases. To exclude the possibility of a sample swap, we used fluorescence in situ hybridization (FISH) with the CEP XY alpha satellite probe set on a blood smear to confirm the blood tube received contained no Y centromere material. With no evidence of a Y centromere, we continued with the SRY/CEP X FISH probe set to determine if the SRY locus could be detected on the X chromosome. One case, with a clinical indication of gynecomastia and hypogonadism, was positive for this translocation resulting in a derivative X chromosome containing the SRY gene. Individuals with this rearrangement are almost always phenotypically male, but may experience hypogonadism and gynecomastia, and are normally infertile. Cases resulting in SRY positive XX males typically result from sporadic errors in paternal meiosis, and therefore are not recurrent in families, although extremely rare cases of familial translocations have been reported. Case 2 had ambiguous genitalia. In this case, the SRY gene was not detected by FISH analysis. This outcome was consistent with the elevated 17-Hydroxyprogesterone found during the newborn screening of the patient and working clinical diagnosis of CAH. These two cases emphasize the utility of cytogenetic testing, together with other clinical information, to help determine the etiology of DSD and clarify the clinical course.

30

VARIANT DETECTION AND DIAGNOSIS OF RASOPATHIES UTILIZING A NEXT GENERATION SEQUENCING PANEL

Jessica Cooley, BS, MB(ASCP)CM; Devin Rhodenizer, BS; Stephen McGee, MS; Katharine Kubiak, MS; Frank Bartel, PhD, FACMG; Michael J. Friez, PhD, FACMG; Jennifer A. Lee, PhD, FACMG, Greenwood Genetic Center The RASopathies make up a large group of syndromes that occur from alterations in the genes of the RAS-MAPK pathway. This pathway is responsible for cell proliferation, cell differentiation, and cell death, and alterations of the pathway cause a variety of effects across many of the body systems. Common features of the RASopathies include dysmorphic features, short stature, skin lesions, heart defects, developmental delay, intellectual disability, and often times an increase in cancer risk. While the RASopathies share several features, each of the different syndromes also has its own set of unique features. In an effort to provide testing more rapidly and cost effectively, a next generation sequencing (NGS) panel has been developed that includes 23 genes (A2ML1, MAP2K1, RASA2, BRAF, MAP2K2, RIT1, CABIN1, NF1, RRAS, CBL, NF2, SHOC2, HRAS, NRAS, SOS1, KAT6B, NSUN2, SOS2, KRAS, PTPN11, SPRED1, LZTR1, RAF1) known to be associated with the different RASopathies. Eighteen samples were included in the RAS panel validation: 14 had (abnormal) variants previously detected by Sanger sequencing, three were not previously tested, and one (Coriell sample NA12878) was used for concordance analysis for comparison to the NIST control. Several of the known validation samples were duplicated for intra-run and interrun comparison. Agilent SureSelect was used to capture the exons and flanking intronic regions of interest, and the prepared samples were run on the Illumina MiSeq. Overall, the average depth of coverage was 820X and the read quality an average of 99.3%. All expected variants were detected in the 14 known abnormal controls. To date, seven diagnostic samples have been sequenced for routine clinical testing, with an average depth of coverage of 478X and an average of 97.8% read quality. Including the three samples from the validation run that were not previously tested, out of ten patients seven had abnormal findings with at least one pathogenic or likely pathogenic alteration or at least one variant of uncertain significance (VUS) detected. Of these seven samples, four pathogenic or likely pathogenic alterations were found (one each in four different patients), as well as six variants of uncertain significance, and one likely benign variant. The four pathogenic alterations included two frameshift mutations in NF1, a synonymous change in NF1 suspected to affect splicing, and one missense mutation in BRAF. The six variants of uncertain significance included one missense alteration in RIT1, one missense and one intronic alteration in CABIN1, two in-frame deletions in KAT6B, and an intronic insertion in A2ML1. The remaining three patients were reported as normal, with no causative pathogenic variant or VUS identified. These results give a diagnostic yield of 40% for the detection of a pathogenic or likely pathogenic variant. All reported alterations were confirmed by Sanger sequencing.

Given the high detection rate of pathogenic alterations (40%), and the ability to analyze multiple genes in a short time period by NGS, it can be concluded that NGS is a cost-effective, time-efficient, and useful method for diagnostic testing of patients suspected of having a defect in the RAS-MAPK pathway.

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

154

Poster Abstracts, AGT 2016

31

DESIGNING A COST-EFFECTIVE NEXT-GENERATION SEQUENCING STUDENT LABORATORY

Ericka Hendrix, PhD, MB(ASCP); Katie Bennett, PhD, MB(ASCP); Trevor Burrow, MS, MB(ASCP), Texas Tech University HSC; Philip Schieble, MS, MB(ASCP); Travis Warmoth, MS, MB(ASCP), Pathogenius Next-generation (NGS) sequencing is a highly sought after technology making its way into clinical laboratories, and particularly all molecular laboratories. The need for skilled graduates to perform and analyze data is climbing as the use of NGS is gaining momentum in the clinical arena. Clinical practice and familiarity with NGS has become paramount in molecular diagnostics programs. Producing graduates that have hands on experience with this technology is imperative; however, incorporation of NGS is a daunting task for most educational programs. The challenges faced by most educational institutions include the high cost associated with NGS and access to this costly equipment. Our DMS program in partnership with its local affiliate has developed a cost effective and relatively simple instructional protocol that provides students with a hands-on NGS experience using the Ion Torrent. The laboratory was designed to give students the experience of the workflow involved in NGS and the analysis of NGS results. The first step of laboratory involved a lecture about the concepts of NGS and library preparation. Next, students were given the protocol to make a DNA library from unknown bacterial colonies by using the Qiagen mini prep kit. PCR was performed with a primer that is specific for 16S and includes a unique barcode, which were supplied by the affiliate laboratory. This allows each student sample to have a unique tag for data analysis. Next, students purified their PCR products using magnetic beads and a magnet supplied by the affiliate, followed by quantification on the Agilent Tapestation and NanoDrop (in house resources). The samples were then pooled and given to the affiliate laboratory for an additional purification and quantitation procedure before loading onto the Ion Torrent chip. Because each student had a unique barcode embedded in the 16S primer, their specific samples were tracked during NGS data analysis and the detection of all bacterial species in their unknown sample was linked to their unique tag. The results of this student laboratory experience revealed that a quality Next Generation sequencing experience is possible and relatively inexpensive for DMS programs to include in their curriculum. The total cost of the laboratory was around $50 per student.

This includes the cost of the sequencing run ($700 for all 20 students) and approximately $300.00 for the primers, DNA isolation kit, PCR reagents and the Tape Station. Suggested improvements include the purchase of another magnet ($500.00), as this was a limited resource, using a better lysing agent such as lysozyme to lyse gram positive bacteria and providing students with access to watch the setup of the NGS instrument. Survey results of the experience will be collected at the end of the semester for further analysis and insight into student satisfaction and learning outcomes.

32

THREE CASES OF LIVE-BORN TRIPLOIDY

Marsha Waters, CLSP, CG(ASCP); Abbas Padeganeh, PhD; Elliot Stolerman, MD; Katie Clarkson, PhD; Chris Houk, Greenwood Genetic Center; Carolyn Lovell, MS; Ravindra B. Kolhe, MD, Georgia Regent University; Alka Chaubey, PhD; Barbara R. DuPont, PhD, Greenwood Genetic Center Triploidy is a common cytogenetic abnormality observed in pregnancy loss and in prenatal diagnosis, especially those pregnancies with abnormal ultrasound findings. Triploidy occurs when three complete haploid (1n) chromosome contribution, two from one parent (2n) and one set from the other parent(1n), are combined to result in a 69(triploid) chromosome conceptus(3n). Because the majority of these triploid conceptions abort early in the pregnancy, it is an unusual cytogenetic finding in live born infants.

Since 1986, the Greenwood Genetic Center Cytogenetic laboratory has analyzed 239 cases of triploidy. Those cases included 217(91%) products of conception/fetal tissue samples, 18(7.5%) prenatal cases, and 4(1.5%) peripheral blood samples. Three of the peripheral blood samples represent live born infants. Chromosome analysis undertaken to evaluate a dysmorphic newborn showed two cases of 69,XXX and one 69,XXY. All three cases were born 9 weeks prematurely. Case 1 was a male with a partial Dandy-Walker malformation observed prenatally by ultrasound. Birth was due to pre-eclampsia. His weight, length, and head circumference were less than 10th percentile. He had ambiguous genitalia, cleft palate, microphthalmia, coloboma on right, and hydronephrosis, as well as anemia, cholestasis, hyperbilirubinemia, intraventricular hemorrhage, patent ductur arteriosis, pulmonary hypertension and respiratory distress syndrome. He lived one month. Case 2, a female, had polyhydramnios, abdominal cysts, and a Dandyâ&#x20AC;&#x201C;Walker malformation observed prenatally by ultrasound. Birth was early due to pre-eclampsia. At birth, weight was 50 th percentile, length 25th percentile and head circumference 75th percentile. She had fusion of the 2nd and 3rd digits on both hands and fusion of the 3rd and 4th digits on right foot and all digits on left. She lived 10 days. Case 3, a female, had an abnormal prenatal quad screen for trisomy 18, as well as a normal non-invasive prenatal screen showing a result of 46,XX. Prenatal ultrasound showed interuterine growth retardation, oligohydramnios and micrognathia. At birth, weight and length was less than 3rd percentile and head circumference was at the 3rd percentile. She had micrognathia, low set ears, microophthalmia (left greater than right), syndactyly of 3rd and 4th fingers on left hand, overlapping 2nd and 3rd toes bilaterally, small left thumb, hypoplastic female genitalia, sacral dimple, 11 pairs of ribs and large patent ductus arteriosus. She lived 7.5 weeks. Live-born triploidy infants are rare and frequently demonstrate multiple anomalies on prenatal ultrasound. Herein we provide further detail on these rare cases and possible mechanisms for triploidy.

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

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Poster Abstracts, AGT 2016

33

THE UTILITY OF MICROARRAY ANALYSIS IN IDENTIFICATION OF THE NUP98-NSD1 FUSION GENE IN PEDIATRIC MIXED LINEAGE LEUKEMIA WITH NORMAL KARYOTYPE

Jane Casey, CG(ASCP); Maureen Sherer, CG(ASCP); Zach Ou, MD; MaryAnn West, CG(ASCP); Sally Kochmar, CG(ASCP); Jie Hu, MD, PhD; Urvashi Surti, PhD; Svetlana Yatsenko, MD, Magee-Womens Hospital of UPMC We report the finding of cytogenetic abnormalities due to a neoplastic process in a thirteen year-old female patient presenting with fatigue, anemia, diarrhea, weight loss, and daily nausea with vomiting. The patient’s health history was unremarkable for birth and developmental defects, physical problems, inherited familial conditions, and exposure to toxins. A CBC showed a white blood cell count of 25,300 with 38% blasts. Flow cytometric immunophenotypic studies indicated involvement by an acute leukemia that was difficult to characterize. Bone marrow evaluation confirmed the diagnosis of mixed phenotype acute leukemia (MPAL), also known as mixed lineage leukemia, comprising 58% of cells in the marrow. Cytogenetic studies on bone marrow showed a 46,XX karyotype.

FISH was negative for monosomy 7, 5q31 and 7q31 deletions, trisomy for chromosomes 4, 10, and 17, the PAX5 gene deletion, and for the BCR/ABL1, KMT2A, ETV6/RUNX1, RUNX1T1/RUNX1, CBFB, TRAD, TRG, and TRB gene rearrangements. Chromosomal microarray analysis on the bone marrow sample revealed a heterozygous 390 kb deletion in the 5q35 region involving part of the NSD1 (nuclear receptor-binding SET-domain protein 1) gene. Cryptic t(5;11) (q35;p15) associated with the NSD1-NUP98 gene fusion and partial deletion of the NSD1 gene has been reported as a rare somatic abnormality in childhood acute myeloid leukemia.

Additional FISH studies on metaphase cells were completed on the diagnostic bone marrow sample to rule out a rearrangement involving 5q; specifically the t(5;11). FISH with the 5q telomere specific probe was positive for a translocation and produced a hybridization signal on the 11p terminus. The NUP98 gene rearrangement has been subsequently identified in 90.8% of interphase cells studied by FISH. The patient started induction chemotherapy and the disease status has been successfully monitored for the percentage of cells positive for the NUP98 rearrangement. Post treatment immunohistochemistry showed no evidence of disease and cytogenetic analysis showed an apparently normal female karyotype. Bone marrow evaluation demonstrated ~1.7% blasts by morphology, but no abnormal blast population by flow cytometry, and FISH was negative for the NUP98 gene rearrangement in 99.5% of the cells. After the third cycle of chemotherapy a bone marrow evaluation revealed 4% blasts by morphology but was negative by flow cytometry; however, FISH was positive for the NUP98 gene rearrangement in 28.3% of the cells. Allogenic stem cell transplant was planned but because of persistent mixed lineage leukemia and hospital admission due to acute cholecystitis, transplantation was postponed. Bone marrow aspiration following cholecystectomy was positive for the NUP98 gene rearrangement in 80.6% of cells and classical cytogenetic analysis showed an abnormal cell line with t(3;16;7). It is unclear whether the NUP98 gene rearrangement is present in this cytogenetically visible abnormal clone. This unusual case illustrates the utility of microarray analysis in the diagnosis of pathogenic alterations in cancer cells that are beyond the detection resolution of traditional cytogenetic techniques. Microarray findings for this patient led to the identification of NSD1-NUP98 gene fusion, facilitating further monitoring of the patient’s bone marrow status during disease progression by interphase FISH using the NUP98 breakapart DNA probe.

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CYTOGENETIC CHARACTERIZATION OF MYELOID NEOPLASMS WITH T(2;3) (P13-25;Q25-29): AN ANALYSIS OF 61 CASES Alexis Dowiak; Carlos A. Tirado, PhD; Justin Buchanan, Department of Pathology and Laboratory Medicine, UCLA School of Medicine

Chromosomal translocations involving the short arm of chromosome 2 and the distal part of the long arm of chromosome 3 are rare yet recurrent findings in patients with myeloid neoplasms, and are associated with a poor prognosis. Such rearrangements can contribute to ectopic expression or formation of fusion genes involving the EVI1 (3q26) (ecotropic virus integration site 1 proton homolog) gene. However, the exact mechanism by which EVI1 and such translocations affect leukemogenesis remains unclear. Herein, we present an analysis of 60 cases with t(2;3)(p13-25;q25-29) in various myeloid diseases that were compiled from the literature, including 37 cases of acute myeloid leukemia, 6 cases of chronic myelogenous leukemia, 3 cases of chronic myeloproliferative disorder, and 13 cases of myelodysplastic syndrome. Among these, t(2;3) existed as a sole abnormality in 22 and with concomitant abnormalities in 38. In the latter group, t(2;3) was a primary abnormality in 39%, a secondary abnormality in 8%, and this could not be determined for 53% of cases. The most common concomitant abnormalities reported were -7, 5q-, and t(9;22), occurring in 28%, 20%, and 12% of cases respectively. Among the 19 cases in which molecular cytogenetic analysis was performed in order to assess the involvement of the EVI1 locus,16 (84%) were shown to involve the gene. This is one of the first large-scale analyses of t(2;3) ((p13-p25;q25-29), an anomaly not extensively molecularly characterized, and provides additional information about the nonrandom incidence of this translocation and its distribution in the spectrum of myeloid neoplasms.

Additionally, most cases had concomitant abnormalities, the majority of which are recurrent and prognostically relevant in myeloid disease. Considering the severe clinical outcome associated with this translocation, further investigation about the molecular mechanisms by which it contributes to carcinogenesis, its interplay with concomitant abnormalities, and its diagnostic and prognostic implications is warranted.

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

156

Poster Abstracts, AGT 2016

35

DISORDERS OF SEX DEVELOPMENTâ&#x20AC;&#x201D;DETECTING SRY POSITIVE 46,XX TESTICULAR DISORDERS OF SEX DEVELOPMENT (TDSD) BY MICROARRAY

Christina Mendiola, CG(ASCP) CM; Veronica Ortega, CG(ASCP) CM; Meiqing Zhu, CG(ASCP) CM; Sheila Hampton, CG(ASCP) CM; Gopalrao Velagaleti, PhD, FACMG, University of Texas Health Science Center-San Antonio

Disorders of sex development (DSD) are defined as congenital conditions associated with the atypical development of chromosomal, gonadal, or anatomic sex. It is estimated that 1:5,500 newborns present with ambiguous genitalia with the majority (60%) being detected prenatally. Congenital adrenal hyperplasia (CAH) and mixed gonadal dysgenesis are the most common causes of ambiguous genitalia, comprising approximately over 50% of all cases of genital ambiguity in newborns. For clinicians and parents, this is a challenging period when treatment and surgical interventions to assign gender become time sensitive. Having a diagnosis and identifying the underlying etiology of ambiguous genitalia is of utmost importance in making the appropriate decisions in management of these children. The first line testing in newborns with ambiguous genitalia includes karyotyping to determine chromosomal sex. Without complete information, parents and eventually the affected child has an inadequate understanding about DSDs and their specific diagnosis. We report a case of a 3-day old newborn presenting with hydrops and ambiguous genitalia. Since time is of essence in determining the chromosomal sex, we implemented our unique microarray reflex testing policy where a 24-hour preliminary karyotype to rule out major chromosome abnormalities is performed and subsequently SNP-oligo microarray if no chromosomal abnormalities are observed on the preliminary study. The preliminary study showed a normal 46,XX karyotype. The reflex microarray using the only FDA cleared CytoScan DX assay from Affymetrix showed loss of a 9 Mb region on X chromosome from Xp22.2 to pter and a simultaneous gain of a 7.1 Mb region on Y chromosome from Yp11.2 to pter. Based on this result, it was interpreted as a possible unbalanced Xp;Yp translocation. Additional FISH studies with SRY probe confirmed presence of SRY on Xp. The newborn is thus diagnosed with SRY-positive 46,XX-TDSD which is a rare condition occurring in 1/20,000 newborn males. Clinical features of this condition include hypospadias, undescended testes, and various degrees of inadequate virilization in external genitalia. Approximately 80-90% of patients have normal male external genitalia with only about 10-20% presenting with undermasculinized external genitalia as seen in our patient. It was suggested that spreading of X-inactivation into SRY or change in the SRY position relative to chromosomal environments (position effect) may compromise SRY expression, leading to undermasculinization. The Xp;Yp translocations can be caused by non-allelic homologous recombination (NAHR) and is thought to be mediated by a paracentric inversion on Yp in some males. The positions of Xp and the Yp breakpoints are variable among patients, and the genomic basis for Xp;Yp translocations remains to be clarified in most patients, especially in those born to Yp inversion-negative males. To determine if the father of our proband is a carrier of this paracentric inv(Y), chromosomal studies on the father are requested as this information is vital for calculating recurrence risks and providing counseling regarding reproductive planning.

36

THE INCIDENCE AND TYPES OF CONSTITUTIONAL MOSAICISM OBSERVED BY SNP MICROARRAY ANALYSIS IN A LARGE COHORT Peri Buus,CG(ASCP)CM; Rachel D. Burnside, PhD, FACMG, LabCorp

The incidence of constitutional mosaicism is not precisely known, but is likely underestimated, as asymptomatic individuals are not likely to be identified and chromosome analysis is not always able to detect mosaicism. Constitutional mosaicism is defined as two or more distinct cell lines in an individual from a single zygote. Recent studies have estimated up to 1% of patients referred for cytogenomic testing show mosaicism by microarray. We undertook a retrospective study of mosaicism observed by SNP array to better estimate the incidence from a large cohort of patients and identify the types and estimate percentages of the types of mosaicism observed. The limitation of the study is the level of detection of mosaicism, which is dependent on both percentage and size of the mosaic segment. We have detected whole chromosome mosaic trisomy as low as 4%, but the reliability of the data decreases in proportion to the size of the mosaic interval, which may result in an underestimate of incidence. From December 1, 2008, we have performed ~100,000 postnatal SNP microarrays, and have identified a cohort of cases with various types of mosaicism demonstrating different mechanisms of formation. Types of mosaicism we have observed include structural mechanisms, such as autosomal trisomies and monosomies; sex chromosome aneusomies; supernumerary ring or marker chromosomes; recurrent microdeletion/microduplication regions; unbalanced translocation derivatives; terminal deletion/inverted duplication with telomere rescue; isodicentrics or isochromosomes; and other terminal or interstitial deletions or duplications, and segmental uniparental disomy (UPD). Such data from array studies show mosaicism can arise from a number of different mechanisms and that it is more common that can be appreciated by chromosome analysis alone. This study also emphasizes the clinical utility of microarray analysis for individuals with abnormal phenotypes.

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

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Poster Abstracts, AGT 2016

38

CLINICAL VALIDATION OF A SIMPLE FISH ASSAY THAT USES 1UL PROBE

James Stanchfield, PhD, SciGene; Eric Crawford, PhD; Mingya Liu; Cynthia Brooks, Genetics Associates; David Wright, Art Robbins Instruments Fluorescent in situ hybridization (FISH) using DNA probes is a common procedure used by all cytogenetics laboratories for detecting chromosomal aberrations in human cancers. However, the high cost of DNA probes and a labor intensive workflow, coupled with the recent dramatic reductions in reimbursements, threaten the economic viability of FISH-based tests. MicroFISH (SciGene; Sunnyvale, CA) is a miniaturized, slide-based FISH assay that reduces probe cost per test up to tenfold and the labor used to prepare and process assay slides by over 60%. The MicroFISH assay uses a multi-well slide for performing up to eight independent microvolume FISH assays using only 1 Îźl of cell sample and probe. Wells are formed with a novel hydrophobic coating that keeps cell and probe solutions within its boundaries. A single coverslip seals all wells by bonding directly to the coating without the need for any sealant, such as rubber cement. Eliminating sealant allows coverslips to be automatically removed in batches of 1 to 24 slides by mechanical agitation using the Little Dipper Processor (SciGene), thereby removing the tedious step of oneby-one removal of sealant and coverslip from each slide. In this report, we present results of a clinical validation study using the MicroFISH assay to process 100 patient samples in parallel with standard methodology. Subjective grading on a 1 to 5 scale by two technologists reading both sets of slides showed comparable hybridization quality. Less than 2% of the hybridization spots required a retest using either method; showing clinical validity of the process. The MicroFISH system is now in routine clinical use processing over 20,000 patient samples annually. Recently, a high-speed liquid handler, Scorpion (Art Robbins Instruments, Sunnyvale, CA), has been applied to automate MicroFISH slide preparation, further streamlining the workflow and increasing quality control by: (1) automating slide dropping and probe application; (2) providing inventory tracking and batch reports with lot and tube numbers, probes and expiration dates; and (3) producing patient-specific score sheets for technologists to use during slide reading. We will show how implementation of the MicroFISH system with workflow automation dramatically reduces reagent cost and labor and make FISH-based tests in cytogenetics laboratories economically viable.

39

RING CHROMOSOME 7: A RARE STRUCTURAL ABNORMALITY IN ACUTE MYELOID LEUKEMIA

Kristie Liu; Ken Siangchin; Carlos A. Tirado, PhD, Department of Pathology and Laboratory Medicine, UCLA School of Medicine Ring chromosomes, often leading to partial deletions, are found in about 2% of cases of acute myeloid leukemia (AML) and are typically associated with a poor prognosis. Herein, we present the case of a 62-year old female who showed a markedly hypercellular marrow with sheets of myeloblasts, monoblasts and promonocytes (90% of marrow and confirmed by flow cytometry), consistent with acute myelomonocytic leukemia. Peripheral blood also showed circulating blasts with predominantly promonocytes (84%), normochromic anemia, and thrombocytopenia. Cytogenetic analysis revealed an apparent monosomy 7 and a ring chromosome in all 20 metaphases analyzed. Concurrent interphase and metaphase FISH studies revealed centromere 7 and 7q31 signals on this ring chromosome. The karyotype was then characterized as: 46,XX,r(7).ish r(7)(p13q32)(CEP7+,D7S486+)[20].

Despite the poor prognostic indication, follow up cytogenetic studies after treatment revealed a normal karyotype. According to the Mitelman Database, 35 other cases of AML with r(7) have been reported. Analysis of these cases demonstrated that r(7) was a sole abnormality in 20%, a primary abnormality in 14%, and in the context of a complex karyotype in 66%. The most common concomitant abnormality, seen in 26% of these cases, was 5q-. Other common abnormalities were constitutional trisomy 21 (20%), -7 (14%), and -17 (14%), though a large variety of other concurrent abnormalities were reported at lower frequencies. The most common r(7) breakpoints were r(7)(p22q22) and r(7)(p11q11), occurring in 20% and 13% of the cases that specified breakpoints, respectively. This case study and analysis of previously reported cases demonstrates the diversity of cytogenetic contexts in which r(7) can occur in AML and underscores the importance of FISH in the characterization of this abnormality. Further investigation of the role of r(7) in AML and other hematological malignancies is warranted in order to properly characterize it and concomitant abnormalities to elucidate its clinical implications.

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

158

Poster Abstracts, AGT 2016

41

CHARACTERIZATION OF FUSION TRANSCRIPTS OF SOFT TISSUE AND BONE TUMORS USING NEXT GENERATION SEQUENCING Ambryn Laubenstein, CG(ASCP)CM, CIncinnati Children’s Hospital Medical Center; Warren Mikako, MD, Department of Pathology, Children’s Hospital of Los Angeles; Li Xia, PhD, Cincinnati Children’s Hospital Medical Center

Next-generation sequencing (NGS) provides high throughput detection of various gene alterations. Presence of a specific fusion transcript is diagnostic for many pediatric bone and soft tissue tumors. To date, there are limited reports using NGS for diagnosing pediatric tumors in this category. We hereby test the robustness of an NGS assay, which simultaneously detect a broad spectrum of fusion transcripts without previous knowledge of the fusion partners. The panel covers 26 genes with over 70 known and novel translocations characteristic of bone and soft tissue tumors. Formalin fixed paraffin embedded (FFPE) samples from 27 patients, 1 cell line, and 2 positive controls were tested. Microscopic examinations, fluorescent in situ hybridization (FISH), RT-PCR (Reverse transcription polymerase chain reaction), and/or conventional cytogenetics were performed to confirm the original diagnosis.

Fusion transcripts were detected in 20 FFPE samples including 10 known fusion transcripts: PDGFB-COL1A1, ALK-CTLC, FOXO1PAX3, EWSR1-FLI1, TFE3-ASPSCR1, EWSR1-WT1, PLAG1-HAS2 and SS18-SSX1 in dermatofibrosarcoma protuberance (DFSP), inflammatory myofibroblastic tumor (IMT), rhabdomyosarcoma, Ewing sarcoma (ES), lipoblastoma and synovial sarcoma (SS), respectively, and ALK-EML4 and ROS1- SLC34A2 in two positive controls. We also identified novel transcripts (USP6-RUNX2 and USP6-PAFAH1B1) in aneurysmal bone cysts (ABC). Our data demonstrated sensitivity of 94.4% (95% CI: 74.2%-99.0%), specificity of 100% (95% CI: 56.6%- 100%) and 100% of reproducibility for this assay. The detection threshold for low level mosaicism was 3.25%. In conclusion, NGS is a robust technology for detection of fusion transcripts from FFPE samples. This technology overcomes the limitation of the conventional methods, allowing multiplexing in a single assay and identifies fusion partners at molecular levels without prior knowledge of the fusion partners. Ultimately, identification of fusion transcripts using NGS technology becomes more important for selecting appropriate treatments for each patient.

42

A T(1;6)(P35;P25) IN CLL

Arielle Majka, CG(ASCP); Kirsten Casavent; Kimberly Machowski; Marina Karamian; Danielle Saffioti; Haiying Meng, PhD, Baystate Health To date there have been few reported cases of chronic lymphocytic leukemia (CLL) patients with a t(1;6)(p35;p25). Patients with this abnormality comprise 0.5% of CLL cases. The gene IRF4 located at 6p25.2 is believed to be involved in this translocation. IRF4 is lymphocyte specific and negatively regulates Toll-like-receptor (TLR) signaling. TLR’s are a class of proteins that activate immune cell response. Although the effect of this translocation on prognosis is not yet known, it is often seen in conjunction with other high risk chromosomal abnormalities such as deletions of 11q and 17p. Here, we report a new case of a t(1;6)(p35;p25) in a CLL patient.

The patient is a 68 year old female with a diagnosis of involvement by low grade B-lymphoproliferative disorder phenotypically distinct from that seen previously. Other abnormalities in this patients karyotype included a deletion of 9(p21) and +12.

43

A COMPLEX KARYOTYPE OF AN UNUSUAL T-PLL CASE INVOLVING MULTIPLE T-CELL RECEPTOR SITES

Nanette Kendall, BS; James Blachly, MD; Carol Cole, BS, CG(ASCP); Cindy Cox, BS, MT(ASCP); Afton Smith, BS, CG(ASCP); Annette Parker, BA, CG(ASCP); Erica Parsley, BS, CG(ASCP); Christie Plickert, BS, MT(ASCP)CG; Lynne Abruzzo, MD, PhD, ABMG; Nyla A. Heerema, PhD, ABMG, FACMG, The Ohio State University Wexner Medical Center A 57-year-old male was referred to oncologists for evaluation after a regional ER visit uncovered an extremely elevated white blood cell count of >700K/μL. The patient was subsequently diagnosed with T-cell Prolymphocytic Leukemia (T-PLL). T-PLL is a very rare, aggressive leukemia of mature T-cells that is primarily found in patients over the age of 30. Routine cytogenetics revealed a highly complex karyotype and atypical abnormal FISH results were seen with the TCL1 (Dako) probe. TCL1 is located at 14q32 and is notoriously rearranged in T-cell disorders. TCL1 interphase FISH results showed 96.7% rearrangement, of which 20.6% of cells had an extra 5’ signal and 73.2% of cells had extra 5’and 3’ signals. With the multiple variant patterns seen and visibly abnormal chromosomes 14, TCL1 and TRA/D(Abbott Molecular) probes were applied to banded metaphases. TRB (Cytocell) was also applied to metaphases as it is another common T-cell receptor site located in chromosome 7 (7q34) and these appeared abnormal as well. Collectively, FISH analyses performed on previously banded metaphases showed an inversion of 14q involving a rearrangement of both TCL1 and TRA/D with the addition of a slightly separated (rearranged) TCL1 and an additional intact TRA/D signal on the p-arm of that same abnormal 14. TRB was discovered to be absent from both chromosomes 7 and relocated to an abnormal chromosome 9. TCL1, TRA/D and TRB rearrangements are common recurring abnormalities seen in patients with T-PLL and other T-cell lymphomas. As of the end of January 2016, the patient has tolerated aggressive chemotherapy treatment with alemtuzumab (Campath) and has the potential to become a candidate for an allogenic stem cell transplant in the future if cardiac issues and other complications are brought under control. There is still the possibility of a complete remission. This particular case demonstrates the importance of in-depth investigation using metaphase FISH to fully decipher a very complex karyotype and ensure the proper treatment route is offered to the patient.

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

159

Poster Abstracts, AGT 2016

45

A CASE STUDY: DEL(11)(Q?23), ETV6/RUNX1+ REARRANGEMENT AND MLL- REARRANGEMENT IN AN ADOLESCENT WITH ALL

Yvoonne Tan, MS; Xiao Li Sun; Constance Chua; Shirley Chan; Michelle Poon, MBBS, MRCP, FRCPath (UK); Leena Gole, PhD, MHGSA(Aus), CLSp(CG),DipACC(UK), Dip Gen Couns(Aus), National University Hospital, Singapore Acute lymphoblastic leukaemia (ALL) accounts for 30% of cancer in children, of which 80-90% are found to have chromosomal abnormalities. Chromosome 11q23 where MLL gene resides is most often disrupted in numerous haematological malignancies.

Translocations involving 11q23 and various partners are common, with 3-7% of ALL having MLL rearranged (MLL+) and this confers a poor prognosis. Therefore, it is helpful to identify MLL rearrangement with a confirmatory test when 11q23 is involved. However in this case study, MLL was deleted. Although patients with del(11) (q23) are generally associated with low-risk features, they are often grouped together with patients with 11q23 translocations/MLL rearrangements at point of disease evaluation. One of the common fusion gene, ETV6/RUNX1 resulting in t(12;21) translocation is found in 20-25% of all ALL cases in children. The overall prognosis of ETV6/RUNX1 is usually favourable across the board of acute leukaemias, both in children and adults. In ~5% of cases with deletion 11q, patients present a t(12;21) (p13;q22). This important association defines a sub-entity of its own, and patients with del(11)(q23)/MLL- rearrangement and ETV6/RUNX1+ ALL can probably be treated with a less intensive protocol since the prognosis is likely more favourable. Here, we present a 19 year old adolescent who described body pain, low-grade fever and lethargy at the haematology clinic after an initial finding of 28% blasts in his full blood count report (FBC). Further investigations showed more than 90% blasts in his bone marrow aspirate (BMA). Conventional cytogenetics reported 46,XY,del(11)(q?23),-13,-15,+21,+22[17]/46,XY[3], molecular studies showed a positive ETV6/RUNX1 rearrangement and fluorescence-in-situ hybridization (FISH) reported a missing copy of MLL. Our patient was treated with an intermediate-risk chemotherapy regime for adult ALL. Subsequent laboratory investigations a month later showed an excellent response following induction, achieving morphological, cytogenetic remission as well as an absence of minimal residual disease. A suitable drug regimen and concurrent patient monitoring are crucial in managing this disease.

46

THE IMPORTANCE OF MOLECULAR WORKUP IN DELINEATING WOLFHIRSCHHORN SYNDROME

Su Keyau Kee, BSc, CG(ASCP)CM; Soon Tiong Alvin Lim, PhD, MHGSA, CG(ASCP) CM, Singapore General Hospital, Singapore; Min Hwee Yong, MSc, MHGSA; Ai Chien Sharon Ling, BSc; Yit Jun Ng, BSc(Hons), CG(ASCP)CM; Tse Hui Lim, MSc, CG(ASCP)CM, Cytogenetics Lab, KK Hospital, Singapore; Meow Keong Thong, FHGSA, M Paed, MD, MBBS, UM Specialist Centre, Malaysia; Peng Chiong Tan, MRCOG, MBBS, University of Malaya, Malaysia; Sook Kit Hon, MD, MRCOG, Regency Specialist Hospital, Malaysia; Sim Leng Tien, MBBS, M Med Introduction: Wolf-Hirschhorn syndrome (WHS) is a microdeletion syndrome caused by a deletion of the short arm of chromosome 4 involving the critical region at band 4p16.3. The most common features of this syndrome include a characteristic “Greek warrior helmet” facial appearance, growth and development delay, intellectual disability, seizures and congenital heart disease. About 50-60% of WHS is caused by a pure deletion of 4p16. The remaining cases are due to a derivative chromosome 4 that had resulted from either a de novo or inherited rearrangement from a parent with a balanced rearrangement. Case Report: We present a case of a 28-year-old lady who was referred for a chromosome study of her fetus’ chorionic villi (CV) due to a previous child with WHS. The CV was setup according to standard procedures. After harvesting, banding and analysis, an apparently normal 46,XX karyotype was obtained. As the 4p deletion was detected in the first child by FISH (done by another laboratory), we suggested further performing a FISH test on the WHS critical region (WHSCR) to exclude a cryptic deletion of 4p16.3. Karyotyping and FISH were also performed on the parental bloods. The results showed the mother had an apparently normal 46,XX karyotype but the FISH result showed that one of the WHSCR signals had translocated to chromosome 22, delineating a t(4;22) rearrangement. A similar translocation FISH pattern was observed in the CV metaphase preparation, indicating the presence of a cryptic balanced translocation. Thus, a normal phenotype was to be expected in this pregnancy. Genetic counseling was appropriately given.

Discussion: The mother was found to be the heterozygote balanced translocation carrier with a 46,XX,t(4;22)(p16.3;q13) karyotype.

There are risks of her passing viable unbalanced gametes to her offspring through malsegregation during meiosis. Due to the cryptic translocation between chromosomes 4 and 22 beyond the resolution of the light microscope, this rearrangement was not detected by karyotyping. This study recommends the inclusion of the FISH assay for WHSCR in patients who have a previous child with WHS but whose karyotypes are normal. This is to identify the risks of an unbalanced rearrangement in a future pregnancy.

Conclusion: In a high proportion of WHS patients (25-30%), the chromosomal abnormality is cryptic. Our study illustrates the importance of a combination of conventional cytogenetic analysis and FISH analysis for such cases.

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

160

Poster Abstracts, AGT 2016

47

AN UNBALANCED REARRANGEMENT OF CHROMOSOME 9 INVOLVING A DIC(9;17)(P13;P12) IN PEDIATRIC B-ALL Peter Mingjui Lee; Carlos A. Tirado, PhD; David Shabsovich, University of California â&#x20AC;&#x201C; Los Angeles

B-cell acute lymphoblastic leukemia (B-ALL) is a hematologic malignancy characterized by the uncontrolled division of B lymphoblasts. Several chromosomal translocations are commonly associated with B-ALL that aid in prognostic determination, such as t(12;21), t(1;19), and t(9;22). PAX5 (9p13), a gene that encodes a transcription factor involved in B-cell differentiation, is implicated in particular chromosomal rearrangements in B-ALL and fusion genes involving PAX5 encode products that can deregulate B-cell differentiation. Herein we report a two-year old female patient whose peripheral blood showed by flow cytometry 88% CD45 dim-to-negative blasts, positive for CD19, CD10, CD20, CD34, and HLA-DR, consistent with B-lymphoblastic leukemia (B-ALL).

Conventional cytogenetic analysis revealed an abnormal female karyotype with a dicentric chromosome involving chromosomes 9 and 17 leading to partial losses of 17p and 9p in 17 of 20 metaphases examined. Concurrent fluorescence in situ hybridization (FISH) studies on interphase nuclei and G-banded metaphases confirmed the presence of centromeres of both chromosomes 9 and 17 and deletion of CDKN2A (9p21) and TP53 (17p13) signals. In light of the FISH findings, the karyotype was conveyed as 45,XX,dic(9;17)(p13;p12)[17]/46,XX[3]. To the best of our knowledge, rearrangements involving PAX5 (9p13) and NCOR1 (17p11.2) has only been reported once in the literature and there is no prognostic information associated with the presence of this fusion gene. This report may represent another case with possible PAX5-NCOR1 fusion gene. Monoallelic losses of TP53 and CDK2NA are generally associated with a poor prognosis in B-ALL but have not been described in conjunction with a possible PAX5-NCOR1 fusion gene, which encourages further investigation of the clinical significance and the role of PAX5 in pediatric B-ALL.

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

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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 Patricia K. Dowling, PhD Pathline Labs 535 E. Crescent Ave. Ramsey, NJ 07446 PDowling@pathlinelabs.com President-Elect 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 Oxford Gene Technology 520 White Plains Road, Suite 500 Tarrytown, NY 10591 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

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 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 Term: 10/11 – 9/16 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

Representative to NAACLS Term: 9/12 – 9/16 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 Representative to Foundation for Genetic Technology Term: 7/10 – 6/16 Patricia LeMay, MT(ASCP), CG(ASCP) CM Monmouth Medical Center Department of Pathology 300 Second Ave. Long Branch, NJ 07740 732-923-7369 plemay1945@aol.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

Publications AGT Journal Editor Mark D. Terry 1264 Keble Lane Oxford, MI 48371 248-628-3025 markterry@charter.net

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162

Other Contacts Liaison to ASCLS Governmental Affairs Committee Kathryn Sudduth, BA, CG(ASCP) CM, DLMCM 2713 Brookmere Road Charlottesville, VA 22901 434-973-0690 kas3m2@embarqmail.com FGT Board of Trustees President Robin A. Vandergon, CG(ASCP) CM, DLMCM 8767 E. Los Altos Ave. Clovis, CA 93619 559-392-0512 rrink@quixnet.net

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: Monica Evans-Lombe, Executive Director 913-222-8636 mevanslombe@kellencompany.com Christie Ross, Education Program Coordinator 913-222-8626 cbross@kellencompany.com Diane Northup, Administrative Assistant 913-222-8630 dnorthup@kellencompany.com

Association Business

AGT 2017 Call for Abstracts

ABSTRACT DUE DATE: Friday, February 3, 2017

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 42nd Annual Meeting Program Committee invites all interested persons to submit New abstracts for the AGT 42nd Annual Meeting, June 15-17, 2017, in St. Louis, Missouri.

New in 2017: Meeting • A significant increase in the number of submissions selected for a platform presentation Format!

• 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 2017 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 16 and Saturday, June 17.

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.

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/ AGT2017Abstracts.

It is important that you follow all instructions carefully.

Abstracts submitted incorrectly will not be considered for presentation.

ABSTRACTS MUST BE RECEIVED BY FRIDAY, February 3, 2017.

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, 2017.

Abstracts not accepted for platform presentation may be accepted for poster presentation.

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Association Business

AGT 2017 Call for Student Abstracts & Student Research Award Entries ABSTRACT DUE DATE: Friday, March 3, 2017

Student members enrolled in an accredited Cytogenetics or Diagnostic Molecular Science program are invited to submit student abstracts for presentation and as an entry for the 2017 Student Research Award. The award will be presented to the recipient at the 2017 Annual Meeting of the Association of Genetic Technologists in St. Louis, Missouri, June 15-17, 2017. 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

Criteria

• Individuals/students eligible to submit abstracts for the award are:

• 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.

¢

¢

¢

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, 2016 and March 4, 2017.

• Individuals/students graduated prior to March 1, 2016 are not eligible.

• Applicants must be members of the Association of Genetic Technologists at the time of application to be eligible to win the Vicki Hopwood 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.

Submission Requirements Abstracts must be submitted electronically at: https://www. surveymonkey.com/r/AGT2017StudentAbstracts. 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.

• 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.

Student Research Award • The recipient will be notified by April 28, 2017.

• The winner will receive complimentary 2017 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.

• Applications may be submitted throughout the year in order to be considered for the 2017 Student Research Award. All entries received after March 3, 2017 will be considered for the 2018 Student Research Award.

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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 training through grants, scholarships and awards.

As always, behind the scenes, the dedicated members of the Foundation work throughout the year to strive to continue the mission of the FGT. Along with support from AGT, the Foundation is able to fund the scholarships and grants that were presented at the Annual AGT Meeting. None of this would have been possible without the help of the donors, vendor sponsorships and the members-at-large of AGT. Once again, the Foundation is fiscally solvent this year, which is a tribute to our many hard-working and dedicated members.

A major source of income for FGT comes from the sale of the study guides for the Cytogenetic and Molecular exams. These are available year-round and can be purchased by visiting the AGT website, and accessing FGT from the Resources tab. Another financial resource has been regional meetings sponsored by FGT. The West Coast meeting is usually in March, and the East Coast meeting is scheduled in September. Please refer to the website for further meeting information.

The Silent Auction at the AGT Annual Meeting was a tremendous success, with over $600 of donated items. This event has 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 who donated items, the winners of our auction and those who stopped by the FGT booth to inquire about us. Your input is important, and your interest is vital to keep FGT opportunities available. For more information, email Pat LeMay at plemay1945@aol.com.

DO YOU KNOW SOMEONE …?

Having just come off a very successful AGT Annual Meeting in Orange County, California, it is time to look ahead to 2017.

Along with AGT and our corporate sponsors, the Foundation for Genetic Technology has many scholarships and awards that we present at the AGT Annual Meeting every year. A complete list, along with requirements, application forms and submission deadlines is listed in the FGT section on the AGT website. There is a wide range of awards available and something for every AGT member to consider…from the newly certified technologist to the careeroriented professional and the seasoned veteran technologist. We encourage each AGT member to consider nominating someone for these awards. It is a wonderful way to acknowledge the accomplishments and the dedication of 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 may feel out of the loop, but each application is reviewed by a committee, and is not a popular vote.

You owe it to yourselves, as AGT members, to recognize outstanding members of our professional organization. If we fail to promote ourselves, then no one is going to do 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 Blvd, Suite 220 â&#x20AC;˘ Overland Park, KS 66211 FGT website: http://www.AGT-info.org/Pages/fgt.aspx

2017 Grants & Awards Deadline: March 31, 2017 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.

Outstanding Technologist Grant ($500) Honors an outstanding AGT member technologist who is BOC-certified with three or more years of work experience in the genetic industry. Sponsored by Leica Microsystems.

Florence Dowling Genome Award ($500) Acknowledges and honors outstanding technologists in cytogenetics and

molecular genetic technology. This award program contributes to the growth of genetic technology as a profession by recognizing individuals with superior professional commitment. Sponsored by Patricia Dowling.

New Horizons Award ($750) Honors newly certified AGT members who submit an essay about their genetic experience and desire to attend the AGT Annual Meeting. Sponsored by Rainbow Scientific.

EXCEL Award ($500) AGT members enrolled in a formal university/hospital or laboratory-based program in diagnostic 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 42nd Annual Meeting, as well as one full-day or two half-day workshops. Sponsored by Oxford 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 that is BOC-certified and has a minimum of five years of teaching experience. Sponsored by James Waurin.

Best Poster Award ($300) All AGT members attending the AGT Annual Meeting may vote for the poster that fits the

winning criteria (i.e., interesting and informative topic, well-organized, clear and concise data, best illustrations, clinical/ laboratory correlation and cutting-edge technology) submitted by an AGT member by the abstract deadline. Sponsored by Irvine Scientific.

Best Platform Presentation Award ($500) All AGT members attending the platform presentations at the AGT Annual Meeting may vote for the presentation that best fits the winning criteria (i.e., presentation must be given by a technologist, be an interesting and informative topic, have clinical/laboratory correlation and present cutting-edge technology). An AGT member must be among the authors of the abstract submitted by the abstract deadline. Sponsored by FGT. Best Exhibitor Booth Award

Honors exhibitors at the AGT Annual Meeting. AGT members will vote by ballot on the following criteria: 1. Best interaction (quality of interaction) with AGT membership, including availability to meeting participants and answering questions with detail.

2. Most valuable technical information or product information, including presentation of literature available.

3. Best overall booth design, including appearance of exhibit and visual impact of the display.

4. Most innovative gifts/raffles for attendees.

5. Creativity.

6. Only exhibitors listed in the final program will be eligible.

Please refer to the website listed above for the details and the application forms for all of these awards, grants and scholarships.

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Association Business

Foundation for Genetic Technology 2015-2016 Board of Trustees Voting Members President Robin Vandergon NeoGenomics 30 E River Park Place West, Ste. 400 Fresno, CA 93720 559-392-0512 cell 559-433-6601 Fax rvandergon@neogenomics.com Vice President, AGT Representative, Grants Committee Chair Patricia LeMay 301-22 Spring Street Red Bank, NJ 07701 plemay1945@aol.com Secretary DeNesha Criswell NeoGenomics 618 Grassmere Park Drive, Unit 20 Nashville, TN 37211

Treasurer, Chair Capital Management Committee Tara Ellingham MUSC-Childrenâ&#x20AC;&#x2122;s Hospital Cytogenetics Lab 165 Ashley Ave., Suite 309 Charleston, SC 29425 843-792-6873 ellingha@musc.edu tellingham@hotmail.com

Public Member, Corporate Compliance Officer Bob Gasparini Consultant 12701 Commonwealth Dr. Ft. Myers, FL 33913 239-357-4237 bgasparini@neogenomics.com

Non Voting Members

AGT Representative, Awards & Scholarship Chair Denise Juroske Short 219 Timberland Trail Ln. Lake City, TN 37769 832-878-6119 dmj4565@gmail.com

Advisor, AGT President Patricia K. Dowling Pathline Labs 535 E. Crescent Ave. Ramsey, NJ 07446 PDowling@pathlinelabs.com

Public Member, Chair FGT Fundraising Jeff Sanford MetaSystems Group, Inc. 70 Bridge St., Ste. 100 Newton, MA 02458 617-924-9950 jsanford@metasystems.org

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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• 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

PRODUCT ORDER FORM Member

NonMember

**Please supply a credit card number or Federal Express account number for for all International orders below. [Please note that the

$90

$110

The Cytogenetics Symposia, 2nd Edition

$65

$80

$40

$55

$35

$50

Item Description

Quantity

Total $

The AGT Cytogenetics Laboratory Manual, 3rd Edition

Lab Manual is only available on a flash drive. The 4th Edition is scheduled to be released in 2016.]

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

 Dropbox (no shipping cost)  Secure Document Hyperlink (no shipping cost) The Dynamics of Chromosome Spreading Video – CD featuring Jack Spurbeck

Shipping/Handling ($10 per item)*

$

TOTAL

$

*INTERNATIONAL ORDERS – Shipping charged directly to recipient. Please provide a Federal Express account number or the credit card number below will be charged separately for shipping.

Name Shipping Address

Method of Payment: Check Visa MasterCard

AMEX Discover

Account Number Expiration Date

City,State Zip

Name on Account

Phone No.

Signature on Account

Email

AGT Executive Office, 4400 College Blvd, Suite 220, Overland Park, KS 66211 Fax (913) 222-8606 l Email: agt-info@kellencompany.com l Website: www.agt-info.org

Please allow 2-4 weeks for shipping.

AGT accepts classified job advertising for:    

Posting on Jobline page of the web site; Online postings are also included in the monthly e-news blast to members.

E-blast to AGT members For publication in JAGT (Journal of the Association of Genetic Technologists) For publication in AGT e-news only - monthly blast to members

Advertisements submitted for posting on the website or as an e-blast are generally completed within approximately 48 hours of acknowledged receipt. If logos are to be used on the posting, they must be submitted in GIF. or JPG. format. Logos and text must be submitted via e-mail.

Advertisements submitted for publication in the Journal of the Association of Genetic Technologists (JAGT) must be submitted by the deadline date for the requested issue and adhere to the mechanical requirements outlined on the insertion order form.

To place your order for advertising, please complete the appropriate form and submit to the AGT Executive Office via email agt-info@kellencompany.com.

Forms can be found on the AGT website here: http://www.agt-info.org/Pages/pricing.aspx

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:

 Regular Membership  Student Membership  Emeritus Membership

$95 $35 $40

 Collaborative

$40 Organization:________________________ Member ID: ________________________

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.

Recruited by: ______________________________________ Member ID: _____________ Name: ________________________________________________________________________________________________________________ Last

First

MI

Certification

 Home Address: _______________________________________________________________________________________________________ City, State, Zip: ___________________________________________________________________ Phone:______________________________

 Business Name: _______________________________________________________________________________________________________ Business Address: _____________________________________________________________________________________________________ City, State, Zip: ___________________________________________________________________ Phone:______________________________ Fax:______________________________ Preferred Email: ___________________________________________________________________

The supplied address will be published in the directory unless otherwise specified.  Do not provide my address in the online AGT Membership Directory.

Membership Status:

 New Member

[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.]

 Renewal

Referred By_____________________________________________________________________ Membership # ___________________________ Did you use a different name last year:

 Yes

 No

Former Last Name________________________________________ First Name________________________________ MI __________________

Position: (check one)

 Director  Head (Lead, Core) Technologist

 Supervisor  Technologist  Tissue Culture Tech.  Education Coordinator

 Lab Manager  Other

 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)

 over 30

NOTICE: OUR MAILING LIST IS MADE AVAILABLE TO OTHER ORGANIZATIONS AND/OR COMPANIES. IF YOU WISH YOUR NAME NOT TO APPEAR ON THESE LISTINGS, PLEASE CHECK HERE: 

Journal Hard Copy Order: Although The Journal of Genetic Technologists is available online to ALL MEMBERS, only North American members can elect to receive a hard copy via regular mail for an additional fee of $100. This fee covers four issues. If you are a North American resident and would like a hard copy of the Journal, please remit the additional fee with your membership application by checking the box and adding the amount to your total payment.  $100 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.

 VISA  MasterCard  AMEX  Discover

_______________________________________________________________ Account No. Exp. Date _______________________________________________________________ Signature

MAIL APPLICATION AND FEE TO: Association of Genetic Technologists 4400 College Blvd, Suite 220 Overland Park, KS 66211 Phone: 913-222-8665 FAX: 913-222-8606 9/16

Meeting/Workshop Announcements

2017-2018 Scientific Meetings Schedule

If you know of a relevant meeting, please send information to Ephrem Chin, Public Relations Director at nzelfman@gmail.com.

We are always looking to improve AGT’s annual meeting. If you attend a meeting and see something you think would enhance our meeting, please email your ideas to Jennifer Sanmann, Annual Meeting Director at jsanmann@unmc.edu.

Meeting

Location

Dates

Website

2016 American Society for Bone & Mineral Research/IBMS (ASBMR) Annual Meeting

Atlanta, GA

September 16-19, 2016

www.asbmr.org

American Association for the Study of Liver Diseases (AASLD): The Liver Meeting

Boston, MA

November 11-15, 2016

www.aasld.org

European Cancer Organization ECO2017 European Cancer Congress

Amsterdam, Netherlands

January 27-30, 2017

www.ecco-org.eu

HUGO HGM 2017

Barcelona, Spain

February 2017

www.hugo-international.org

Conference on Retrovirus & Opportunistic Infections (CROI)

Seattle, WA

February 12-16, 2017

www.retroconference.org

American Association for the Advancement of Science (AAAS) Annual Meeting

Boston, MA

February 16-20, 2017

www.aaas.org

American Academy of Allergy, Asthma, & Immunology (AAAAI)

Atlanta, GA

March 3-6, 2017

www.aaaai.org

United States & Canadian Academy of Pathology (USCAP) Annual Meeting

San Antonio, TX

March 4-10, 2017

www.uscap.org

Society of Maternal/Fetal Medicine (SMFM) 9th International Symposium on Diabetes, Hypertension, Metabolic Syndrome and Pregnancy

Barcelona, Spain

March 8-12, 2017

www.smfm.org

Association for Gerontology in Higher Education (AGHE) Annual Meeting and Educational Leadership Conference

Miami, FL

March 9-12, 2017

www.aghe.org

Society for Reproductive Investigation (SRI) Annual Scientific Meeting

Orlando, FL

March 15-18, 2017

www.sri-online.org

American College of Cardiology (ACC) Scientific Session & Expo

Washington, DC

March 17-19, 2017

www.acc.org

American College of Medical Genetics (ACMG) Annual Clinical Genetics Meeting

Phoenix, AZ

March 21-25, 2017

www.acmg.net

Clinical Laboratory Management Association (CLMA) KnowledgeLab

Nashville, TN

March 26-29, 2017

www.clma.org

American College of Physicians (ACP) Internal Medicine Meeting

San Diego, CA

March 30 – April 1, 2017

www.acponline.org

The Endocrine Society’s ENDO Annual Meeting

Orlando, FL

April 1-4, 2017

www.endocrine.org

American Association for Cancer Research (AACR) Annual Meeting

Washington, DC

April 1-5, 2017

www.aacr.org

American Chemical Society (ACS) National Meeting & Exposition

San Francisco, CA

April 2-6, 2017

www.acs.org

American Society for Biochemistry & Molecular Biology (ASBMB) Annual Meeting

Chicago, IL

April 22-26, 2017

www.asbmb.org

American Society for Investigative Pathology (ASIP) Annual Meeting at Experimental Biology

Chicago, IL

April 22-26, 2017

www.asip.org

2017

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Meeting/Workshop Announcements Meeting

Location

Dates

Website

American Transplant Congress (ATC)

Chicago, IL

April 29 – May 3, 2017

www.atcmeeting.org

Cambridge Healthtech Institute’s 7th Annual Biomarker World Congress 2011

Philadelphia, PA

May 2-4, 2017

www.biomarkerworldcongress. com

American Association of Clinical Endocrinologists (AACE) Annual Meeting and Clinical Congress

Austin, TX

May 3-26, 2017

www.aace.com

5th Quadrennial Meeting of the World Federation of Neuro-Oncology Societies (WFNOS)

Zurich, Switzerland

May 4-7, 2017

https://www.eano.eu/wfnos2017-meeting/welcome/

American Gastroenterological Association (AGA) Digestive Disease Week (DDW)

Chicago, IL

May 6-9, 2017

www.ddw.org www.gastro.org

Pediatric Academic Society (PAS) and Asian Society for Pediatric Research (ASPR) Joint Meeting

San Francisco, CA

May 6-9, 2017

www.pas-meeting.org

Clinical Virology Symposium & Annual Meeting of the Pan American Society for Clinical Virology

Savannah, GA

May 7-10, 2017

www.clinicalvirologysymposium. org

American Association of Immunologists (AAI) Annual Meeting

Washington, DC

May 12-16, 2017

www.aai.org

American Urological Association (AUA) Annual Meeting

Boston, MA

May 12-16, 2017

www.aua2017.org

American Association of Bioanalysts (AAB) Annual Meeting and Educational Conference/CRB Symposium

Houston, TX

May 18-20, 2017

www.aab.org

American Geriatrics Society (AGS) Annual Scientific Meeting

San Antonio, TX

May 18-20, 2017

www.americangeriatrics.org

International Society for Antiviral Research (ISAR) Antiviral Conference on Antiviral Research (ICAR)

Atlanta, GA

May 21-25, 2017

www.isar-icar.com

Bio-IT World (Cambridge Health Tech Institute)

Boston, MA

May 23-25, 2017

www.healthtech.com www.bio-itworldexpo.com

American Society of Clinical Oncology (ASCO) Annual Meeting

Chicago, IL

June 2-6, 2017

www.asco.org

American Society for Mass Spectrometry (ASMS) Annual Conference

Indianapolis, IN

June 4-8, 2017

www.asms.org

Cambridge Healthtech Institute (CHI) World 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

American Association for Clinical Chemistry (AACC)’s Annual Meeting

San Diego, CA

July 30 – August 3, 2017

www.aacc.org

American Society of Clinical Laboratory Science (ASCLS) Annual Meeting

San Diego, CA

July 30 – August 4, 2017

www.ascls.org

Society for Inherited Metabolic Disorders (SIMD) Annual Meeting

Rio de Janeiro, Brazil

September 5-8, 2017

www.simd.org

International Congress of Pediatric Laboratory Medicine (ICPLM)

Durban, South Africa

October 20-22, 2017

www.icplm2017.org

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Meeting/Workshop Announcements Meeting

Location

Dates

Website

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

www.aacr.org

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

Kyoto, Japan

July TBD, 2018

www.iuphar.org

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

www.aabb.org

American Society of Human Genetics (ASHG) Annual Meeting

San Diego, CA

October 16-20, 2018

www.ashg.org

Job placement ads are online at http://www.AGT-info.org The Journal of the Association of Genetic Technologists 42 (3) 2016

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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 $5/each and by non-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