JAGT - Issue 4 2017

The Journal

of the Association of Genetic Technologists

Volume 43 • Number 4 • Fourth Quarter 2017

Brain Tickler

Column Editor: Helen Lawce

Brain Tickler

Bone marrow was received on a 53-year-old male with newly diagnosed leukemia.

Representative cells from the two related abnormal clones are shown.

Submitted by: Helen Lawce, Anthony Minero, Craig Davis, Nancy Unsworth and Shannon Wooton Knight Diagnostic Cytogenetics Laboratory Oregon Health & Science University Portland Oregon

The answer to this Brain Tickler appears on page 217.

The Journal of the Association of Genetic Technologists Fourth Quarter 2017 Volume 43, Number 4

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

The official journal of the AGT

Column Editors and Review Board . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196 A Note from the Editor. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197 Erratum The t(12;21)(p13;q22) in Pediatric B-Acute Lymphoblastic Leukemia: An Update Maximilian Becker, Kristie Liu and Carlos A. Tirado. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198 Review A Molecular and Cytogenetic Update on Non-Small Cell Lung Carcinoma Maximilian Becker, Lori Ryan, Alexis Dowiak and Carlos A. Tirado. .. . . . . . . . . . . . . . . . . . 199

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 Book Review Editor Helen Lawce, BSc, CLSp(CG) Copyright © 2017 by the AGT. All rights reserved. Contents are not to be reproduced or reprinted without permission of the AGT Editor.

The Journal of the Association of Genetic Technologists is published four times a year and is available to individuals and libraries at a subscription rate of $115 per year. The subscription rate for members of the AGT is included in the annual membership dues.

Back issues can be purchased for members at $25 per issue as long as supplies are available.

Material intended for publication or correspondence concerning editorial matters should be sent to the editor.

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

Profiles and Perspectives Dr. Carlos Tirado, PhD, FACMG Interviewed by Mark Terry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213 Genetics in the News A Breakthrough in Gene Editing of Human Embryos to Correct Pathogenic Gene Mutations Jaime Garcia-Heras . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215 Brain Tickler Summary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217 Continuing Education Opportunities Test Yourself #4, 2017. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218 AGT Journal Clubs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221 Association Business Letter from the President. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227 Peru Symposium. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228 Association of Genetic Technologists BOD Contacts . . . . . . . . . . . . . . . . . . . . . . . . . 229 AGT 2018 Call for Abstracts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230 Student Research Award . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231 FGT Letter. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232 2018 FGT Grants and Awards Deadlines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233 FGT Board of Trustees . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234 Product Order Form . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235 2017 AGT Outstanding Achievement Award Nomination Form . . . . . . . . . . . . . . . . . . . 236 New Membership Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237 2017–2018 Scientific Meetings Schedule .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238 Information for Authors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inside Back Cover

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ISSN 1523-7834

The Journal of the Association of Genetic Technologists is indexed in the life sciences database BIOSIS and in the National Library of Medicine’s PubMed. The Journal of the Association of Genetic Technologists 43 (4) 2017

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The Journal of the Association of Genetic Technologists Staff

Column Editors Abstract Reviews/Genetics in the News Genetics, Government & Regulation Helen Bixenman, MBA, CLSup, CLSp(CG) Jaime Garcia-Heras, MD, PhD San Diego Blood Bank Adjunct Assistant Professor 3636 Gateway Center Avenue, Suite 100 Cytogenetic Technology Program San Diego, CA 92102 School of Health Professions 619-400-8254 UT MD Anderson Cancer Center hbixenman@sandiegobloodbank.org 1515 Holcombe Blvd., Unit 2 Houston, Texas 77030 Jennifer Crawford-Alvares jgarciaheras@hotmail.com Cytogenetic Technologist II Section of Hematology/Oncology Brain Tickler/Book Review Editor The University of Chicago Medicine Helen Lawce, BSc, CLSp(CG) 5841 S. Maryland Ave., Rm. I-304 Clinical Cytogenetics Laboratory Chicago, IL Oregon Health Sciences University jen.crawford34@gmail.com 3181 SW Sam Jackson Parkway Office: 773-702-9153 MP-350 Letters to the Editor Portland, OR 97201 Mark Terry, JAGT Editor 503-494-2790 1264 Keble Lane 503-494-6104 FAX Oxford, MI 48371 lawceh@ohsu.edu 586-805-9407 (cell) markterry@charter.net

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

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

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

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

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

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

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

Nancy Taylor, BSc, CLSp(CG), MT(ASCP) (Cytogenetics, Cancer cytogenetics) Stephen R. Moore, PhD, ABMG (Clinical cytogenetics, radiation biology, Thomas Wan, PhD toxicology; clinical molecular genetics) (Cytogenetics, Molecular genetics, Lynn Hoyt, BSc, CLSp(CG), CLSup 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 (Cytogenetics, Molecular, Education) Heather E. Williams, MS, CG(ASCP)CM Jonathan P. Park, PhD (Cytogenetics, Molecular Genetics) (Cytogenetics, Molecular genetics, Julia Kawecki, BSc, CLSp(CG) Cell biology) (Cytogenetics, Molecular genetics) Su Yang, BSc, CLSP(CG) (Education, Traditional Cytogenetics) David Peakman, AIMLT, CLSp(CG) Turid Knutsen, MT(ASCP), CLSp(CG) (Prenatal diagnosis) (Cancer cytogenetics, CGH, SKY) Jason A. Yuhas, BS, CG(ASCP)CM (Cytogenetics, Molecular cytogenetics) Carol Reifsteck, BA Brandon Kubala, BSc, CLSp(CG) (Breakage syndromes, Fanconi’s (Traditional Cytogenetics) James Zabawski, MS, CLSp(CG) anemia, Prenatal diagnosis) (Education, Traditional Cytogenetics) Anita Kulharya, PhD Gavin P. Robertson, PhD (Molecular genetics, Clinical (Cytogenetics, Molecular genetics, cytogenetics) Somatic cell genetics, Tumor suppressor Helen Lawce, BSc, CLSp(CG) genes, Cancer genes) (Prenatal diagnosis, Solid tumors, FISH, Laurel Sakaluk-Moody, MSc, MLT(CG) Chromosome structure, Evolution) (Cytogenetics, Developmental biology, Prenatal cytogenetics) Michelle M. Hess, MS, CLSp(CG) (Cytogenetics, Cancer cytogenetics)

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A Note from the Editor

A Slightly Different Point of View

CRISPR

As I’ve mentioned before, my “day job,” if you will, is as a freelance writer, editor and author. Currently my bread-and-butter writing is about the biopharmaceutical industry. It was only a few years ago that it was mostly focused on clinical diagnostics.

Things change.

The point, however, is that two of our articles tweaked my interest a bit more than usual because they are topics that are big in the biopharmaceutical industry. So I thought I’d provide a little back story, or a little bit different point of view, about these topics.

Jaime Garcia-Heras contributed a “Genetics in the News” piece titled “A Breakthrough in Gene Editing of Human Embryos to Correct Pathogenic Gene Mutations.” This article presents some background on a recent groundbreaking article in Nature about researchers that used CRISPR-Cas9 gene editing technology to correct a disease-causing mutation in viable human embryos.

There’s still some controversy over this topic, and I doubt that controversies over use of CRISPR-Cas9 and other technology on human embryos is going to go away anytime soon. It’s a good example of science outpacing ethics.

I’ve written quite a bit about CRISPR, but more from the business point of view. CRISPR-Cas9 as a technology was discovered by a University of California Berkeley professor Jennifer Goudna and Emmanuelle Charpentier of the Helmholtz Centre for Infection Research in Braunschweig, Germany.

There’s a big patent battle ongoing with Feng Zhang, a researcher at the MIT-Harvard Broad Institute, who filed a broad U.S. patent claim on the technology. The patent battle revolves quite a bit around changes in U.S. patent law in the last few years.

For a long time it was “first to invent,” meaning that whoever was the first person to invent or discover a technology was the patent owner. They had to prove it, but there you go. Then, in late 2011, the U.S. switched to “first-to-file,” and it went into effect on March 16, 2013.

Anyway, Doudna founded a biotech company called Caribou Biosciences, which had a collaboration deal with Novartis Institute for Biomedical Research. Caribou then founded another firm, Intellia Therapeutics. Charpentier sold her part of the rights of the CRISPR-Cas9 platform to another company, CRISPR Therapeutics. CRISPR Therapeutics also has a licensing/collaboration deal with Vertex Pharmaceuticals. Zhang co-founded a biotech company, Editas Medicine.

Is there money involved? C’mon, folks! There are millions, potentially billions of dollars, involved.

Lung Cancer Max Becker, Lori Ryan, Alexis Dowiak and Carlos Tirado contributed an article, “A Molecular and Cytogenetic Update on Non-Small Cell Lung Carcinoma.” Non-small cell lung cancer (NSCLC) is something I write about quite regularly these days because it’s a hot area of drug development.

For example, on Oct. 18, I reported on a company, Ignyta, which had presented data from its STARTRK-2 clinical trial of entrectinib in NSCLC. (Yes, I thought the STARTRK designation was sort of clever.) The drug is a CNS-active, selective tyrosine kinase inhibitor for tumors with NTRK fusions or ROS1 fusions, which if you read the article in this issue, will be discussed quite a bit. In the trial so far, the drug showed a 78% confirmed objective response rate (ORR) and a 69% ORR, using two separate metrics in 32 patients with locally advanced or metastatic NSCLC that harbored ROS1 fusions. The median duration of response (mDOR) was 28.6 months for these patients.

Investors were clearly interested, because after the news the stock jumped about 5%.

Back in May, I wrote about AstraZeneca and its MedImmune division, which indicated positive results in its Phase III clinical trial of Imfinzi (durvalumab) in NSCLC. The PACIFIC trial looked at patients with locally-advanced, unresectable (Stage III) NSCLC in patients who had not progressed after standard platinum-based chemotherapy concurrent with radiation therapy.

An Independent Monitoring Committee (IMC) analyzed the interim data and decided it had already met its primary endpoint, a statistically significant and clinically meaningful progressionfree survival (PFS). The study will continue to assess overall survival, the other primary endpoint of the trial.

The company stock jumped at the news to $33.46 per share. It traded at the end of trading day Oct. 20 at $34.62, for people who are into that sort of thing.

And in case you were wondering, science giveth and science taketh away. Back in August, Bristol-Myers Squibb reported that its CheckMate-026 clinical trial of Opdivo (nivolumab) as a monotherapy for NSCLC whose tumors expressed PD-L1 at more than 5% failed its trial, and did not meet its primary endpoint of progression-free survival. (Opdivo’s a pretty great cancer drug, but apparently not effective for every type of cancer.)

The news didn’t affect the company’s stock too much, since it was trading around then for about $56.30 per share, but is currently trading for $64.42.

Changes Also, a reminder. The Association of Genetic Technologists is ending its relationship with its management company, Kellen, at the end of this year. Communications with the executive director and the board (although you can still communicate directly with the board) should be through Denise Juroske-Short after January 1, 2018.

Cheers, Mark Terry, Editor

The Journal of the Association of Genetic Technologists 43 (4) 2017

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Erratum

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

2.

3.

4.

5.

6.

Centro de Investigaciones Tecnológicas, Biomédicas y Medioambientales, Lima, Peru Centro Nacional de Salud Publica, Instituto Nacional de Salud, Lima, Peru Allina Health, Minneapolis, MN 55407 HPS, Minneapolis, MN 55407 The University of Minnesota, School of Medicine. Department of Laboratory Medicine and Pathology, Minneapolis, MN 55407 The International Circle of Genetics Studies, Los Angeles, CA 90024 Leukemia Study Group (SCLSG); NOPHO Leukemia Cytogenetic Study Group (NLCSG). Cytogenetic patterns in ETV6/RUNX1-positive pediatric B-cell precursor acute lymphoblastic leukemia: A Nordic series of 245 cases and review of the literature. Genes, Chromosomes Cancer.

2007;46(5): 440–450.

Stams WA, den Boer ML, Beverloo HB, Meijerink JP, van Wering ER, Janka-Schaub GE, Pieters R. 2005. Expression levels of TEL, AML1, and the fusion products TEL-AML1 and AML1-TEL versus drug sensitivity and clinical outcome in t(12;21)-positive pediatric acute lymphoblastic leukemia. Clin Cancer Res. 2005;11(8): 2974–2980.

Erratum Figure 1 on the last edition The Journal of the Association of Genetic Technologists. 2017;43(3): 113-127 does not contain the derivative 21. We are replacing this figure with the present one.

In the section Secondary genetic aberrations we would like to add that: Deletions of 11q23 are observed in 5-6% of cases (Raynaud et al., 1999; Attarbaschi et al., 2004; Alvarez et al., 2005; Forestier et al., 2007).

The authors regret this.

In the References section we are adding:

Corresponding author:

Forestier E, Andersen MK, Autio K, Biennow E, Borgstrom G, Golovleva L, Heim S, Heinonen K, Hovland R, Johannsson JH, Kerndrup G, Nordgren A, Rosenquist R, Swolin B, Johansson B; Nordic Society of Pediatric Hematology and Oncology (NOPHO); Swedish Cytogenetic

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

Figure 1: A. Ideograms of chromosomes 12 and 21 indicating the position of the ETV6 and RUNX1 genes on 12p13 and 21q22 respectively, and the translocation on the derivative chromosomes der(12)(t(12;21) and der (21)(t12;21). The fusion transcript ETV6/RUNX1 on der(21) is the crucial one for leukemogenesis as the reciprocal RUNX1/ETV6 on der(12) is expressed in only 70% of the cases (Kempski et al., 1999; Al-Shehhi et al., 2013; Baughn et al., 2014;). B. Overview of the functional domains and main protein interactions of ETV6, RUNX1 and ETV6/RUNX1. The main functional domains are indicated as follows: For ETV6, the N-terminal pointed domain (PNT) and the C-terminal E26Transforming Specific domain (ETS). For RUNX1, the N-terminal Runt homology domain (RHD), the C-terminal transactivation domain (TD) and inhibition domain (ID). The translocation t(12;21)(p13;q22) fuses the N-terminal 336 amino acids of ETV6 to the majority of the RUNX protein. It converts RUNX1 into a constitutive transcriptional repressor, which binds to DNA through the RHD domain. Recruitment of HDACs (via N-CoR) by the ETV6 component and recruitment of the corepressors mSin3A by both components changes the patterns of gene expression of target genes. Based on: Bakshi et al., 2010; Boccuni et al., 2003; Chakrabarti & Nucifora 1999; De Braekeleer et al., 2009; Fenrick et al., 1999; Guidez et al., 2000; Gunji et al. ,2004; Guo et al., 2011; Golub et al., 1995; Hart & Foroni 2002; Hiebert et al., 1996; Imai et al., 1998; Kim et al., 2001; Kitabayashi et al., 1998; Kitabayashi et al., 2001; Levanon et al., 1998; Romana et al., 1996; Stams et al., 2005; Wang & Hiebert 2001; Yu et al., 2012; Zhao et al., 2007.

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Review

A Molecular and Cytogenetic Update on Non-Small Cell Lung Carcinoma Maximilian Becker1, Lori Ryan2,3, Alexis Dowiak1 and Carlos A. Tirado1,2,3,4 1.

2.

3.

4.

The International Circle of Genetic Studies, Los Angeles, CA Allina Health, Inc., Minneapolis, MN Hospital Pathology Associates, Minneapolis, MN The University of Minnesota School of Medicine, Department of Laboratory Medicine and Pathology. Minneapolis, MN

Abstract Lung cancer is one of the leading causes of cancer-related death worldwide. Among patients with lung cancer, approximately 85% have non-small cell lung carcinoma (NSCLC). The discovery of oncogenic driver mutations in NSCLC opened new personalized treatment options. Several methods that can identify these biomarkers are used routinely in a clinical setting to stratify patients for targeted therapy. In this review, we summarize the most clinically relevant driver genes, discuss the advantages and limitations of current clinical detection methods, and highlight the benefits of personalized treatment over standard chemotherapy.

Lung cancer epidemiology

like polonium-210 (Dela Cruz et al., 2011). Other risk factors include occupational exposure to the carcinogenic radioactive gas Radon (222Ra), exposure to asbestos, as well as long-term exposure to fine particulate air pollution generated by combustion in the environment (Cohen et al., 2005; Dela Cruz et al., 2011).

Lung cancer has been the most commonly diagnosed type of cancer in the world for many decades with an estimated 1.82 million new cases (13.0% of total new cancer cases) and 1.59 million deaths (19.4% of total cancer deaths) in 2012 (Ferlay et al., 2015). In males, the incidence rates are generally high in Central and Eastern Europe and East Asia (53.5 and 50.4 per 100,000, respectively), and low in Middle and West Africa (2.0 and 1.7). The rates for lung cancer tend to be generally lower in women than in men; they are highest in North America and Northern Europe (33.8 and 23.7), followed by East Asia (19.2) and are lowest in Middle and West Africa (0.8 and 1.1) (Dela Cruz et al., 2011).

Molecular testing in NSCLC The College of American Pathologists (CAP), the International Association for the Study of Lung Cancer (IASLC), and the Association for Molecular Pathology (AMP) recommend molecular testing for EGFR sensitizing mutations or ALK rearrangements to select patients for targeted TKI therapy in all patients with advanced lung adenocarcinoma, or tumors with an adenocarcinoma component, regardless of clinical characteristics (i.e., smoking status, age). In limited tumor specimens, where an adenocarcinoma component cannot be excluded due to sample heterogeneity, molecular testing is recommended in the presence of suggestive clinical parameters like a young age or lack of smoking history (Lindeman et al., 2013) (See Table 1).

Lung cancer pathology Lung cancer arises from the respiratory epithelium and can be divided into two main histopathologic types: small cell and nonsmall cell carcinoma. Small cell lung cancer (SCLC) is a highly malignant tumor that originates from neuroendocrine cells and accounts for 15% of lung cancer cases. The remaining 85% are made up of non–small cell lung cancer (NSCLC), which is further divided into two major pathologic subtypes: adenocarcinoma and squamous cell carcinoma. Adenocarcinoma represents 38.5% of all lung cancer cases and is the most common type in lifelong nonsmokers. Twenty percent of lung cancer patients are diagnosed with squamous cell carcinoma (SCC), which is closely related to a history of tobacco smoking (Dela Cruz et al., 2011).

Driver genes Non-small lung carcinomas are characterized by complex numerical and structural cytogenetic abnormalities that affect many chromosomes, but only a few of these have been related to diagnosis or prognosis (Heighway and Betticher, 2004).

However, a subset of lung cancer patients contains recurrent gene mutations that are essential to proliferation and cell survival, and thereby contribute to the neoplastic transformation (Cooper et al., 2013) (See Table 2).

Genes that harbor these mutations are called “driver genes,� defined as those whose mutations increase net cell growth under the specific microenvironmental conditions that exist in the cell in vivo (Stratton et al., 2009). Aberrations in driver genes such as EGFR, ALK and ROS1 have been routinely tested in clinical practice (Lindeman et al., 2013). The clinical relevance of several emerging driver genes, including RET, BRAF, MET, HER2, KRAS and FGFR1 is still under investigation, and these genes are not yet indicated in routine stand-alone tests. However, in cases where no mutations have been found in EGFR/ALK/ ROS1 and if sufficient tumor tissue is available, a panel of these driver genes may be analyzed. The current literature does not

Risk factors The geographic pattern of lung cancer incidence reflects the different historical exposure to risk factors. Smoking tobacco is the principal risk factor for lung cancer, and has been estimated to account for 20% of all lung cancer deaths worldwide (Parkin et al., 1994).

The causal relationship between lung cancer and smoking has been demonstrated since the 1960s, and since then more than 70 carcinogenic components have been identified in tobacco smoke: polycyclic aromatic hydrocarbons (PAHs), tobacco-specific nitrosamines (TSNAs), as well as a multitude of other volatile organic and inorganic compounds, and even radioactive elements

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Review A Molecular and Cytogenetic Update on Non-Small Cell Lung Carcinoma support evidence for clinical use of other driver genes including CDKN2A (Jiang et al., 2016), AKT1 (Malanga et al., 2014), MEK1 (Arcila et al., 2015), PIK3CA (Samuels, 2004; Kawano et al., 2006) and PTEN (Lee et al., 2010).

mutations in specimens composed of as few as 10% cancer cells. In case of TKI resistance, the test should detect secondary mutations in specimens with as little as 5% of cancer cells. Testing for EGFR mutations may incorporate different methods, including PCRbased methods (like allele-specific PCR, real-time PCR), Post-PCR mutation detection (Capillary electrophoresis, RFLP) as well as mutation screening (denaturing HPLC of high-resolution melting analysis). Apart from the most common exon 19 deletions and L853 point mutations, clinical EGFR mutation testing should also detect other less common mutations that have been reported with a frequency of at least 1% of EGFR-mutated lung adenocarcinoma (Lindeman et al., 2013). In order to monitor EGFR mutations, invasive tissue biopsy may not be possible at each follow-up. The analysis of ctDNA from the patient’s bloodstream has become more and more available in recent years (Villaflor et al., 2016).

EGFR

The epidermal growth factor receptor (EGFR) gene, located at the short arm of chromosome 7 at position 7p11, encodes a transmembrane receptor from the tyrosine kinase family. Binding of a ligand activates the signaling pathways like RAS-RAF-MEKERK and PI3K-AKT-mTOR that promote cell proliferation and survival. Mutations like exon 19 deletions or exon 21 L858R mutations induce a ligand-independent activation of downstream pathways and are therefore called activating mutations (Gazdar et al., 2009). These mutations involve the ATP-binding pocket of the tyrosine kinase domain, which is also the binding site of reversible Tyrosine Kinase Inhibitors.

Activating EGFR mutations are the most extensively studied mutations in NSCLC, and occur in average of around 35% of patients. However, the prevalence is considerably higher in the Asia-Pacific population (up to 67.0%) compared to the Caucasian population (4-18%) (Midha et al., 2015).

Aberration of downstream genes of EGFR has also been associated w it h NS C LC , i nd ic at i n g a n important role in the neoplastic transformation. Activating EGFR mutations affect residues in the ATP-binding pocket, which on one hand enhance the ligand-dependent activation of EGFR, but also increase the inhibition through TKIs (Bell et al., 2005). However, most patients Figure 1: Proposed algorithm for molecular testing in NSCLC. Adapted from Lindeman et al., 2013; treated with these TKIs develop Bubendorf et al., 2016. Abbreviations. AC: Adenocarcinoma, ALK: anaplastic lymphoma kinase, resistance in less than one year EGFR: epidermal growth factor receptor, ROS1: Proto-oncogene tyrosine‑protein kinase ROS, IHC: Immunohistochemistry, FISH: Fluorescence in situ hybridization, NGS: Next-generation sequencing.

(Wang et al. 2016). Several cases that relapsed showed the secondary mutation T790M (Kobayashi et al., 2005; Pao et al., 2005). Since then, the T790M mutation has ALK been constantly found in more than half of TKI-resistant NSCLC The anaplastic lymphoma kinase (ALK) is a member of the cases (Ma et al., 2011). One possible mechanism suggests that the insulin receptor superfamily and is located on 2p23.1. Activating T790M mutation restores the affinity of the L858R mutant to mutations or translocations of ALK have been described in many ATP to wild-type levels, and thereby decreases the effect of TKIs types of cancer (Kwak et al., 2010). Soda et al identified the fusion (Yu et al., 2013). of ALK to the echinoderm microtubule-associated protein-like Patients with acquired TKI resistance are now routinely tested 4 (EML4) in a adenocarcinoma specimen, and confirmed the for EGFR T790M mutation. Once the T790M mutation has carcinogenicity in mice (Soda et al., 2007). EML4-ALK fusions can been confirmed, the therapy can then switch to specific TKIs be formed in a variety of ways, most commonly fusion of the entire that target the T790M mutation (Ma et al., 2011). Other less ALK tyrosine kinase domain (exons 20-29) to various truncations frequent mechanisms of acquired EGFR TKI resistance have been of EML4. This leads to a constitutively expressed protein kinase described, including MET amplification, HER2 amplification and and therefore increased cell growth, proliferation, and decreased histologic transformation to small cell carcinoma (Yu et al., 2013). apoptosis (Morán et al., 2013). EML4-ALK fusions have been Laboratories may use any validated EGFR testing method with identified in 4% of lung cancers, mostly in adenocarcinoma, in acceptable performance characteristics and a sensitivity to detect The Journal of the Association of Genetic Technologists 43 (4) 2017

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Review A Molecular and Cytogenetic Update on Non-Small Cell Lung Carcinoma young adult patients, and in patients who have never smoked or are light smokers (Table 1). In a majority of these cases, ALK fusions are mutually exclusive with other oncogenic mutations found in NSCLC (Pillai and Ramalingam, 2012). Patients tested positive for ALK rearrangements constitute a molecular subgroup that may respond to ALK-targeted therapy.

Several methods can be used to determine ALK status in NSCLC patients (Soda et al., 2007). Fluorescence in situ hybridization (FISH) using dual-labeled break-apart probes was the first clinically validated and recommended method to select patients for ALK inhibitor therapy (Lindeman, 2013; Thorne-Nuzzo et al., 2017). As FISH is technically challenging, immunohistochemistry-based detection of ALK rearrangements has been proposed as a quicker and cheaper alternative to FISHbased detection. Several immunohistochemistry (IHC) methods have been published, but the tested antibodies had varying sensitivity and specificity, likely due to the low expression levels of the fusion protein (Lindeman, 2013). However, the FDAapproved, fully automated Ventana ALK (D5F3) CDx Assay (Ventana Medical Systems, Tucson, AZ) that uses a highly sensitive detection system demonstrated high concordance with FISH, and may be used as a stand-alone test to select patients for ALK inhibitor treatment (Thorne-Nuzzo et al., 2017). RT-PCR, in contrast, is not recommended for ALK testing due to the high variability of ALK fusion partners (Lindeman et al., 2013).

by several mechanisms: translocations, activating mutations, and the widely studied gene amplifications (Miao et al., 2016).

Amplification of FGFR1 is more frequently found in SCC (18.%) than ADC (4%) (Table 1). FGFR1 amplification can be detected using FISH, where the FGFR1 locus is labeled with a green fluorochrome and the centromeric reference probe (CEN8) is labeled with an orange fluorochrome. Most studies suggest a cutoff value to define a positive result of FGFR1 amplification as a FGFR1/ CEN8 ratio ≼2.0 or FGFR1 gene copy number of more than 6.2 (Miao et al., 2016). The association between FGFR1 amplification and clinical characteristics (i.e., smoking status, disease stage) and the prognostic significance of FGFR1 amplification remains controversial. Although some studies reported a significant increase of FGFR1 amplification in current smokers compared to former and never smokers (Craddock et al., 2013; Kim et al., 2013), others did not find a significant correlation between FGFR1status and clinicopathological parameters (Kohler et al., 2012; Tran et al., 2013). Regarding the prognosis, some studies report a shorter overall survival of patients with FGFR1 amplification (Kim et al., 2013; Cihoric et al., 2014), while others found longer survival of FGFR1-positive patients (Heist et al., 2012; Toschi et al., 2014). Therefore, more investigation needs to be done to provide sufficient evidence for routine molecular testing of FGFR1 in lung cancer (Miao et al., 2016).

TP53

ROS1

The tumor suppressor gene TP53 (17p13) regulates key pathways including growth arrest, apoptosis, DNA repair and metabolism through transcriptional activation of a large number of downstream genes (Mogi and Kuwano, 2011). In NSCLC, mutation of TP53 is more common in squamous cell carcinomas (~70-80% of cases) than in adenocarcinomas (~50%), particularly in patients with smoking history. Missense mutations of TP53 often have a gain of function that leads to high expression levels in tumor cells, while deletions and insertions often cause a truncated and inactive protein (Goldstein et al., 2010). Generally, TP53 mutations are associated with severe disease, poor prognosis and resistance to chemotherapy and radiation treatment (Mogi and Kuwano, 2011; Halvorsen et al., 2016).

Although it was discovered in the early 1980s, little is known about the biology of the orphan receptor tyrosine kinase family member ROS1. The protein consists of a large glycoproteinrich extracellular domain, a helical transmembrane domain and a cytoplasmic tyrosine kinase domain, but a ligand that activates ROS1 still has to be found (Bubendorf et al., 2016).

Rearrangements of the ROS1 gene, located at 6q22.1, were initially identified in a glioblastoma cell line, and subsequently in several other types of cancer including ovary, gastric and NSCLC (Gainor and Shaw, 2013). In NSCLC, ROS1 rearrangements occur in 1-2% of cases (Table 1). Recurrent fusion partners include CD74 (in 32% of cases), SLC34A2 (17%) and SDC4 (11%), among others. Of note, the ROS1 tyrosine kinase domain remains intact in all of the identified fusion events (Bubendorf et al., 2016). The products of these rearrangements have been reported to up-regulate signaling pathways such as PI3K-AKTmTOR, JAK-STAT, and RAS-RAF-MEK/ERK (Davies et al., 2012). ALK, ROS1 rearrangements can be detected using duallabeled break-apart FISH probes and immunohistochemistry, and also with RT-PCR, multiplex PCR or NGS platforms that cover the fusion genes (Bubendorf et al., 2016).

HER2

The human epidermal growth factor receptor 2 (HER2 or ERBB2) is frequently deregulated in cancer. Located on 17q12, amplification of HER2 occurs in up to 30% of breast cancer patients. Treatment with the anti-HER2 monoclonal antibody trastuzumab improved the survival of breast cancer patients with high levels of HER2 expression (Peters and Zimmermann, 2014). In NSCLC patients, HER2 protein overexpression and gene amplification has been reported in 6-35% and in 10-20%, respectively (Mazieres et al., 2013). While IHC is used to quantify the amount of expressed HER2 protein, FISH is used to identify HER2 gene copy numbers (Mar et al., 2015). Unfortunately, NSCLC patients with overexpressed HER2 as detected by IHC did not benefit from treatment with trastuzumab in clinical trials (Clamon et al., 2005). Mutations of HER2 occur in 2-3% of adenocarcinoma NSCLC patients, and are mostly in-frame insertions of exon 20, which activate the downstream AKT and

FGFR1

The fibroblast growth factor receptor 1 (FGFR1), located on 8p11.23, belongs to the highly conserved FGFR family and consists of an extracellular ligand-binding domain, a hydrophobic transmembrane domain and a cytoplasmic tyrosine kinase domain (Miao et al., 2016). The signaling pathways of FGFR1 are involved in cellular processes like angiogenesis, anti-apoptosis and proliferation. Constitutive activation of FGFR1 can be caused

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Review A Molecular and Cytogenetic Update on Non-Small Cell Lung Carcinoma MEK pathways (Li et al., 2016). Cells harboring HER2 mutations have been shown to respond to TKIs that target both EGFR and HER2 like afatinib (Shimamura et al., 2006). However, clinical trials did not show the expected potential of afatinib to control disease in pretreated patients with advanced NCSLC harboring HER2 exon 20 mutations (Smit et al., 2017)

present in ~3-4% of NSCLC cases whereas amplification of the MET receptor is more frequent, occurring in ~25-75% of cases (Benedettini et al., 2010). In vitro studies recently demonstrated sensitivity to MET inhibitors in cells with alterations in exon 14 (Frampton et al., 2015). Patients harboring more than five copies of MET gene assessed by FISH had increased progression-free survival when using combined EGFR and MET inhibitor therapy (Dziadziuszko et al., 2012).

BRAF

BRAF, located at 7q34, encodes for a Ser-Thr kinase, which activates MAPK in the RAS-RAF-MEK-ERK-MAPK pathway and mediates cellular responses to growth signals. Mutations in the BRAF gene are associated with increased kinase activity and lead to constitutive activation of MAPK2 and MAPK3. BRAF is frequently mutated in malignant melanomas and, to less extent, in many other human cancers like NSCLC (3-4% of cases) (Brose et al., 2002; Cardarella et al., 2013). Of these cases, 60% of mutations are found on exon 15 that cause a V600E amino acid substitution.

Tumors harboring the BRAF V600E mutation are associated with an aggressive histology and decreased disease-free and overall survival rates. A retrospective study of 1046 NSCLC patients found non-V600E mutations in 40-50% of cases. These mutations were detected within exons 11 and 15 and were associated with smoking history, but not with any clinical-pathologic parameters or prognosis (Marchetti et al., 2011).

RET

A member of the cadherin superfamily, the RET protooncogene is located at 10q11.21 and encodes the receptor tyrosine kinase rearranged during transfection (RET), which has been shown to be mutated or rearranged in thyroid cancer (Lira, 2014).

Several studies have identified fusions of RET in 1-2% of NSCLC (Table1), most notably the transforming fusion gene KIF5B-RET (Kohno et al. 2012). Patients with RET fusions tend to be neversmokers and to have poorly differentiated adenocarcinomas, with a median relapse-free survival of 20 months (Wang et al., 2012).

KRAS/NRAS

Members of the Ras family of GTPases, KRAS and NRAS, regulate cell differentiation, proliferation and apoptosis. The KRAS gene (homologous to the oncogene from the Kirsten rat sarcoma virus), located at 12p12, codes for a GTPase that mediates signaling of growth factor receptors and plays a critical role in cell differentiation, proliferation, and survival. KRAS was one of the first characterized oncogenes (Rodenhuis et al., 1987). Mutations of NRAS in NSCLC were initially found by screening for mutations in cancer cell lines and confirmed in clinical samples of adenocarcinoma (Davies et al., 2002; Ohashi et al., 2013). In the western countries, mutations of KRAS are found in around 25% of adenocarcinomas and are associated with a smoking history (Riely et al., 2008; Dogan et al., 2012).

The majority of mutations are concentrated in hotspots spanning codons 12 and 13 of exon 2, and less frequently in codon 61 of exon 3 (Karachaliou et al., 2013). The high prevalence and easy identification through PCR covering the hotspot region makes it an attractive therapeutic target, but to date there is no targeted therapy for KRAS-positive tumors.

MET

The mesenchymal epithelial transition factor (MET) gene located on 7q31.2 encodes a tyrosine receptor kinase, which is activated through binding of the hepatocyte growth factor (HGF). Mutation or amplification of this proto-oncogene increases receptor phosphorylation activity and activates downstream pathways such as PI3K-AKT-mTOR and RAS-RAF-MEK-ERK, leading to tumor cell growth, proliferation, invasion, survival, migration, and metastasis (Solomon et al., 2014). Two mechanisms by which MET can be activated in NSCLC have been described: Exon 14 splicing alteration, and gene amplification. Exon 14 splicing mutations are

Figure 2: Key pathways in NSCLC. Tumor suppressors are shown in green, oncogenes in red. Transcription factors are depicted in orange.

P=phosphorylation. Adapted from Park, et al., 2017.

As the prevalence of some driver mutations significantly differs between ethnic groups, comprehensive cancer genome sequencing studies in different populations will be needed to identify new molecular targets in the treatment of NSCLC.

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Review A Molecular and Cytogenetic Update on Non-Small Cell Lung Carcinoma Table 1: Prevalence of recurrent gene aberrations of driver genes in NSCLC. Gene mutation

Frequency in NSCLC

Features

References

ALK

rearrangement

3.0–5.0%

Young age, light smoking, AC histology

Takeuchi et al., 2008 Gainor et al., 2013

AKT1

mutation

0.7–1.0%

More common in SCC

Malanga et al., 2007 Sasaki et al., 2008

BRAF

mutation

1.6–4.9%

No predominant histologic pattern

Naomi et al., 2002 Marchetti et al., 2011

EGFR

mutation

5.6–67.0%

AC histology, more common in Asia-pacific population

Midha et al., 2015

FGFR1

amplification

5.3–24.8%

More common in SCC

Gadgeel et al., 2013 Tran et al., 2013

HER2

overexpression amplification mutation

6.0–35.0% 10.0–20.0% 2.0–4.0%

AC histology, never-smokers, women

Mar et al., 2015 Mazières et al., 2013

KRAS

mutation

9.4–26.5%

AC histology, smoking history

Lee et al., 2010 Dogan et al., 2012

MEK1

mutation

0.4–1.0%

AC histology, smoking history

Sasaki et al., 2010 Marks et al., 2008

MET

mutation

1.5–3.0%

AC histology, never-smokers

See et al., 2012 Frampton et al., 2015

NRAS

mutation

0.8%

AC histology

Brose et al., 2002

PIK3CA

mutation

0.9–7.1%

SCC histology

Kawano et al., 2006 Lee et al., 2010

PTEN

mutation

1.7–10.7%

SCC histology, never-smokers

Lee et al., 2010

RET

rearrangement

0.9–1.9%

Mostly AC histology

Takeuchi et al., 2010 Kohno et al., 2012

ROS1

rearrangement

0.9–1.8%

Younger age, never-smokers

Takeuchi et al., 2010 Bergethon et al., 2012

Detection Methods

gene mutations (Sanger and Coulson, 1975). It has been used in the clinic to detect EGFR mutations to identify patients for targeted therapy (Kobayashi et al., 2005). The method requires a sequencing primer, thermostable DNA polymerase, nucleotides (dNTPs) and fluorescently labeled dideoxynucleotides (ddNTPs) to terminate the DNA strand elongation. DNA fragments can then be resolved through capillary electrophoresis and detection of fluorophores. However, this method requires high tumor content, a high amount of starting material and it is limited to detect genetic variants with mutation rates less than 15% (Gao et al., 2016) (See Table 2).

Pre-analytic considerations Molecular testing in non-small cell carcinoma requires high quality and quantity of tissue specimens. The source of the sample, amount of tumor cells, and type of specimen fixation are critical in molecular testing. Formalin-fixed paraffin-embedded (FFPE) tissue blocks have been the standard sample source for standard testing, but degradation nucleic acids due to formalin is an important limitation which can lead to false negative test results. Cytological samples can also be used, but require separate validation (Ellison et al., 2013; Schmid-Bindert et al., 2013) . In order to increase the amount of tumor cells in the sample, laser capture microdissection has proven useful (Chowdhuri et al., 2011). The specimens should be analyzed for cancer cell content and DNA quantity/quality, and subjected to molecular testing in a turnaround time of 10 working days (Lindeman et al., 2013).

FISH

Fluorescence in situ hybridization (FISH) is the most common clinical assay to identify chromosomal and genomic changes in lung cancer such as rearrangements and amplifications. Until now, FISH has been considered the gold standard to identify ALK rearrangements (Shackelford et al., 2014), and the Abbot Vysis ALK break-apart FISH Probe Kit was approved by the FDA in 2011. The ALK break-apart probes label the fusion breakpoint with an orange fluorophore on the telomeric end, and a green fluorophore on the centromere. The proximity of the probes creates a fused signal in normal cells, while rearrangement splits

Molecular Assays Chain termination DNA sequencing

For decades, the chain termination DNA sequencing method developed by F. Sanger has been the standard in detecting

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the signal in affected cells. In this case, FISH can accurately and reliably detect all ALK rearrangements regardless of the fusion partner. FISH is also the most common technique to detect MET amplifications, where a red probe detects MET on chromosome 7, and a green probe detects the centromere of the chromosome (CEP7). Normal tissue shows an average of two MET signals and two control probes. An increased MET/CEP7 ratio indicates multiple copies of the chromosomal region that contains MET (Cappuzzo et al., 2009). The advantage of FISH over molecular methods like NGS, qPCR or array CGH is that it allows a single cell analysis and thus a precise analysis of copy number variations and detection of intratumoral copy number heterogeneity. Challenges of FISH include technical complexity, high cost and a lengthy turnaround time. Also, the interpretation of data requires expertise (Shackelford et al., 2014) (See Table 2).

Reverse-transcriptase PCR (RT-PCR) is a highly sensitive specialized technique that allows the detection of gene fusions based on extracted RNA in tumor tissue. In RT-PCR, primers hybridize with the chimeric transcripts, and reverse transcription of the RNA produces DNA that can be amplified by PCR. The amplicons can then be sequenced to identify the specific fusion variant. The advantages of RT-PCR include a low volume of required cells, a rapid turnaround time, and a high sensitivity.

(Shackelford et al., 2014). However, RT-PCR assays not only require high-quality RNA, but also a specific set of primers for each translocation and are therefore only suitable to detect known fusion genes. For example, the high variability of the ALK fusion partners and breakpoints can cause high false-negative rates. This is why RT-PCR is currently used for screening of recurrent gene fusions only, for example in the case of the known ROS1 fusion partners (Bubendorf et al., 2016). But in recent years, the development of genomic target enrichment methods like anchored multiplex PCR (AMP), has enabled the detection of clinically relevant gene rearrangements without prior knowledge of the fusion partners or specific breakpoints (Zheng et al., 2014).

Immunohistochemistry (IHC)

Immunohistochemistry (IHC) is a rapid and inexpensive method that can easily be implemented in laboratories. It combines anatomical, immunological and biochemical techniques to identify antigens in tissue sections using specific labeled antibodies. For NSCLC molecular testing, antibodies are available against EGFR point mutations, ALK rearrangements and ROS1 rearrangements. EGFR mutation-specific IHC antibody clones for the L858R substitution and exon 19 deletions have reported sensitivities of 76% and 83%, and positive predictive values of 73% and 94%, respectively. Although the poor sensitivity limits their use in EGFR mutation screening, these antibodies may overcome the challenges of other molecular methods such as high cost, variable sensitivity/specificity due to poor DNA quality and/or low tumor cellularity, and contamination with the nonmutated allele (Allo et al., 2014).

For the detection of ALK rearrangements in NSCLC patients, the use of ALK IHC in relation to the gold standard ALK FISH has been widely debated. Some authors suggest ALK IHC as an economic and rapid screening method prior to FISH confirmation (Hutarew et al., 2014). Others recommend ALK IHC in combination with FISH to increase sensitivity and specificity of detection of ALK-rearranged lung cancers (Sholl et al., 2013). These assays use different antibodies, scoring methods, and detection systems of the assays, and their variable sensitivity and specificity is likely due to the low expression levels of the fusion protein (Lindeman et al., 2013; ThorneNuzzo et al., 2017). Novel monoclonal antibodies recognizing the human ALK protein, including clone D5F3, showed high sensitivity, specificity and reproducibility as compared to FISH (Rogers et al., 2015).

A fully automated ALK IHC assay using the D5F3 clone, the ALK (D5F3) CDx Assay (Ventana Medical Systems, Tucson, AZ), uses a highly sensitive detection system and obtained FDA approval in 2016. The assay demonstrated high concordance with FISH, and is the first to be used as a stand-alone test to select patients for ALK inhibitor treatment (Thorne-Nuzzo et al., 2017).

Concerning the detection of ROS1-rearrangements, IHC may provide cost-effective screening prior to confirmation by FISH, due to the high cost of FISH and the low prevalence of ROSIrearrangements in NSCLC (Bubendorf et al., 2016) (See Table 2).

Multiplex testing

As the numbers of known genomic alterations in NSCLC increased in the last decade, many algorithms incorporated multiplex assays to simultaneously detect multiple DNA or cDNA targets in one reaction. The FDA approved the cobas® EGFR mutation tests (Roche Basel, Switzerland), which identifies 41 mutations across exons 18, 19, 20 and 21 in FFPE specimens of NSCLC tumors. The overall concordance between the cobas® test and the gold standard Sanger sequencing was 96.7% (O’Donnell et al., 2013). Other tests like the SNaPshot assay can detect up to 50 mutations in several key NSCLC genes. The SNaPshot assay is based on multiplexed PCR and single-base extensions that generate allele-specific fluorescently labeled probes.

The products can then be resolved and analyzed by capillary electrophoresis. This technique has been integrated into clinical routine as a CLIA-certified test (Sequist et al., 2011). Limitations of multiplexed testing arise through the nature of the mutations.

As mutations in oncogenes like EGFR, KRAS, BRAF and ERBB2 (HER2) are limited to a narrow range, multiplex testing is feasible in clinical practice. However, inactivating mutations in tumor suppressor genes involve deletions or point mutations in much larger loci, making multiplex testing for inactivating mutations inefficient in clinical practice (Sequist et al., 2011) (See Table 2).

Next-Generation Sequencing (NGS)

Because next-generation sequencing (NGS) continues to become more affordable, it has surpassed several single-gene PCR assays in a clinical setting. NGS includes comprehensive sequencing of genomes (whole-genome sequencing, WGS), exomes (whole-exome sequencing, WES) and transcriptomes (whole-transcriptome sequencing, RNA-seq) and targeted sequencing of both DNA and RNA (Lu and Lu, 2017). NGS has been applied to detect point mutations, InDels, rearrangements as well as amplifications. The NGS workflow consists of DNA library preparation from patient sample followed by PCR amplification

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Therapies

and sequencing of the templates in massively parallel fashion.

The average number of independent reads, called depth of coverage, indicates the certainty of a detected DNA variant.

The proportion of sequence reads matching a variant in relation to the overall coverage at that locus, called mutation rate or Variant Allele Frequency (VAF), then identifies the percentage of DNA molecules on the specimen that carry the variant. For example, homozygous loci generally should be near 100% VAF; heterozygous loci around 50% VAF; and reference loci at 0%.

However, factors such as contamination of normal tissue and intrinsic tumor heterogeneity may cause uncertainty in VAFs. Generally, NGS routinely identifies clinically relevant mutations in tumor samples from 5-10% VAF (Strom, 2016).

In contrast, Sanger sequencing usually has a sensitivity to detect mutant alleles from 10-20% VAF, which makes NGS more efficient than first-generation sequencing. However, next-generation sequencing also has limitations. The complex validations to ensure a high level of accuracy and sensitivity, and the complex bioinformatic analysis make it difficult to implement in the clinical setting. Also, it is still uncertain to which degree certain genomic variants are clinically relevant (Strom, 2016). One successful application of NGS to molecular testing of NSCLC patients comes from the application of targeted NGS. In one study, a panel of commonly mutated genes detected somatic mutations in 46% of 209 samples, including KRAS (28%), EGFR (14%), PIK3CA (4%), PTEN (1%), and BRAF (1%). A targeted therapy was initiated on the basis of NGS data in 11% of the sequenced patients (Hagemann et al., 2014) (See Table 2).

Current treatment options for non-small cell lung cancer include surgery, radiation therapy, chemotherapy, and targeted therapy, but despite the advances in diagnosis and therapy made during the past decades, the prognosis for NSCLC patients remains unsatisfactory.

The heterogeneity and genomic complexity makes NSCLC a challenging disease to treat. Understanding the underlying biological causes is critical to developing better treatment options.

Radiation therapy Depending on the disease stage, radiation therapy can be used as a main treatment, before or after surgery, alongside chemotherapy or for palliative purposes. The two main types of radiation therapy to treat NSCLC include external beam radiation, and internal radiation (brachytherapy). While the conventional external radiation involves a beam of high-energy x-rays, internal radiation is based on a local delivery of a radioactive source to the area of interest (Parashar et al., 2013). A recent review of fourteen randomized controlled trials with 3576 patients concludes that short-course radiotherapy improves symptoms, but found no evidence that a long-course radiotherapy improves the prognosis.

In contrast, prolonged radiotherapy leads to immediate side effects, especially sore swallowing (Stevens, 2015).

Chemotherapy and Targeted therapy Standard first-line chemotherapy for patients affected by advanced NSCLC often includes platinum-based doublet of cisplatin or carboplatin, which cause cytotoxicity by targeting

Table 2: Overview of common molecular diagnostic techniques, their advantages/limitations and applications.

Advantages

Disadvantages

Applications Point mutations

InDels

Rearrangements

Amplification

Non-recurrent alterations

Sanger seq.

Gold standard

Requires high tumor content and mutation rates (Gao, 2016)

EGFR T790M (Kobayashi 2005)

–

–

–

Only if inside sequenced loci

FISH

Single-cell resolution

High cost, technical complexity

–

–

Break-apart FISH (Schackelford, 2014)

MET (Capuzzo, 2009)

–

IHC

Rapid, cost-effective

Poor sensitivity

EGFR L853R

EGFR exon 19del

ALK, ROS1

–

–

RT-PCR

Highly sensitive

High falsenegative rates

–

–

Screening for recurrent gene fusions

–

–

Multiplex testing

Many targets

Only for hotspot mutations

Detection of key gene mutations

–

–

–

–

NGS

High sensitivity

High cost, complex analysis

x

x

x

x

x

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Anti-ALK/ROS1 therapy

DNA replication and transcription and eventually induce apoptosis (Rossi and Di Maio, 2016). Despite a slight increase in toxicity profile, platinum-based regimens significantly improve the 1-year overall survival (OS) from 10-20% up to 20-30% in comparison with best supportive care (Rossi and Di Maio, 2016).

Approximately 30% of the patients experience disease progression following first-line chemotherapy and require subsequent therapy.

This second-line chemotherapy often consists of the cytotoxic agents pemetrexed and docetaxel (Weiss and Stinchcombe, 2013). When molecular testing has identified genetic aberrations in driver genes such as EGFR, ALK or ROS1, the patient may receive targeted treatments using TKI inhibitors (TKIs) like erlotinib and gefitinib. Many second-line trials have demonstrated an improvement in progression-free survival and OS (Weiss and Stinchcombe, 2013).

In 2011, the FDA approved the oral ALK inhibitor crizotinib for treatment of ALK-positive NSCLC patients. Crizotinib showed increased progression-free survival and objective response rate compared to standard first-line and second-line chemotherapy for advanced ALK-positive NSCLC patients (Shaw et al., 2013; Solomon et al., 2014). However, several mechanisms of acquired resistance of crizotinib have been demonstrated. These can occur through ALK dependent manners, in which ALK gene amplifications shift the intracellular balance between the TKI and the kinase in favor of the kinase, or secondary mutations in the kinase domain confer differential sensitivity to the drug (Gainor et al., 2013). Other possible mechanisms include ALKindependent manners, for example the emergence of secondary oncogenic drivers such as KRAS (Doebele et al., 2012). This led to the development of second-generation ALK inhibitors such as ceritinib and alectinib, that were approved in 2014 as second-line treatment for advanced NSCLC in the U.S. and Japan, respectively (Liao et al., 2015). Crizotinib is also effective in a subset of ALKnegative NSCLC patients. In case reports, two patients that were tested positive for ROS1 rearrangement by FISH showed tumor shrinkage after treatment with crizotinib (Bergethon et al., 2012; Davies et al., 2012). In the Phase I study of crizotinib, a cohort of patients that was found positive for ROS1 by FISH had an ORR of 72% and a median PFS of 19.2 months (Shaw et al., 2014). In a retrospective study of 32 adenocarcinoma patients that tested positive for ROS1 rearrangement by FISH and confirmed

by NGS, treatment with crizotinib showed a response rate of 80% and a median progression-free survival interval of 9.1 months (Mazières et al., 2015). Testing for driver genes in NSCLC in order to select patients for targeted therapies were shown to improve both progression-free survival and overall survival in comparison to standard chemotherapy, as well as better toxicity profile and patient convenience (Sellmann et al., 2015).

Anti-EGFR therapy The inhibition of the EGFR through tyrosine-kinase inhibitors (TKIs) was the first successful example of targeted therapy in non-small cell lung cancer. Several clinical trials have proven superiority of EGFR TKIs over first-line chemotherapy in EGFR-mutated NSCLC patients in terms of overall response rate (ORR), progression free survival (PFS), but not in overall survival (OS). In two Phase III studies, EGFR mutation positive patients with advanced stage lung adenocarcinoma were randomly assigned to treatment with receptor tyrosine kinase inhibitor afatinib or standard doublet chemotherapy. Interestingly, the afatinib group did not show increased median overall survival in comparison to the chemotherapy group. However, when patients were stratified by EGFR mutation, patients with del19positive tumors showed an increase of median overall survival of more than 12 months when treated with afatinib (Sequist et al., 2013; Yang et al., 2015). These results suggest that patients with EGFR del19 mutation might be distinct from patients with other mutations and suggest that these subgroups should be independently investigated in further trials.

However, almost all patients suffer disease progression due to acquired resistance. The most common mechanism involves the T790M mutation that decreases binding affinity of the TKI to EGFR (Wang et al., 2016). Therefore, second- and third generation EGFR inhibitors were designed that irreversibly bind to EGFR. The orally delivered irreversible TKI rociletinib specifically targets the common EGFR mutations including T790M, with minimal activity towards the wt receptor (Walter et al., 2013). In a phase I-II study, T790M-positive patients that were treated with rociletinib had an objective response rate of 59%, in comparison to only 29% in patients with T790-negative disease.

The median progression-free survival was longer in patients with the EGFR T790M mutation in comparison to those without (13.1 months vs 5.6 months). The safety profile was acceptable.

The most common grade 3 adverse event was hyperglycemia in 20% of patients, which could be managed with oral hypoglycemic agents (Sequist et al., 2015). Despite these promising results, novel resistance mutations to third-generation EGFR inhibitors including EGFR L718Q, L844V and C797S have been described, which will lead to the development of new strategies to inhibit EGFR (Ercan et al., 2015).

Anti-angiogenesis therapy Another strategy to treat NSCLC targets angiogenesis, a key process of tumor growth, progression and metastasis (Hanahan and Weinberg, 2011). Anti-angiogenic therapy aims to cut off the tumor’s supply of nutrients and oxygen. Multi-targeted antiangiogenic tyrosine kinase inhibitors (MATKIs) target a key mediator of tumor angiogenesis, the vascular endothelial growth factor receptor (VEGFR), as well as other key pathways.

Recent trial of the effectiveness of MATKI agents in NSCLC found an increased objective response rates (ORR) and prolonged progression free survival (PFS) in patients who received MATKI treatment in combination with standard treatments and in patients who had previously undergone chemotherapy (Liang et al., 2014). These studies provide evidence for the effectiveness of MATKI as both monotherapy and as co-therapy for patients with advanced NSCLC.

PD-1/PD-L1 Immunotherapy Evading immune destruction of tumor cells is another cancer hallmark of therapeutic interest (Hanahan and Weinberg, 2011).

The complex and not entirely understood interplay between tumor cells and the immune system involves the interaction of

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membrane-bound ligands and receptors such as PD-L1 and PD-1.

In physiological conditions, these immune checkpoint proteins regulate the immune system to prevent autoimmune response.

However, many cancer cell types express PD-L1, which then binds to PD1 on tumor infiltrating lymphocytes (TILs). This ultimately inhibits T-cell activity and the cancer cells escape the immune response (O’Donnell et al., 2017). The rationale to stimulate the immune response against cancer cells via the PD-1/PD-L1 pathway opens promising opportunities for targeted therapy.

Several drugs are being tested in clinical trials including the PD-1 inhibitors nivolumab and pembrolizumab, and the PD-L1 inhibitors atezolizumab, durvalumab, and avelumab. For example, pembrolizumab showed promising results in a Phase III trial in treatment-naïve NSCLC patients with at least 50% PD-L1 expression. Patients treated with pembrolizumab had a higher response rate (45% vs 29%), and a longer duration of response than patients treated with chemotherapy (Reck et al., 2016). Atezolizumab obtained FDA approval in 2016 as secondline treatment for advanced NSCLC, following a randomized phase III trial showing an increase of median overall survival of 2.9 months compared to chemotherapy, and a better safety profile. Interestingly, the improvement of overall survival correlated with increasing PD-L1 expression, supporting the hypothesis of PD-L1 expression as a predictive biomarker (Fehrenbacheret al., 2016). Therefore, it is crucial to accurately detect PD-L1 expression in tumor samples in order to select patients for checkpoint inhibitor treatment. PD-L1 expression is detected by immunohistochemistry. Each PD-L1 drug has been co-developed with its own PD-L1 immunohistochemical PD-L1 clone on a specific testing platform. This creates challenges as the assays involve different antibody clones, target different PD-L1 domains, employ different technical protocols/platforms, and show different cutoffs for positivity (Sholl et al., 2016).

Recent studies compared commercially available, trial-validated assays and demonstrated analytical equivalence (Ratcliffe et al., 2017; Rimm et al., 2017). Interestingly, the assays showed higher concordance for PD-L1 expression in tumor cells than in immune cells (Rimm et al., 2017).

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Conclusions Patients with advanced non-small cell lung carcinoma (NSCLC) still have poor prognosis, but the increased knowledge of the molecular basis and the discovery of targetable mutations in driver genes have progressively improved patient outcomes. Molecular testing of driver gene aberrations like EGFR mutations and ALK/ROS1 rearrangements has already been established in guidelines for patients with lung adenocarcinoma and is routinely performed in clinical laboratories.

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Conflict of interest The authors declared no conflict of interest.

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

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Profiles and Perspectives

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

Dr. Carlos Tirado, PhD, FACMG Interviewed by Mark Terry Dr. Carlos Tirado should be well known to readers of The Journal of the Association of Genetic Technologist, because of his numerous articles co-written with technologists and graduate students that appear here. Born and raised in Peru, Dr. Tirado received his Bachelor of Science degree from the Universidad Nacional Pedro Ruiz Gallo in Lambayeque, Peru. In June 1991, he was a Fullbright Scholar.

He then attended Arizona State University where he received his Master’s of Science degree, completing his thesis titled, “Cytogenetics Findings of Pancreatic Cancer” in 1993. He then stayed at Arizona State University to complete his PhD. His doctoral dissertation was titled “Molecular Cytogenetics of Pancreatic Cancer.” Dr. Tirado completed his Fellowship in Clinical Cytogenetics at Duke University Medical Center in 2004. He then joined Quest Diagnostics Nichols Institute in Chantilly, Virginia, followed by an Assistant Professor position at University of South Carolina School of Medicine in Columbia, South Carolina. In 2008, Dr. Tirado became an Associate Director of Cytogenetics at University of Texas Southwestern Medical Center in Dallas, where he also ran the ABMG Fellowship in Clinical Cytogenetics. In 2011, he joined UCLA as an Associate Director until 2016. He was also an Adjunct Professor at MD Anderson Health Sciences and UT San Antonio. Currently, Dr. Tirado is Director of the Cytogenetics Laboratory at Allina Health System in Minneapolis, Minnesota, and Adjunct Associate Professor in the Department of Laboratory Medicine & Pathology at the University of Minnesota Medical School. He is also an Honorary Professor with the Universidad Nacional, Pedro Ruiz Gallo, in Lambayeque, Peru. Single, Dr. Tirado lives in Minneapolis. His research interests are in cancer cytogenetics, specifically solid tumors and hematological malignancies.

MT:

What is your current position?

CT:

I have been the Director of the Cytogenetics Lab at Allina Health since Nov. 7, 2016. I also have an appointment as an Associate Professor at the University of Minnesota School of Medicine, Department of Laboratory Medicine and Pathology.

MT:

When did you become interested in genetics? When did you become interested in clinical cytogenetics?

CT:

I became interested in genetics in 1988 when I got a position as a TA in the course of General Genetics at a National University in Peru. I was doing research in cytogenetics of Buffo species in Peru.

MT:

What changes have you seen in the field since you entered it?

CT:

When I started my research during the Master’s program at Arizona State University, I was taking photographs of chromosomes and I was cutting them with scissors. I have seen the beginning of FISH with ONCOR probes that I used for my project. Then CHG at the beginning to chromosomal microarrays.

Dr. Carlos Tirado, PhD, FACMG

MT:

Please describe your training.

CT:

I started my Masters Project at ASU under Dr. John F. Stone at the Southwest Biomedical Research Institute.

The medical director was Dr. Avery Sandberg, one of the pioneers in Cancer Cytogenetics. Then I kept working with them for my PhD. After I finished my studies I became an Associate Professor at the Department of Biology at Universidad Peruana Cayetano Heredia in Lima, Peru. I came back to the U.S. to do a Fellowship at Duke University Medical Center under Dr. Barbara K. Goodman. In 2004 I started to work as an Assistant Director of Cytogenetics at Quest Diagnostics Nichols Institute in Chantilly, Virginia. I got my first faculty position at University of South Carolina School of Medicine. After passing the boards in Cytogenetics, I moved to The University of Texas Southwestern Medical Center in 2008 as an Associate Director of Cytogenetics.

In 2011, I got an excellent position at UCLA where I was the Director of the training in Cytogenetics by the state of California. In November 2016, I became the Director of the Cytogenetics Lab at Allina Health in Minnesota and Associate Professor at the Department of Laboratory Medicine and Pathology at University of Minnesota School of Medicine.

MT:

You’ve worked for big clinical labs, like Quest Diagnostics, as well as at Universities and major medical systems. Can you discuss the differences you’ve found in practicing clinical genetics in the different settings?

CT:

Of course, a clinical lab has a different environment than a reference lab or a commercial lab. Both places have their good and bad things. I really enjoy academia.

MT:

Please tell me about the International Circle of Genetic Studies and your involvement with it.

CT:

The International Circle of Genetic Studies is a project where we are looking for new leaders in Genetics at the level of undergraduate and high school. We are more than 20 professors working with undergraduates, including for example, Dr. Christine F. Stephenson, Dr. Rodney Wiltshire, Dr. Guillermo Martinez de Tejada, Dr. Daniel Paoli, Dr. Carlos Santa Maria, Dr. Elio Quiros, Lic. David Palencia, Dr. Francisco Javier Novo, Dr. Carlos Romani, Dr. Franklin Fuda, Dr. Esteban Dell’Angelica, Dr. Katrina Dipple, Mr. Francisco Castillo, Dr. Nyla Heerema, etc., who are helping me in this big project where I am the PI. We have three chapters, one in Los Angeles, one in Pamplona, Spain and another one in

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Profiles and Perspectives

Dr. Carlos Tirado, PhD, FACMG

Chiclayo, Peru. We are trying to form new leaders in Genetics. We are focused on forming new scientists with integrity. So far we have been very successful and all of our students have been involved in research. They have also presented in national and international meetings. Most of them are now in medical school, graduate programs in Spain and the USA. MT:

You are, I believe, one of the organizers of the V International Symposium of Genetics in Peru. Tell me about that conference and how you got involved in it?

CT:

Yes, I am the Chair of this conference. My students’ project has a challenge to organize an international meeting in a foreign country. This year it will be also in Peru. The students do not speak Spanish, but they try really hard.

They look for the speakers, ask for topics and work on the logistics of these meetings. Up to now there have been four previous meetings and we will have the next one in 2018.

MT:

What are your research interests? Tell me a little bit about your research.

CT:

I am a Clinical Cytogeneticist with interests in hematological malignancies, as well as solid tumors. I have continued working in pancreatic cancer. But I am also am very interested in doing research in other solid tumors as well as hematological malignancies.

MT:

Do you have any advice for technologists considering pursuing higher education in the field?

CT:

I really encourage people to go back to school if this is what they want to do for life. Experience is an excellent teacher.

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Genetics in the News

A Breakthrough in Gene Editing of Human Embryos to Correct Pathogenic Gene Mutations Jaime Garcia-Heras The prevention of genetic disease has been feasible using a wide variety of diagnostic and screening tools. The first were the screening for Tay-Sachs disease (O’Brien et al., 1970) and sickle cell disease (Schneider et al., 1967), the traditional prenatal screening for chromosome abnormalities (Chasen, 2014) and open neural tube defects (Krantz et al., 2016) through analytes in maternal serum, and the standard invasive prenatal diagnosis of gene disorders and chromosome abnormalities in amniotic fluid (Robinson and Henry, 1985) and chorionic villi (Brambati and Tului, 1998). Much more recent are the uses of expanded pre-conceptional screening of carriers of constitutional mutations associated with gene disorders (Langlois et al., 2015; Holtkamp et al., 2016), non-invasive prenatal diagnosis for gene disorders (Drury et al., 2016) or screening for the most common chromosome aneuploidies (Palomaki et al., 2012) in cell-free fetal DNA that circulates in maternal blood, and preimplantation genetic diagnosis in embryos generated by assisted reproductive technology (ART) (Geraedts and De Wert, 2009; Brezina and Kutteh, 2014; Garcia-Herrero et al., 2016).

These approaches have substantially reduced the burden of genetic diseases, especially in families with high risk and previously affected individuals. But sometimes couples can only conceive offspring with genetic disorders caused by mutations transmitted by either parent. For such unfortunate circumstances, genomic editing of embryos in the setting of assisted reproductive technology (ART) has been proposed as a reasonable alternative so that these couples can have their own healthy children.

Recent research in early human embryos generated by ART reported the correction of a pathogenic gene mutation that caused familial hypertrophic cardiomyopathy (HCM) (Ma et al., 2017).

This development in gene editing was accomplished with CRISPRCas9 technology which allowed the excision of the mutated gene from the DNA and replacing it with a normal copy.

HCM is the most common heritable cardiac disease. It is frequently responsible for sudden heart failure and death in individuals >50 years, particularly young athletes. The disease is characterized by left ventricular hypertrophy and by contractile dysfunction, myocardial fibrosis and arrhythmias as the disease progresses (Dadson et al., 2017). HCM is an autosomal dominant disease caused by mutations of the MYBPC3 gene in ~40-50% of the cases (Carrier et al., 2015). The offspring of patients have a 50% risk of inheriting the disease. HCM may have late onset of the clinical manifestations and often by the time of diagnosis the MYBPC3 mutation was already transmitted to descendants because it is not subject to natural selection. There is no cure for HCM at this time. The only options are palliative care of the symptoms.

Given the severe clinical features and hereditary pattern of HCM, prevention is extremely important and is frequently offered to couples at risk of having affected offspring. One option is preimplantation genetic diagnosis (PGD) in embryos generated by ART followed by a transfer of embryos without pathogenic mutations. For the worst scenario of only retrieving embryos with heterozygous pathogenic mutations associated with HCM, corrective gene editing was explored to recover mutant embryos (Ma et al., 2017).

The research study of Ma et al accomplished the correction of a heterozygous dominant four-base-pair deletion in exon 16 of MYBPC3 that was associated with familial HCM in embryos that were not intended for implantation. Gene editing was performed in human zygotes generated by fertilizing healthy donor oocytes with sperm from a male patient heterozygous for the MYBCPC3 mutation. These embryos were collected at the 4-8-cell stage and were subsequently disaggregated into individual blastomeres for processing and analysis at the singlecell level. The first experiments were conducted on 54 embryos 18 hours after fertilization, injecting into pronuclear zygotes a CRISPR-Cas9 cocktail (a sgRNA to direct the excision of the mutated gene, a recombinant Cas9 protein to induce single strand breaks at the site of the mutation, and an exogenous single-stranded oligonucleotide (ssODN) with the normal MYBPC3 gene as template). It was feasible to correct the paternal mutated MYBCP3 gene since a significant number of embryos carried two normal copies of the MYBPC3 gene in all the blastomeres (n=36, 67%). These embryos were predicted to be healthy. However, there were 18 (33%) other embryos which were uniformly heterozygous for the paternal MYBPC3 mutation in all blastomeres (n=5, 9%), or were mosaics (n=13, 24%) carrying the mutation in some blastomeres while other blastomeres were normal. These initial results indicated the need to improve the efficiency of gene editing with CRISPRCas9 because the embryos with the MYBPC3 mutation (33%) would not be free from disease, unsuitable for transfer, and it would be difficult to identify them by pre-implantation diagnosis.

The conclusion was that this protocol performed 18 hours after fertilization was not acceptable for clinical applications.

A different gene-editing procedure of a simultaneous injection by intra cytoplasmic sperm injection (ICSI) of sperm and the same CRISPR-Cas9 cocktail to M-phase oocytes at the time of fertilization was consequently tested in 58 other embryos. This procedure yielded a higher number of completely normal homozygous embryos with two normal copies of MYBPC3 (n=42, 72%). In addition, the number of embryos heterozygous for the MYBPC3 mutation was reduced (n=16, 28%), and the mosaicism of normal and abnormal blastomeres in embryos was eliminated almost entirely (only a single mosaic embryo). This markedly improved method was considered more appropriate for an eventual application in patients.

It was quite a surprise that all the gene editing experiments consistently showed a predominant correction of the paternal MYBPC3 mutation through homology-directed repair (HDR) using the normal (wild-type) maternal allele as a template instead of the supply of a synthetic oligonucleotide DNA. This outcome suggests that human embryos and zygotes have very stringent DNA repair mechanisms to keep the fidelity of the genome in the germline. HDR is a highly efficient and accurate DNA-repair pathway that can correct a mutant allele using as a template the normal homologous copy or an exogenous source of synthetic DNA.

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Genetics in the News A Breakthrough in Gene Editing of Human Embryos to Correct Pathogenic Gene Mutations The gene correction achieved with CRISPR-Cas9 did not impair the preimplantation development and survival of the treated embryos since they progressed normally to the stages of 8-cells and blastocysts. This observation implied that they had the potential for a normal full development if they were transferred in clinical practice.

There has been concern that gene editing with CRISPRCas9 might induce unintended off-target mutations at regions of high homology with the targeted sequences. If this is the case, the risks of secondary genetic damage might offset the benefits expected from corrective gene editing. It was very reassuring to see that the gene editing performed did not induce off-target mutations detectable by Digenome-seg, whole genome sequencing and whole exome sequencing. Furthermore, the genome stability of the corrected embryos was maintained after the gene correction with CRISPR-Cas9.

with disabilities or normal skills. There is also a controversy of sharply opposing views, while some consider genome editing a desirable and welcomed advance, while others believe it is an unacceptable risk that should be avoided. Hopefully a proper balance of all the issues will be achieved, clinical trials will be successfully completed, and finally proper medical use takes place in the future. Many families struck by devastating genetic diseases would benefit from this innovative approach of genome editing.

References Brambati B, Tului L. Prenatal genetic diagnosis through chorionic villus sampling. In: Genetic disorders and the fetus: Diagnosis, Prevention and Treatment, 4th ed. John Hopkins University Press, 1998.

Brezina PR, Kutteh WH. Clinical applications of preimplantation genetic testing. BMJ 2014; 349: g7611.

Carrier L, Mearini G, Stathopoulou K, Cuello F. Cardiac myosin-binding protein C (MYBPC3) in cardiac pathophysiology. Gene. 2015;573: 188-197.

Chasen ST. Maternal serum analyte screening for fetal aneuploidy. Clin Obstet Gynecol. 2014;57: 182-188.

Dadson K, Hauck L, Billia F. Molecular mechanisms in cardiomyopathy.

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Drury S, Hill M, Chitty LS. Celll-free fetal DNA testing for prenatal diagnosis. Adv Clin Chem. 2016;76: 1-35.

Garcia-Herrero S, Cervero A, Mateu E, Mir P, Póo ME, Rodrigo L, Vera M, Rubio C. Genetic analysis of human preimplantation embryos. Curr Top Dev Biol. 2016;120: 421-447.

Geraedts JPM, De Wert GMWR. Preimplantation genetic diagnosis. Clin Genet. 2009;76: 315-325.

Holtkamp KCA, Mathijssen IG Lakeman P, van Maarle MC, Dondorp WJ, Henneman L, Cornel MC. Factors for successful implementation of population-based expanded carrier screening: learning from existing initiatives. Eur J Publ Health. 2016;27: 372-377.

Krantz DA, Hallahan TW, Carmichael JB. Screening for open neural tube defects. Clin Lab Med. 2016;36: 401-406.

Langlois S, Benn P, Wilkins-Haug L. Current controversies in prenatal diagnosis 4: pre-conception expanded carrier screening should replace all current prenatal screening for specific single gene disorders. Prenat Diagn. 2015;35: 23-28.

Liang P, Xu Y, Zhang X, Ding C, Huang R, Zhang Z, Lv J, Xie X, Chen Y, Li Y, Sun Y, Bai Y, Songyang Z, Ma W, Zhou C, Huang J. CRISPR/ Cas9-mediated gene editing in human tripronuclear zygotes. Protein Cell. 2015;6: 363-372.

Ma H, Marti-Gutierrez N, Park SW, Wu J, Lee Y, Suzuki K, Koski A, Ji D, Hayama T, Ahmed R, Darby H, Van Dyken C, Li Y, Kang E, Park AR, Kim D, Kim ST, Gong J, Gu Y, Xu X, Battaglia D, Krieg SA, Lee DM, Wu DH, Wolf DP, Heitner SB, Izpisua Belmonte JC, Amato P, Kim JS, Kaul S, Mitalipov S. Correction of a pathogenic gene mutation in human embryos. Nature. 2017;548: 413-419.

National Academy of Sciences and National Academy of Medicine.

Huma n Genome E diting : Science, Et hics a nd Gover na nce.

Washington, DC; National Academies Press; 2017.

O’Brien JS, Okada S, Chen A, Fillerup DL. Tay-Sachs disease. Detection of heterozygotes and homozygotes by serum hexosaminidase assay. N Engl J Med. 1970;283: 15-20.

Palomaki GE, Deciu C, Kloza EM, Lambert-Messerlian GM, Haddow JE, Neveux LM, Ehrich M, van den Boom D, Bombard AT, Grody WW, Nelson SF, Canick JA. DNA sequencing of maternal plasma reliably identifies trisomy 18 and trisomy 13 as well as Down syndrome: An international collaborative study. Genet Med. 2012;14: 296-305.

Robinson A, Henry GP. Prenatal diagnosis by amniocentesis. Ann Rev Med. 1985;36: 13-26.

Schneider RG, Alperin JB, Lehman H. Sickling tests. Pitfalls in performance and interpretation. JAMA. 1967;202: 419-421.

Tang L, Zeng Y, Du H, Gong M, Peng J, Zhang B, Lei M, Zhao F, Wang W, Li X, Liu J. CRISPR/Cas9-mediated gene editing in human zygotes using Cas9 protein. Mol Genet Genomics. 2017;292: 525-533

Concluding Remarks The results published by Ma et al are a major milestone in human gene editing for the prevention of heritable genetic diseases caused by gene mutations. This highly successful gene editing of an HCM mutation in early embryos supersedes previously published research (Liang et al., 2015; Tang et al., 2017), it successfully addressed the concerns about the safety and efficacy, particularly for ART, and could be expanded to other genetic conditions caused by inherited gene mutations. To mention just a few of the most frequent gene disorders in humans, it could be applied to BRCA1 and BRCA2 mutations linked to risk for breast and ovarian cancer, Huntington’s disease, as well as other autosomal recessive conditions (e.g. Tay-Sachs disease, thalassemia, sickle cell anemia, cystic fibrosis). Although, in theory, the current prospects of gene editing in embryos seem promising, Ma et al stated the need to assess if their proposed methodology is applicable to other heterozygous mutations. They also cautioned about the challenges to repair homozygous mutations (both genes mutated) because gene-editing by homology directed repair (HDR) is not doable due to a lack of a normal gene as template.

This exciting research by Ma et al was published a few months after a scientific committee from the National Academy of Sciences and the National Academy of Medicine from the USA issued a report about the use of genome editing (National Academy of Sciences and National Academy of Medicine, 2017). Regarding germline (heritable) genome editing, a set of specific criteria that should be met were recommended. These include the absence of reasonable alternatives, a restriction to preventing a serious disease or condition, and restriction to editing genes that cause or predispose to a disease or condition.

The consensus of this committee was that clinical trials should be permitted within a robust and effective regulatory framework.

Now that human genome editing has been endorsed under proper guidelines and regulations (National Academy of Sciences and the National Academy of Medicine, 2017), there are important societal issues that should be discussed and resolved with ethical and legal matters at the top of the list. Furthermore, there has been a lingering concern that human gene editing might be improperly used for some form of eugenics—that is the design of individuals with enhanced traits such as intelligence or athletic capabilities, which may end up devaluating individuals

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Brain Tickler

Brain Tickler Summary (see inside front cover)

The pathology report specified that the leukemia was an acute myelogenous leukemia.

Karyotype: 47,XY,der(5)t(5;17)(q12;q12),+8,+15,der(15)t(11;15)(q22;q24)dup(11)(q25q22),del(16)(q22q24), -17,der(20;21)(p10;q10),+mar[16]/46,idem,der(10)t(10;11)(q25;q23)t(5;11)(q12;q25),-mar[3]/46,XY[1] Clone 1 had a deletion of chromosome 5q, trisomy 8, derivative 15 with 11q, duplication of 11q, deletion 16q, loss of 17p due to a derivative 5;17, a complicated der(20;21) and a marker chromosome.

Clone 2 had the same findings plus a translocation between chromosomes 10 and 11.

FISH confirmed the deletion of EGR1 and the deletion of CBFB (data not shown). In addition, MLLÂ was amplified in most interphase cells (see FISH images). In clone 1 the amplified MLL signals were located on the der(15) and on a marker chromosome; in clone 2 the amplified MLL signals clustered both on the der(15) and on the der(10).

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Continuing Education Opportunities

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

Test Yourself #4, 2017 Readers of The Journal of the Association of Genetic Technologists are invited to participate in this “open book test” as an opportunity to earn Contact Hours.

AGT offers 3 Contact Hours for this Test Yourself based on articles in Volume 43, Number 3, Third Quarter 2017 of the Journal.

Test Yourself is free to AGT members and $30 for non-members. To take this exam, send a copy of your completed Answer Sheet along with the completed Contact Hours Reporting Form to the AGT Education Committee Representative in your region. The list of representatives can be found on the AGT website. Non-members should submit a check payable to AGT for $30 with their answer sheet. Entry material must be post-marked on or before March 2, 2018.

(Passing score is 85% or 17/20 correct answers.) Compiled by Doina Ciobanu and Sally Kochmar.

The following questions are from Becker M et al. The t(12;21) in Pediatric B-Acute Lymphoblastic Leukemia: An update.

J Assoc Genet Technol. 2017;43(3): 99-109.

7. The translocation t(12;21): a. is a nonrandom rearrangement occurring in 75% of cases of B-cell childhood ALL. b. is in itself sufficient to generate an overt leukemia. c. can occur in utero. d. does not need additional secondary aberrations for disease development.

1. According to this article, pediatric B-cell acute lymphoblastic leukemia: a. is the second most common hematological malignancy in children. b. is a malignant disorder of T cells. c. is attributed to about 80% of pediatric leukemias. d. has t(12;21) occurring in 75% of cases.

The following questions are from Book Review: The AGT Cytogenetics Laboratory Manual. J Assoc Technol.

2017;43(3): 110.

2. All of the following are true except:

8. The AGT Cytogenetics Laboratory Manual:

a. The gene ETV6 is located at band 12p13 b. ETV6 encodes a transcription factor containing three functional domains c. The N-terminal domain acts as a strong transcriptional repressor d. The RUNX1 gene was previously known as TEL

I. is a reference text.

II. is essential for all cytogenetic and molecular professionals.

III. addresses many cytogenetic practices.

IV. presents many basic cytogenetic concepts. a. b. c. d.

3. ETV6/RUNX1 increases self-renewal and survival properties of pre-leukemic human cord blood cells. a. True b. False

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

The following questions are from Mah M. Molecular Diagnostics: A high school experience. J Assoc Genet Technol. 2017;44(3): 111-112.

4. Secondary genetic alterations:

9. All of the following are true except: a. Sequencing starts with amplification of target regions b. Target regions of interest are most often a couple hundred base pairs in length c. Sanger sequencing has templates of the same length with different fluorescence d. In Sanger sequencing, templates are denatured and run through capillary electrophoresis

a. are present in 60% of ETV6/RUNX1 positive ALL patients. b. consist of a duplication of the der(21)t(12;21) chromosome in 70% of cases. c. occur mostly in initial disease. d. occur most likely postnatally.

5. Choose the correct statement: a. PAX5 gene is involved in cell-cycle control. b. BMF gene is located on 5q31.3. c. CDKN2A is involved in cell-cycle control and located at 9p13. d. ETV6 is responsible for hematopoiesis.

10. In semiconductor sequencing, DNA fragments are attached to beads using molecular tags. a. True b. False

6. Of the 363 cases compiled: a. b. c. d.

About 224 had a complex karyotype About half had deletion 12p 82.2% had trisomy 21 5.51% had trisomy 10

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

The following questions are from Shabsovich et al. NGS: Elucidation of Novel Chromosomal Abnormalities in Pancreatic Cancer: Conventional and Molecular Cytogenetic Characterization of 16 Pancreatic Cell Lines. J Assoc Genet Technol. 2017;43(3): 113-127.

The following questions are from Gardner J et al. Myelodysplastic Syndrome with Isolated del(5q). J Assoc Genet Technol.

2017;43(3): 128.

16. All of the following are true, except: a. MDS with isolated del(5q) has a female predominance. b. The patient’s complete blood count revealed anemia and thrombocytosis. c. The biopsy revealed a hypercellular marrow. d. MDS with del(5q) has an unfavorable prognosis.

11. Pancreatic carcinoma: a. has a five-year survival rate of about 15%. b. uses cytogenetic analysis in its clinical management. c. is diagnosed by histopathologic methods. d. is treated with surgery only.

The following questions are from Garcia-Heras J. The milestone of non-invasive prenatal identification of chromosomal abnormalities in fetal trophoblasts recovered from maternal blood. J Assoc Genet Technol. 2017;43(3): 129-133.

12. A break-apart probe set targeting TGFBR2 showed evidence of a biallelic rearrangement in cell lines: a. one b. two c. five d. fifteen

17. According to this article, confined placental mosaicism is observed in % of cytogenetic studies in CVS performed at 9–12 weeks gestation: a. 10–12 b. 1–2 c. 3–4 d. 25

13. Among all the cell lines analyzed in this study: a. The most common structural abnormalities involved chromosomes X, 9, 18 and marker chromosomes. b. All lines showed distinct cytogenetic changes ranging in complexity. c. Very few marker chromosomes were noted. d. Four key genetic events discussed by Iacobuzzio-Donahue were analyzed.

18. Confirmation of all positive results generated by NIPT technology with cell-free DNA is an improvement achievable with genomic analysis of trophoblasts. a. True b. False

14. Novel cytogenetic targets in pancreatic cancer assessed in this study are: a. TGFBR2 and KRAS b. MYC and KRAS c. TGFBR2 and ARID1A d. SMAD4 and TP53 15. HER2 FISH analysis revealed amplification in cell lines analyzed. a. twelve b. four c. three d. sixteen

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

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

19. All of the following statements are true, except: a. NIPT screening for microdeletions was recommended in 2015. b. Trophoblast analysis may be able to evaluate rare autosomal trisomies. c. Genomic analysis of trophoblasts might be able to confirm all positive results generated by NIPT. d. Trophoblasts analysis may be able to rule out or confirm sex chromosome aneuploidies.

of the 16

20. How many false negative results did the Breman study have? a. one b. seven c. two d. three

Test Yourself #3, 2017 Answer Key

Please Print Clearly 11.____ 12.____ 13.____ 14.____ 15.____

(Passing score is 85% or 17/20 correct answers)

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

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

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

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11. a 12. b 13. d 14. d 15. c

16. c 17. b 18. d 19. d 20. c

Continuing Education Opportunities

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

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Continuing Education Opportunities

The AGT Education Committee’s Journal Club Journal Clubs are a great way to earn Contact Hours without leaving your home or lab! Journal Clubs can be completed as a group or individually. Each Journal Club includes a reading list, several discussion questions and a post-test. The discussion questions provide a starting point for group discussion and give individuals taking a Journal Club questions to consider while reading the articles. The post-test is taken after reading the articles and is returned to the regional representatives of the Education Committee to be graded.

Each successfully completed Journal Club is worth 4.0 Contact Hours. Journal Clubs can be ordered through the AGT Executive Office.

READING LIST 54 – General Content Area: Chromosome Breakage Syndromes–2006

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

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

1. Methylthioadenosine Phosphorylase Gene Deletions are Frequently Detected

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

READING LIST 74 – General Content Area: Role of Short Interfering RNA in Gene Silencing–2011 1. Highly Specific Gene Silencing by Artificial miRNAs in Rice.

2. Gene silencing by RNAi in mouse Sertoli cells.

3. Retrovirus-delivered siRNA.

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

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

3. Epigenetics at the Epicenter of Modern Medicine

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

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

READING LIST 79 – General Content Area: Whole Genome Amplification & 1986 Chernobyl, Ukraine Nuclear Power Plant Accident–2010 1. BAC-FISH assays delineate complex chromosomal rearrangements in a case of post-Chernobyl childhood thyroid cancer.

2. Whole Genome Amplification Technologies - Eliminating the Concern Over Running Out of DNA Samples Mid Experiment.

3. A break-apart fluorescence in situ hybridization assay for detecting RET translocation in papillary thyroid carcinoma.

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Continuing Education Opportunities READING LIST 80 – General Content Area: Expression of miRNA in Diffuse Large B-Cell Lymphoma–2010 1. Differentiation stage specific expression of microRNAs in B lymphocytes and diffuse large B-cell lymphomas.

2. Coordinated Expression of MicroRNA-155 and Predicted Target Genes in Diffuse Large B-cell Lymphoma.

3. Specific expression of miR-17-5p and miR127 in testicular and central nervous system diffuse large B-cell lymphoma.

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

3. Genetic and Environmental Influences on Pro-Inflammatory Monocytes in Bipolar Disorder

READING LIST 82 – General Content Area: Role and Detection of Human Endogenous Retroviruses in Rheumatoid Arthritis–2011 1. Increase in Human Endogenous Retrovirus HERV-K(HML-2) Viral Load in Active Rheumatoid Arthritis.

2. A role for human endogenous retrovirus-K (HML-2) in rheumatoid arthritis: investigating mechanisms of pathogenesis 3. Lack of Detection of Human Retrovirus-5 Proviral DNA in Synovial Tissue and Blood Specimens From Individuals With Rheumatoid Arthritis or Osteoarthritis.

READING LIST 83 – General Content Area: Roles of Oncogenes in Breast Cancer–2010 1. The Nuclear Receptor Coactivator Amplified in Breast Cancer-1 Is Required for Neu (ErbB2/HER2) Activation, Signaling, and Mammary Tumorigenesis in Mice.

2. Dysregulated miR-183 inhibits migration in breast cancer cells.

3. Current and emerging biomarkers in breast cancer: prognosis and prediction

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

2. Endogenous Retrovirus Type W GAG and

Envelope Protein Antigenemia in Serum of Schizophrenic Patients.

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

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

READING LIST 86 – General Content Area: p53 Family and Its Role In Cancer–2010 1. Telomere dysfunction suppresses spontaneous tumorigenesis in vivo by initiating p53-dependent cellular senescence.

2. Shaping genetic alterations in human cancer: the p53 mutation paradigm.

3. p53 polymorphisms: cancer implications.

READING LIST 87 – General Content Area: Proteins Involved with Chronic Myleloid Leukemia and Other Myleoprolifertive Disorders–2011 1. Gain-of-Function Mutation of JAK2 in Myeloproliferative Disorders.

2. Kinase domain mutants of Bcr enhance Bcr-Abl oncogenic effects.

3. Destabilization of Bcr-Abl/Jak2 Network by a Jak2/Abl Kinase Inhibitor ON044580 Overcomes Drug Resistance in Blast Crisis Chronic Myelogenous Leukemia (CML).

READING LIST 88 – General Content Area: DNA Topology–2010 1. The why and how of DNA unlinking.

2. Bacterial DNA topology and infectious disease.

3. DNA topoisomerase II and its growing repertoire of biological functions.

READING LIST 89 – General Content Area: LPL Waldenstrom Macroglobulinemia–2010 1. Spontaneous splenic rupture in Waldenstrom's macroglobulinemia.

2. How I Treat Waldenstrom's Macroglobulinemia.

3. International prognostic scoring system for Waldenström Macroglobulinemia.

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

sequencing technologies.

2. Combining Next-Generation Sequencing Strategies for Rapid Molecular Resource Development from an Invasive Aphid Species.

3. Evaluation of next generation sequencing platforms for population targeted sequencing studies.

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

READING LIST 92 – General Content Area: Severe Combined Immunodeficiency Screening and Patient Studies–2011 1. Long-term Outcome after Hematopoietic Stem Cell Transplantation of a Singlecenter Cohort of 90 Patients with Severe Combined Immunodeficiency.

2. Why Newborn Screening for Severe Combined Immunodeficiency Is Essential: A Case Report.

3. Development of a Routine Newborn Screening Protocol for Severe Combined Immunodeficiency.

READING LIST 93 – General Content Area: Biological and Physical Hazards Encountered in the Laboratory–2011 1. Lab Safety Matters.

2. Virus Transfer from Personal Protective Equipment to Healthcare Employees’ Skin and Clothing. Emerging Infectious Diseases.

3. Prevalence of Hepatitis C Virus Infection Among Health-Care Workers: A 10-Year Survey.

READING LIST 94 – General Content Area: Rapid whole-genome mutational profiling using nextgeneration sequencing technologies–2011 1. Comparison of next generation sequencing technologies for transcriptome characterization.

2. ShortRead: a bioconductor package for input, quality assessment and exploration of highthroughput sequence data.

3. Next-Generation Sequencing: From Basic Research to Diagnostics.

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

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

2. Truncated forms of BNIP3 act as dominant negatives inhibiting hypoxiainduced cell death.

3. Hypoxia-Induced Autophagy Is Mediated through Hypoxia-Inducible Factor Induction of BNIP3 and BNIP3L via Their BH3 Domains.

READING LIST 96 – General Content Area: Genetic Associations of Cerebral Palsy–2011 1. Mannose-binding lectin haplotypes may be associated with cerebral palsy only after perinatal viral exposure.

2. Methylenetetrahydrofolate Reductase Gene Polymorphisms and Cerebral Palsy in Chinese Infants.

3. Apolipoprotein E genotype and cerebral palsy.

READING LIST 97 – General Content Area: Treatments for HIV/AIDs–2011 1. Early Antiretroviral Therapy Reduces AIDS Progression/Death in Individuals with Acute Opportunistic Infections: A Multicenter Randomized Strategy Trial.

2. Asia can afford universal access for aids prevention and treatment.

3. Trends in reported aids defining illnesses (adis) among participants in a universal antiretroviral therapy program: an observational study.

READING LIST 98 – General Content Area: Myosin Light Chain Kinase (MYLK) Gene Mutation Affect in Smooth Muscle Cells–2012 1. Myosin light chain kinase is central to smooth muscle contraction and required for gastrointestinal motility in mice.

2. Mutation in myosin light chain kinase cause familial aortic dissections.

3. Chemical genetics of zipper-interacting protein kinase reveal myosin light chain as a bona fide substrate in permeabilized arterial smooth muscle.

READING LIST 99 – General Content Area: Chromosome 6 and Its Associated Diseases–2011 1. Novel Cleft Susceptibility Genes in Chromosome 6q.

2. A susceptibility locus on chromosome 6q greatly increases risk lung cancer risk among light and never smokers.

3. The identification of chromosomal translocation, t(4;6)(q22;q15), in prostate cancer.

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

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

1. Genetics of Alzheimer Disease.

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

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

1. Activation of the NLRP3 inflammasome by islet amyloid polypeptide provides a mechanism for enhanced IL-1 2 in type 2 diabetes.

2. ER stress in Alzheimer’s disease: A novel neuronal trigger for inflammation and Alzheimer’s pathology.

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

READING LIST 101 – General Content Area: Multiplex PCR and Emerging Technologies for the Detection of Respiratory Pathogens–2011 1. A multiplex one-step real-time RT-PCR assay for influenza surveillance.

2. Taking New Tack, PrimeraDx Offers MDx Tech as Open Platform for Test Developers.

3. Comparison of Automated Microarray Detection with Real-Time PCR Assays for Detection of Respiratory Viruses in Specimens Obtained from Children.

READING LIST 102 – General Content Area: Single Nucleotide Polymorphism (SNP) Array Analysis–2011 1. A fast and accurate method to detect allelic genomic imbalances underlying mosaic rearrangements using SNP array data.

2. SAQC: SNP array quality control.

3. Calibrating the performance of SNP arrays for whole-genome association studies.

READING LIST 103 – General Content Area: Research of BRAF Gene Related to Cancer–2011 1. Kinase-Dead BRAF and Oncogenic RAS Cooperate to Drive Tumor Progression through CRAF. 2. Distinct patterns of DNA copy number alterations associate with BRAF mutations in melanomas and melanoma derived cell lines.

3. Pharmacodynamic Characterization of the Efficacy Signals Due to Selective BRAF Inhibition with PLX4032 in Malignant Melanoma.

READING LIST 104 – General Content Area: Microarray Single Nucleotide Polymorphism (SNP) Troubleshooting–2011 1. Model-based clustering of array CGH data.

2. Application of a target array comparative genomic hybridization to prenatal diagnosis.

3. A model-based circular binary segmentation algorithm for the analysis of array CGH data.

READING LIST 106 – General Content Area: DNA Barcoding–2011 1. Commercial Teas Highlight Plant DNA Barcode Identification Successes and Obstacles.

2. Mutational Patterns and DNA Barcode for Diagnosing Chikungunya Virus.

3. The Barcode of Life Data Portal: Bridging the Biodiversity Informatics Divide for DNA Barcoding.

READING LIST 107 – General Content Area: HERV-K and Its Correlation With Melanoma Cells–2011 1. Expression of human endogenous retrovirus K in melanomas and melanoma cell lines Cancer.

2. Expression of HERV-K correlates with status of MEK-ERK and p16INK4A-CDK4 pathways in melanoma cells cancer.

3. An endogenous retrovirus derived from human melanoma cells.

READING LIST 108 – General Content Area: Refractory Myeloma–2011 1. Pomalidomide plus low-dose dexamethasone in myeloma refractory to both bortezomib and lenalidomide: comparison of 2 dosing strategies in dual-refractory disease.

2. Relapse/Refractory Myeloma Patient: Potential Treatment Guidelines.

3. Emerging role of carfilzomib in treatment of relapsed and refractory lymphoid neoplasms and multiple myeloma.

READING LIST 109 – General Content Area: Short Tandem Repeat (STR) Technology in Forensic Community–2011 1. An integrated microdevice for highperformance short tandem repeat genotyping.

2. A comparison of the effects of PCR inhibition in quantitative PCR and forensic STR analysis.

3. Generating STR profile from "Touch DNA".

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Continuing Education Opportunities READING LIST 110 – General Content Area: Methods of Screening and Evaluation of Hepatitis C Virus–2011 1. Hepatitis c virus: prevention, screening, and interpretation of assays.

2. Serial follow-up of repeat voluntary blood donors reactive for anti-hcv elisa.

3. Comparison of fibrotest-actitest with histopathology in demonstrating fibrosis and necroinflammatory activity in chronic hepatitis b and c.

READING LIST 111 – General Content Area: Pharmacogenomics–2011 1. Pharmacogenomic testing: Relevance in medical practice: Why drugs work in some patients but not in others.

2. Clinical assessment incorporating a personal genome.

3. Genomics and drug response.

READING LIST 112 – General Content Area: Adrenoleukodystrophy–2011 1. Novel exon nucleotide deletion causes adrenoleukodystrophy in a Brazilian family.

2. X-linked adrenoleukodystrophy: ABCD1 de novo mutations and mosaicism.

3. Identification of novel SNPs of ABCD1, ABCD2, ABCD3, and ABCD4 genes in patients with Xlinked adrenoleukodystrophy (ALD) based on comprehensive resequencing and association studies with ALD phenotypes.

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

2. Comparison of familial and sporadic chronic lymphocytic leukaemia using high resolution array comparative genomic hybridization.

3. Microarray-based comparative genomic hybridization.

READING LIST 114 – General Content Area: mFISH–2012 1. Human interphase chromosomes: a review of available molecular cytogenetic technologies.

2. Establishment of a new human pleomorphic malignant fibrous histiocytoma cell line, FU-MFH-2: molecular cytogenetic characterization by multicolor fluorescence in situ hybridization and comparative genomic hybridization.

3. CD5-negative Blastoid Variant Mantle Cell Lymphoma with Complex CCND1/ IGH and MYC Aberrations.

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

READING LIST 116 – General Content Area: Autism - 2015 1. Intellectual disability and autism spectrum disorders: Causal genes and molecular mechanisms.

2. Aberrant tryptophan metabolism: the unifying biochemical basis for autism spectrum disorders?

3. Decreased tryptophan metabolism in patients with autism spectrum disorders

Copyright law prohibits AGT from supplying readers with the actual journal articles (electronically or otherwise). Availability of articles online does not imply the service is free.

Some journals require a subscription or impose a fee. The web addresses are included for the convenience of those wishing to obtain the articles in this way.

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Continuing Education Opportunities

AGT Journal Club Question Order Form

To order the AGT Journal Club Questions, please fill in the requested information below. Make check or money order payable to AGT. Copyright law prohibits AGT from supplying readers with the actual journal articles (electronically or otherwise).

Participants must obtain articles themselves.

Discussion and Question Set for Reading List No. (Please enter the number of copies requested next to each Journal Club Number)

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AGT Members: $10 each ____________

Non-Members: $40 each ____________

Total ____________

Name of Institution_____________________________________________________________________________________________ Person or Department__________________________________________________________________________________________ Address________________________________________________________________________________________________________ City________________________________________________ State__________________ Zip Code__________________________ Phone No.________________________________________________ FAX_________________________________________________ Method of Payment:

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AGT Executive Office 4400 College Blvd, Suite 220 Overland Park, KS 66211 Phone: 913-222-8665

Please make check payable to AGT.

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

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Association Business

Letter from the President AGT—What We Need Apprehension: anticipation of adversity or misfortune; suspicion or fear of future trouble. (Dictionary.com) As our membership is aware, uncertainty of our future (apprehension) has been the recent “soup of the day” at AGT. For the past 42 years, AGT has tried to be the foremost professional organization for genetic professionals. We began as a volunteer-based organization with a focus on the science of Cytogenetics and the greater community who performed said genetic testing. As we grew, we needed greater help in maintaining our organization and thus utilized a professional management company. This proved to be both a professional blessing and a financial curse. Our membership has slowly been declining as our cost of operations continued to increase.

We couldn’t detract services from our membership and hoped, year after year, to once again grow like we did in the beginning. Our apprehension began to grow…

The AGT Board of Directors (BOD) and Council of Representatives (COR) has been meeting weekly since late August to find the way to best preserve AGT and its legacy for the future. They are trying to be both optimistic and realistic for AGT, which has proven to be a struggle for many (especially myself). They have run budgetary projections multiple times to only rerun them again with new factors or updated membership numbers. I firmly believe that our BOD and COR are many of the best fit individuals to redesign and prepare AGT for the future.

The question is, can we do it with what is left? Can we break even, or even pay our bills, for next month or the month after? The apprehension has begun to run wild.

The decision to close AGT, or the potential to try and revert to a volunteer organization, has been placed in the hands of AGT’s members. I won’t lie to you and say that the membership has been active in the vote and had the greatest passion to preserve AGT. However, it’s the members who need be present and active to ensure AGT’s future. The apprehension concerning our future has been unbearable.

Can we fix AGT to be better suited and prepared for the future? I don’t know. Is it too late to be able to fix? I don’t know. I wish I knew more, but the apprehension is too thick for many of us to even see clearly.

What does AGT need? AGT needs you (members). AGT needs active members who are involved and supportive of AGT’s mission. We need donations and financial support. Without funds, we cannot afford representation for our membership. Our resources and publications need funds to survive. Our annual meeting needs funds and attendance to ensure we can keep bringing high-quality meetings to our membership. AGT needs more from every single one of us (me included). Will it be enough? I don’t know, but I do know that we need you to survive.

—Jason Yuhas, President

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Association Business The Association of Genetic Technologists is proud and pleased to be a sponsor of the V Journada Internacional De Genetica to be held in August 8–10, 2018 Chiclayo, Peru.

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Association Business

AGT, The Organization for Cytogenetic & Molecular Professionals AGT, originally founded in 1975 as the Association of Cytogenetic Technologists, serves to: • promote the scientific and professional development of all areas of genetics; • foster the exchange of information between those interested in genetics; • encourage cooperation between those persons actively or formerly engaged in genetics; and • stimulate interest in genetics as a career.

AGT has approximately 1,000 members. Membership is open to all who are employed or interested in genetics. All regular members are entitled to hold office, vote in elections, attend all AGT meetings, and receive The Journal of the Association of Genetic Technologists and access the AGT International Online Membership Directory.

Board of Directors Officers President Jason A. Yuhas, BS, CG(ASCP)CM Mayo Clinic Division of Laboratory Genetics Cytogenetics Lab 200 First St. SW Rochester, MN 55905 507-538-7634 yuhas.jason@mayo.edu Secretary-Treasurer Denise Juroske Short, MSFS, MB(ASCP)CM 219 Timberland Trail Lane.

Lake City, TN 37769 dmj4565@gmail.com Public Relations Director Ephrem Chin MBA, MB(ASCP)CM, QLC 1907 Woodlawn Terrace Ct. Sugar Land TX 77479 404-579-9995 nzelfman@gmail.com Education Director Sally J. Kochmar, MS, CG(ASCP)CM Magee-Womens Hospital Pittsburgh Cytogenetics Lab 300 Halket St., Room 1233 Pittsburgh, PA 15213 412-641-4882 skochmar@upmc.edu Annual Meeting Director Jennifer N. Sanmann, PhD, FACMG UNMC Human Genetics Laboratory 985440 NE Med. Center Omaha, NE 68198-5440 402-559-3145 jsanmann@unmc.edu

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

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

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

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

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

Publications

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

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

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

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

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

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

Visit AGT’s Website at www.AGT-info.org The Journal of the Association of Genetic Technologists 43 (4) 2017

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Association Business

ABSTRACT SUBMISSION DEADLINE: Friday, February 2, 2018

AGT 2018 Call for Abstracts

Dialogue and the sharing of ideas is critical to the success of our field, so we want to hear about the work being done in your laboratories. The 43rd Annual Meeting Program Committee invites all interested persons to submit abstracts for the AGT 43rd Annual Meeting.

Returning in 2018: • A significant increase in the number of submissions selected for a platform presentation • Multiple platform presentation sessions throughout the scientific meeting • Focused platform presentation sessions, such as cytogenetics and molecular genetics • Expanded areas of interest, to include topics such as clinical genetics, genetic counseling and regulatory affairs Abstracts will be printed in the Final Program Book and in the 2018 Third Quarter issue of the Journal of the Association of Genetic Technologists if they are presented at the meeting.

All abstracts must contain: • a description of the study design, • a statement of results (including data), and • an informative conclusion.

Abstracts will be assigned to platform or poster presentations by a Review Committee. The Committee will consider the author’s preference and the time constraints of the meeting.

Poster Presentations

Submission Requirements

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

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

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

It is important that you follow all instructions carefully.

Abstracts submitted incorrectly will not be considered for presentation.

Platform Presentations Platform presentations are limited to 15 minutes each: twelve minutes for the presentation and three minutes for questions/discussion. If AGT accepts your abstract for platform presentation, you will receive notification by March 31, 2018.

All platform presentations presented by AGT members are eligible to win the Best Platform Award at the Annual Meeting.

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

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

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Association Business

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

Eligibility:

•

• • •

• •

•

Criteria

Individuals/students eligible to submit abstracts for the award are:  Those enrolled in a NAACLS “Serious Applicant Status” or “Accredited” undergraduate or certificate program in Cytogenetics or Diagnostic Molecular Science (U.S. Programs).  Those enrolled in a Canadian Society of Laboratory Technology Approved undergraduate or certificate program in Cytogenetics or Molecular Biology (Canadian programs).  Those attending formal undergraduate or certificate programs outside of the United States and Canada if the program is recognized at the national level of the country in which the program is located.

Applicants MUST be enrolled in the program between March 1, 2017 and March 4, 2018.

Individuals/students graduated prior to March 1, 2017 are not eligible.

Applicants must be members of the Association of Genetic Technologists at the time of application to be eligible to win the Student Research Award, but nonmembers may submit an abstract in the student category.

Information regarding AGT membership is available from your program director or the AGT Executive Office.

The applicant will be required to be the first author on the abstract.

The applicant and a program official will be required to validate that: the research was conducted during enrollment and completed prior to graduation from the program, the applicant was the primary investigator, and the abstract submitted is the work of the applicant.

Applications may be submitted throughout the year in order to be considered for the 2018 Student Research Award. All entries received after Friday, March 16, 2018 will be considered for the 2019 Student Research Award.

• • • • • •

•

A purpose for the experiment or investigation where the scientific merit or contribution is stated.

A hypothesis, which indicates scientific expectations of the investigation and should be appropriate for the purpose.

Scientific methods which are appropriate to the investigation, concise and organized.

Data resulting from the investigation/experiment should be concise, specific and organized.

The interpretation should be consistent with the data.

The conclusions should be consistent with the data and interpretation. They may be intertwined with the interpretation, should support or refute the hypothesis, and should include the need for further investigation or suggest how other variables may influence the results.

References should be used when appropriate. The institution name or any other identifying information should not be included in the abstract.

Submission Requirements

Abstracts must be submitted electronically at: https://www.surveymonkey.com/r/AGT2018StudentAbstracts.

If you are submitting more than one abstract, you must submit each abstract separately.

It is important that you follow all instructions within the online submission site carefully. Abstracts submitted incorrectly will not be considered for presentation.

Student Research Award:

• The recipient will be notified by April 27, 2018.

• The winner will receive complimentary 2018 Annual Meeting registration and expenses to travel to the meeting.

• The recipient will be invited to present his/her research in a 10-minute platform presentation.

• All other submissions will be considered for poster presentation.

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Association Business Association Business

The Foundation for Genetic Technology (FGT) was organized exclusively for scientific and educational purposes. It is a non-profit organization whose funds and assets are used to promote education in genetic technology and provide professional opportunities for The Foundation for Genetic Technology (FGT) was organized exclusively for scientific and educational purposes. It is a non-profit training through grants, scholarships organization whose funds and assets and are awards. used to promote education in genetic technology and provide professional opportunities for As always, behind the scenes, the dedicated members of the Foundation work throughout the year to strive to continue the mission of training through grants, scholarships and awards. the FGT. Along with support from AGT, the Foundation is able to fund the scholarships and grants that were presented at the Annual AsMeeting. always, behind thethis scenes, dedicated members of the Foundation, work throughout year to strive to the continue the mission AGT None of wouldthe have been possible without the help of the donors, vendorthe sponsorships and members-at-large of the FGT. Along with support from AGT, the Foundation is able to fund the scholarships and grants that were presented at the of AGT. Once again, the Foundation is fiscally solvent this year, which is a tribute to our many hard-working and dedicated members.

Annual Meeting. None of this would have been possible without the help of the donors, vendor sponsorships and the members-at-large major source of income for FGTiscomes the this sale year, of thewhich studyisguides for to theour Cytogenetic and Molecular exams. These are of A AGT. Once again, the Foundation fiscallyfrom solvent a tribute many hard-working and dedicated members. available year-round and can be purchased by visiting the AGT website, and accessing FGT from the Resources tab. Another financial A major sourceregional of income for FGT comes from the The sale of the Coast study meeting guides foris the Cytogenetic These are resource has been meetings sponsored

by FGT. West usually in March,and andMolecular the East exams.

Coast meeting is available year-round and Please can berefer purchased visiting AGTmeeting website,information. and accessing FGT from the Resources tab. Another financial scheduled in September. to the by website forthe further resource has been regional meetings sponsored by FGT. The West Coast meeting is usually in March, and the East Coast meeting is The Silent Auction atPlease the AGT Meeting a tremendous success, with over $600 of donated items. This event has scheduled in September. refer Annual to the website for was further meeting information. proven to be a great way for the FGT to raise money and also promote interaction among the attendees. Thank you to everyone who participated…those of you whoAGT donated items, the winners of our auction and those who stopped by the FGT booth tofor inquire about The Silent Auction at the Annual Meeting was a tremendous success.

This event has proven to be a great way the FGT to raise money also promote among thetoattendees.

Thank you to everyone of youPat whoLeMay donated us.

Your inputand is important, andinteraction your interest is vital keep FGT opportunities available.who Forparticipated…those more information, email at items, the winners of our auction and those who stopped by the FGT booth to inquire about us. Your input is important, and your plemay1945@aol.com. interest is vital to keep FGT opportunities available. For more information, email Robin Vandergon at rvandergon@neogenomics.com.

Do You Know Someone…?

Having just come off a very successful AGT Annual Meeting in St. Louis, DO YOU SOMEONE …?

Missouri, it is timeKNOW to look ahead to 2018.

Having justAGT comeand off aour very successful AGT Annual Meeting infor Orange Along with corporate sponsors, the Foundation Genetic County, California, is time to look 2017.

Technology has many itscholarships and ahead awardstothat we present at the AGT Annual Meeting every year.

A complete list, along sponsors, with requirements, application forms and Along with AGT and our corporate the Foundation for Genetic submission deadlines is listed in the FGT section on that the AGT website.atThere is a wide Technology has many scholarships and awards we present the AGT range of awards available something for every member consider…from Annual Meeting everyand year.

A complete list,AGT along with to requirements, the newly certified to the career-oriented professional and theon seasoned application formstechnologist and submission deadlines is listed in the FGT section the veteran technologist. encourage each AGT member consider nominating AGT website.

There We is a wide range of awards available andtosomething for every someone for thesetoawards.

It is a wonderful waycertified to acknowledge the accomplishments AGT member consider…from the newly technologist to the careerand the dedication of those individuals we know work so We hardencourage for the field oriented professional and the seasonedwho veteran technologist. ofeach genetics. aretomany qualified candidates, manyfor of these whomawards. work inIt small AGTThere member consider nominating someone is laboratories feel out of the but each application reviewed ofby a a wonderfulthat waymay to acknowledge theloop, accomplishments and the is dedication committee, and is not a popular vote. those individuals who we know work so hard for the field of genetics. There are many qualified candidates, many of whom work in small laboratories that Youfeel oweout it of to the yourselves, AGTapplication members, istoreviewed recognize may loop, butaseach byoutstanding a committee,members and ofisour organization. If we fail to promote ourselves, then no one is notprofessional a popular vote. goingYou to do us. So, if you knowmembers, an AGTtomember who demonstrates passion, oweititfor to yourselves, as AGT recognize outstanding members knowledge and a commitment to genetics, please check out the various of our professional organization.

If we fail to promote ourselves, thenawards no onethat our is organization going to do sponsors. it for us. So, if you know an AGT member who demonstrates passion, knowledge and a commitment to genetics, please check out the various awards that our organization sponsors.

The Journal of the Association of Genetic Technologists 43 (4) 2017

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

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s

Association Business Foundation for Genetic Technology

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

nology (FGT) was organized exclusively for scientific and educational purposes. It is a non-profit 2018 Grants & Awards Deadline: Friday, March 30, 2018 are used to promote education in genetic technology and provide professional opportunities for and awards.

For more information, guidelines, criteria and application forms for the grant and awards listed below visit the FGT website at

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

Sponsored

by Leica Microsystems. is fiscally solvent this year, which is a tribute to our many hard-working and dedicated members.

T comes from the sale of the study guides for the Award Cytogenetic and Molecular exams.

Florence Dowling Genome ($500) Acknowledges andThese honorsareoutstanding technologists in cytogenetics and hased by visitingmolecular the AGT genetic website,technology. and accessing from the Resources tab.toAnother financial ThisFGT award program contributes the growth of genetic technology as a profession by recognizing sponsored by FGT. The West Coast meeting is usually in March, and the EastbyCoast meeting is individuals with superior professional commitment.

Sponsored Patricia Dowling. to the website for further meeting information.

New Award ($750) newly certified items.

AGT members whohas submit an essay about their genetic experience Annual Meeting was Horizons a tremendous success, with Honors over $600 of donated This event to attendinteraction the AGT Annual Meeting.

Sponsored Rainbow Scientific.

T to raise moneyand anddesire also promote among the attendees.

Thankby you to everyone who

ted items, the winners of our auction and those who stopped by the FGT booth to inquire about EXCEL Award ($500) AGT members enrolled in a formal university/hospital or laboratory-based program in diagnostic interest is vital to keep FGT opportunities available. For more information, email Pat LeMay at molecular technology or a NAACLS-accredited certificate or undergraduate cytogenetics or molecular program may be eligible to compete for free student registration to the AGT 43rd Annual Meeting, as well as one full-day or two half-day workshops.

Sponsored by Oxford Gene Technology.

Barbara J. Kaplan Scholarship ($1,000) AGT members enrolled in a formal training program, including university/ hospital, laboratory-based or a NAACLS-accredited program for molecular genetics or cytogenetics may be eligible for the $1,000 scholarship. Program directors may visit the FGT website for more information. Sponsored by FGT. Joseph Waurin Excellence in Education Award ($500) Honors an outstanding AGT member educator in the genetic community who is BOC-certified and has a minimum of five years of teaching experience. Sponsored by James Waurin.

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

the winning criteria (i.e., interesting and informative topic, well-organized, clear and concise data, best illustrations, clinical/ correlation cutting-edge g just come off laboratory a very successful AGTand Annual Meetingtechnology) in Orange submitted by an AGT member by the abstract deadline. Sponsored by Irvine California, it is time to Scientific. look ahead to 2017. with AGT and our corporate sponsors, the Foundation for Genetic Best Platform Presentation Awardat($500) All AGT members attending the platform presentations at the AGT Annual gy has many scholarships and awards that we present the AGT vote forlist, the presentation best fits the winning criteria (i.e., presentation must be given by a technologist, be an Meeting every Meeting year. Amay complete along with that requirements, interesting and informative havesection clinical/laboratory correlation and present cutting-edge technology). An AGT member on forms and submission deadlines is listed intopic, the FGT on the must be among the authors of the abstract submitted by the abstract deadline. Sponsored by FGT. bsite. There is a wide range of awards available and something for every mber to consider‌from the newly certified technologist to the careerBoothtechnologist.

Award professional andBest the Exhibitor seasoned veteran We encourage Honors exhibitors at the AGT Annual T member to consider nominating someone for theseMeeting. awards. AGT It is members will vote by ballot on the following criteria: 1.

Best interaction (quality of interaction) ful way to acknowledge the accomplishments and the dedication of with AGT membership, including availability to meeting participants answering with detail. ividuals who we know work and so hard for thequestions field of genetics.

There qualified candidates, many of whom work in small laboratories 2. Most valuable technical informationthat or product information, including presentation of literature available. out of the loop, but each application is reviewed by a committee, and 3. Best overall booth design, including appearance of exhibit and visual impact of the display. opular vote.

Most innovative formembers attendees. we it to yourselves, as AGT4. members, to recognizegifts/raffles outstanding ofessional organization. If5. we Creativity. fail to promote ourselves, then no one to do it for us. So, if you know an AGT member who demonstrates 6. Only exhibitors listed in the final program will be eligible. knowledge and a commitment to genetics, please check out the various hat our organization sponsors.

Please refer to the website listed above for the details and

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

The Journal of the Association of Genetic Technologists 43 (4) 2017 The Journal of the Association of Genetic Technologists 42 (3) 2016

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233

Association Business

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

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

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

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

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PRODUCT ORDER FORM Item Description

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The AGT Cytogenetics Laboratory Manual, 3rd Edition

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

Click here to order publication now through the publisher.

The Cytogenetics Symposia, 2nd Edition

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

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

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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 Boulevard, Suite 220, Overland Park, KS 66211 Fax (913) 222-8606  Email: agt-info@kellencompany.com  Website: www.agt-info.org

Please allow 2-4 weeks for shipping.

2018 AGT Outstanding Achievement Award Nomination Nominations are open for the 2018 AGT Outstanding Achievement Award. We are looking for those who are committed to furthering the field of genetics as demonstrated by their work, attitude and AGT activities. If you have a colleague who performs above and beyond the call of duty, nominate him or her for the AGT Outstanding Achievement Award today. All nominees must be AGT members, however they cannot be current AGT Board members or on its Council of Representatives.

NOMINATIONS MUST BE POSTMARKED NO LATER THAN FEBRUARY 5, 2018.

Requirements 1. The nominee works as or has worked as a genetic technologist (this does not include post-doctoral fellowships).

2. Current membership in AGT 3. Current certification in cytogenetics, molecular genetics or biochemical genetics 4. A letter from the nominee stating accomplishments and contributions to the field of

Return to: AGT Executive Office 4400 College Blvd., Ste. 220 Overland Park, KS 66211 AGT-info@kellencompany.org

genetic technology with reference to the award criteria. The letter should state specifically how the nominee’s expertise and contributions have furthered genetic technology.

5. Two letters of support from individuals familiar with the nominee’s accomplishments 6. Copies of relevant publications authored by the candidate (maximum of five) 7. A current résumé

Criteria 1.

2.

3.

4.

5.

6.

7.

8.

Employment in the field of genetic technology (include length of time and positions held) Consideration of total length of time as AGT member AGT service (past service on the AGT Board of Directors, Council of Representatives, committees and task forces, editorships, etc.).

Presentations at the AGT annual meeting (featured speaker, workshop or breakout session presenter, platform presenter) Other educational and continuing education activities (teaches genetic technology; gives seminars, workshops or lectures; other genetic technology-related educational activities) Publications relevant to genetic technology, education and/or management (list all publications, including journal articles, books, posters, abstracts, etc.)

Development and/or implementation of new methods or procedures Other contributions to the fields of genetic technology

Nominee’s Name: Company/Institution: Address: City, State, Zip: Phone:

Email:

I am nominating this person for the AGT Outstanding Achievement Award because:

Nominated by (Name): Address: City, State Zip Phone:

Email:

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

 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: 

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

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

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

9/14

Association Business

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.

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 43 (4) 2017

238

The Journal of the Association of Genetic Technologists The Journal of the Association of Genetic Technologists is a peer-reviewed journal, and scientific materials for publication containing original research will be reviewed by independent referees. Manuscripts that require revision or that contain major editorial changes will be returned to the senior author of the article. Materials submitted will not be retained following publication nor will photographs, disks, or hard copies of manuscripts be returned to authors. Rejected manuscripts will not normally be returned, although an effort will be made to return original photographs and prints.

Manuscript content is the responsibility of the author(s). All articles published, including editorials, letters, book reviews, invited articles, Brain Ticklers, columns, and reviews, represent the opinions of the authors and do not reflect the official policy of AGT or the institution with which the author is affiliated unless specified by the author. AGT, its members, and the editor of The Journal of the Association of Genetic Technologists make no warranty and assume no liability with respect to the information contained herein.

Information For Authors The Journal of the Association of Genetic Technologists is pleased to consider manuscripts that describe experience with cytogenetics, molecular genetics, or biochemical genetics and the application of these disciplines.

Submitted manuscripts must be typed, preferably double-spaced, using a 12 point font and 1” margins. In addition to the original, three copies of the manuscript and camera-ready illustrations must be submitted to the editor-in-chief. Items to be italicized or enhanced (bold, underlined) should be clearly indicated. The conversion factor for print equivalency is as follows: two double-spaced typed pages equal approximately a one-half typeset page.

Authors may supply the material on a 3½” disk, preferably in Microsoft Word, WordPerfect, or ASCII format, along with the hard copy. Macintosh disks are also acceptable, but conversion costs will be assessed accordingly to AGT and a delay in processing may occur. Materials may alternatively be supplied to the editor via email at the address shown on inside front cover. Email submission is preferred.

Illustrations must be original photographs, computer-generated digitized files (preferably saved as a .tif, .eps, or .bmp file), or black and white line drawings, professionally prepared. The cost of separating and printing color photographs or illustrations will be charged to the author.

Photographs must be properly identified on the back, including the author’s name, title of article, and top direction. A ball point pen should not be used for labeling. The affixing of a typewritten label to the illustration or table will prevent damage.

Notation & References Authors’ titles must be accompanied by a position description of less than 15 words, which will be printed with the article.

Textual citations to the referenced literature should be parenthetically noted by author’s surname followed by year of publication, and arranged chronologically and then alphabetically, as demonstrated in the following example: (Lese and Ledbetter, 1998; Reilly, 1998a; Morgan et al., 1999).

In situations with more than two authors, the first author’s surname should be followed with et al. When references are made to more than one paper published in the same year by the same author, a lower case a, b, etc. should be appended to the date of publication and should be included in both textual citations and the reference list.

References should be listed completely at the end of the paper in alphabetical order by surname of first author, and then by year of publication.

When more than one publication appears with the same first author, listings will be alphabetized by the first varying co-author. Irrespective of the number of authors, et al. should not be used in the reference list. Journal titles should be abbreviated according to Index Medicus and book titles should be italicized. Use the following format for references: Journal Article Brothman AR, Zhu XL, Maxell T, Cui J, Derbler DA. Advances in the cytogenetics of prostate cancer. J Assoc Genet Technol. 1999;25(1):1-6.

Book Chapter Barch MJ and Lawce HJ. The cell and cell division. In: Barch MJ, Knutsen T, Spurbeck JL (eds). The AGT Cytogenetics Laboratory Manual, 3rd ed.

Philadelphia: Lippincott-Raven; 1997:1-18.

Book Mark HFL. Medical Cytogenetics. New York: Marcel Dekker; 2000.

All references should be complete. Accuracy is the responsibility of the authors. Only published articles and those in press may be included in the reference list. If necessary, unpublished data and submitted manuscripts should be cited parenthetically within the text.

Reprint Orders Reprints of articles can be purchased by authors at cost within two years after publication. On the order request, specify the journal’s volume and issue numbers, year of publication, page numbers, article title, author(s), and quantity requested. Include the contact name(s), address(es) and phone number(s) to be used for either shipping purposes or related questions. Payment should accompany the order. Checks must be made payable to AGT. Minimum order is 50 copies.

Reprints are produced on 60# white offset paper, saddle-stitched (unless under four pages), and will appear exactly as they do in the journal.

Price is based on article length, quantity ordered, and color requirements. Orders are not processed until payment is received. Once payment is received, allow four weeks for printing and shipping. Prices quoted include shipping by UPS ground; expedited shipping is available at an additional charge. Journal copies can be purchased by AGT members for $25/each, if copies are available.

Please forward reprint orders or questions regarding price quotations to the AGT Executive Office (see inside front cover for address).

ISSN 1523-7834