39th 39th - Association of Genetic Technologists

Transcription

39ANNUAL
th
MEETING
June 12-14, 2014
Louisville, Kentucky
Louisville Marriott Downtown
Final
Program
Book
Louisville photos courtesy of Louisville Convention & Visitors Bureau
Genetics
Inside!
AGT 39th
ANNUAL MEETING
39 AGT Annual Meeting
th
Marriott Louisville Downtown
Louisville, Ky.
Copyright 2014 by the Association of Genetic Technologists
Association of Genetic Technologists
P.O. Box 19193
Lenexa, KS 66285-9193
Overnight Only:
18000 W. 105th St.
Olathe, KS 66061
Phone: 913-895-4605
Fax: 913-895-4652
Website: www.agt-info.org
Email: [email protected]
All rights reserved. No part of this publication may be reproduced, stored in
a retrieval system, or transmitted, in any form or by any means, electronic,
mechanical, photocopying, recording, or otherwise, without the prior written
permission of the copyright owner.
Printed in the United States of America
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Welcome to the AGT 39th Annual Meeting
June 12-14, 2014
Marriott Louisville Downtown • Louisville, Ky.
Table of Contents
Hotel Map. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
GENERAL INFORMATION
Registration Desk, Meeting & Reception Rooms, Exhibit Hall Hours . . . . . 9
Mobile Devices, Name Badges/Tickets, Session Handouts . . . . . . . . . . . 10
Special Events . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Continuing Education Credits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Awards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Sponsors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Board of Directors 2013-2014. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Council of Representatives 2013-2014. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
EXHIBITOR INFORMATION. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
DAY-AT-A-GLANCE
Thursday, June 12, Pre-Conference Workshops . . . . . . . . . . . 31
Friday, June 13, Scientific Sessions . . . . . . . . . . . . . . . . . . . . . 33
Saturday, June 14, Scientific Sessions . . . . . . . . . . . . . . . . . . . . 51
Business Meeting Agenda and Minutes . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Platform Presentations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Student Research Award. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
ABSTRACTS
Poster Abstracts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Student Posters. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
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Second Level
MARRIOTT LOUISVILLE DOWNTOWN HOTEL MAP
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Automated Imaging
Platforms for Genetic Analysis
Quality Clinical Results
Booth #19P
FDA cleared for the following
GenASIs applications: ALK, BandView,
FISHView, UroVysion, CEP XY &
HER2/neu FISH
www.spectral-imaging.com • [email protected]
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Dear AGT Annual Meeting Attendee:
Welcome to the 39th Annual Meeting of the Association of Genetic Technologists and to the city of
Louisville, Kentucky. Thanks to our Annual Meeting Planning Committee, chaired by our Meeting
Director, Jason Yuhas, and Co-Director, Adam Sbeiti, for being diligent and resourceful leaders
in arranging an outstanding program with an exciting, educational and socially packed three
memorable days. The program is filled with a variety of workshops and scientific sessions that
will meet your needs for biochemical genetics, cytogenetics and molecular genetics knowledge.
Those of you fortunate to attend one or more of the workshops on Thursday will have the
opportunity to focus on topics of your interest to improve your knowledge, skills and practice.
It is a great opportunity and forum for all of us to explore, learn and share vast experiences
across laboratories within and outside the United States. The workshops were carefully selected
to ensure that they provide insights on useful and pertinent practical applications as well as new
technology.
In addition to the Welcome Reception Thursday evening – a place to begin making contacts and
meet old and new friends – you will find a wealth and variety of educational sessions, platform
presentations, posters and the exhibit hall, with laboratory and vendor representatives. Our
vendors are there to support us and provide us with an opportunity to learn more about their
products.
Throughout the meeting we will honor the achievements of our colleagues with recognitions and
awards. This meeting will present a forum filled with tremendous opportunities for us to explore,
learn and share our passion and love for the genetics field, and most importantly, will renew and
establish ties to develop friendships to strengthen our community.
The 39th Annual Meeting of the Association of Genetic Technologists will offer a wide variety of
genetics content while you enjoy the beauty and history of Kentucky. Louisville is best known for
being the location of the Kentucky Derby horse race. You can also tour historic “Old Louisville.”
This Victorian neighborhood is the largest historic preservation of these types of homes and
buildings in the United States. Louisville also offers a wide variety of museums and cultural
centers. Enjoy the stunning waterfront skyline, over 120 parks, the nation’s largest urban forest,
Bourbon country and urban Bourbon trail and many more!
Organizing an annual meeting is inevitably a group effort, and I am glad and proud to be able
to work with everyone involved in organizing this event. Final thanks must go to the people who
bring ideas to help make a meeting a memorable and successful event. My personal thanks and
most appreciation goes to Jason, Adam and Christie Ross and the staff at the AGT Executive
Office for their assistance with the planning of this meeting.
Also, I am most grateful and thankful to the elite presenters and the vendors for their willingness
to support and share their experience and knowledge with all of us! I encourage you to provide
feedback so that future meetings will continue to meet and exceed your needs and expectations.
I hope your will find time to experience Louisville before your return home safely.
Please tell your colleagues what it means to you to be a laboratory professional and a member
of AGT and encourage them to become members. With your continued support, AGT will be the
voice for genetic technologists for many years to come!
Sincerely,
Mervat S. Ayad, BS, EMBA, CG(ASCP)CM, DLMCM, CCS
AGT President
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General Information
2014 ANNUAL MEETING DIRECTORS
Jason Yuhas
Annual Meeting Director
Adam Sbeiti
Annual Meeting Co-Director
Registration Desk
AGT staff and volunteers will be available during the following hours:
Wednesday, June 11
5:00 p.m. – 7:00 p.m.
Thursday, June 12 7:00 a.m. – 3:00 p.m.
6:30 p.m. – 8:00 p.m.
Friday, June 13 7:00 a.m. – 4:00 p.m.
(closed 12:15 p.m. – 1:15 p.m.)
Saturday, June 14
7:00 a.m. – 6:00 p.m.
(closed 12:30 p.m. – 1:30 p.m.)
Meeting & Reception Rooms
Meeting rooms are noted throughout this program. All AGT meeting rooms are
located on the 2nd Floor.
Exhibits and Posters
Marriott Ballroom I-V
Scientific Sessions Marriott Ballroom VI
Pre-Conference Workshops Bluegrass, Thoroughbred, Rose
Business Meeting Breakfast Marriott Ballroom VI
Job Fair Marriott Ballroom Foyer
Awards Reception Marriott Ballroom Foyer
Awards Banquet and Dance Marriott Ballroom V
Exhibit Hall Hours
The Exhibit Hall will be open during the following hours:
Thursday, June 12
7:00 p.m. – 9:00 p.m.
Friday, June 13
9:00 a.m. – 4:00 p.m.
Saturday, June 14
9:00 a.m. – 11:30 a.m.
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Mobile Devices
Please be considerate of other attendees and speakers by turning all mobile
devices off or to vibrate. Thank you for your cooperation.
Name Badges/Tickets
Name badges are provided for all registered meeting attendees, exhibitors,
speakers, Board members and staff. Please wear your name badge at all
times. Badges are required for entrance to the exhibit hall, poster
area and all educational sessions and social events. Drink tickets for
the Welcome Reception and Awards Reception/Banquet are included in your
packet. It is recommended that you place your tickets in your name badge holder
to ensure you will not forget to bring them.
Session Handouts
Annual Meeting handouts are provided on the AGT web site. All attendees have
access to the site, and the final presentations will be posted within three weeks of
the Annual Meeting.
Visit with the Exhibitors
Play to Win! Join in the fun by visiting with all of the exhibitors listed on your
“Play to Win” card. Your card must be complete to be entered into the prize
drawing that will take place during the Saturday evening banquet. You could win
a complimentary 2015 membership, registration for the 2015 Annual Meeting
and much more. YOU MUST BE PRESENT TO WIN.
Smart Phone App
AGT has an app for that! We encourage you to download the mobile guide to
enhance your experience at the 39th Annual Meeting. You will be able to plan
your day with a personalized schedule (including alerts), browse exhibitors and
sponsors, view maps, upload pictures and much more! The App is compatible
with iPhones, iPads, iPod Touch and Android devices. Follow the simple steps
below and go mobile!
To get the guide, choose one of the methods below:
1. Download “Guidebook” from the Apple App Store or the Android
Marketplace.
2. Visit http://guidebookapp.com/getit/ from your phone’s browser.
3. Or, scan the image that will appear on a separate flyer with your meeting
materials with your mobile phone (QR Code reader required, e.g., “Red
Laser,” “Barcode Scanner”).
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Special Events
Thursday, June 12
Welcome Reception in the Exhibit Hall – Marriott Ballroom I-V
7:00 p.m. – 9:00 p.m.
Don’t miss the opening of the AGT 39th Annual Meeting with a Welcome
Reception in the Exhibit Hall. The reception is an excellent opportunity to learn
more about the latest services and products from our exhibitors and also get a
sneak peek at the posters. AGT will provide a variety of hors d’oeuvres and one
drink ticket (beverages can be purchased after you have used your drink ticket).
Attendance at the reception is included with your registration fee. If you would
like a guest to attend the Welcome Reception, tickets can be purchased for $35.
Business casual attire is appropriate.
FGT Silent Auction – Marriott Ballroom I-V
FGT is hosting a silent auction to take place in the Exhibit Hall during all open
Exhibit Hall hours. All proceeds will benefit the Foundation for Genetic Technology,
which will utilize the funds to promote education in genetic technology through
provision of professional opportunities for training through grants, scholarships
and awards. Participating in the auction is a great way to show your support for
the Foundation and AGT. All donations are tax deductible.
The Silent Auction will close at 11:20 a.m. on Saturday, June 14. Winners will be
announced at 3:25 p.m. before the refreshment break on Saturday. Items can
be picked up at the AGT Registration Desk between 3:30 p.m. and 4:00 p.m.
on Saturday.
Friday, June 13
Job Fair – Marriott Ballroom Foyer
5:30 p.m. – 7:30 p.m.
AGT has invited laboratories and other organizations with employment
opportunities for technologists to participate at its annual Job Fair. You may find
the job that you have been looking for or just satisfy your curiosity by discovering
the other employment opportunities available. The Job Fair will allow you to
network in a casual atmosphere with potential employers and fellow professionals
one-on-one. Attendance at the Job Fair is complimentary and open to the public.
As of May 7, the following companies have signed up to participate in the Job
Fair
•• Allied Search Partners
•• NeoGenomics
•• Staff Icons
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Saturday, June 14
Annual Business Meeting Breakfast – Marriott Ballroom VI
7:00 a.m. – 7:45 a.m.
This complimentary breakfast provides the opportunity for both members and
non-members to learn more about the Association, meet the incoming Board of
Directors, get the latest updates on AGT activities and hear updates from other
organizations.
Awards Reception – Marriott Ballroom Foyer
6:00 p.m. – 7:00 p.m.
Banquet and Dance – Marriott Ballroom V
7:00 p.m. – 11:00 p.m.
Please join us to celebrate the last evening of the meeting by honoring our award
winners and thanking our sponsors for their support. AGT will provide dinner and
one drink ticket (beverages can be purchased after you have used your drink
ticket).
This is a wonderful opportunity to network with your colleagues from across the
country one last time!
Prize drawings will also be held.
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CONTINUING EDUCATION CREDITS
Contact
Hours
Awarded for:
15.5
Attending the Scientific Sessions on both Friday and Saturday
3.0
Attending a three-hour pre-conference workshop
2.0
Attending a two-hour pre-conference workshop
2.0
Poster viewing (1.0 contact hour per five posters viewed)
Contact
Hours
Awarded for Abstract Authors/Presenters
10.0
First authors of abstracts
5.0
Additional authors of abstracts
10.0
Platform presentations
An all-in-one certificate for Workshops, Scientific Sessions and poster viewing
contact hours is included with your Annual Meeting materials. To receive contact
hours, enter the number of CEs for each session that you attend in the “Total CEs
Earned” column at the far right of the form. Only report CEs for the sessions you
attended. If you did not attend an entire session, report only the portion you did
attend. Enter the total number of continuing education hours earned during the
meeting at the bottom of the form. The white copy of this form must be
turned in at the AGT registration desk at the end of the program
and will be kept on file at the AGT Executive Office. Please retain
the YELLOW copy as this is your actual CE CERTIFICATE. IN ORDER
TO BE VALID IT MUST BE STAMPED.
AGT is a registered accrediting agency for the State of Florida and the State of
California. Therefore, you must comply with these requirements to receive CEs.
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Awards
The following awards will be presented at the Awards Banquet on Saturday,
June 14, 2014.
Association of Genetic Technologists Awards
2014 Student Research Award
A review panel has selected an AGT student member, enrolled in an approved
cytogenetic or molecular program, as the recipient of this award, based on the
research abstract submitted.
2014 Outstanding Achievement Award
The Outstanding Achievement Award is the highest honor that AGT bestows. It
is presented to an AGT member who holds a current certification in cytogenetics
or molecular genetics and has proven his or her commitment to furthering the
field of genetics as demonstrated by his or her work, attitude and AGT activities.
This award is underwritten by Martha Keagle.
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AGT thanks the Foundation for Genetic Technology for its ongoing support
of AGT and the field of genetic technology. Our meeting would not be as
successful without the support of the FGT.
The following awards will be presented at the
Awards Banquet & Dance on Saturday, June 14, 2014.
AGT Student Research Award
Barbara J. Kaplan Scholarship
EXCEL Award
Genome Award
Joseph Waurin Excellence
in Education Award
Best Poster Award
Best Platform Presentation Award
Best Exhibit Booth Award
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Sponsors
The support and assistance received by the following organizations greatly
enhances this year’s meeting. Please take a moment to thank representatives of
these companies for their contributions.
Silver Sponsors
Tote Bags Name Badge Lanyards
Hotel Keycards
BRONZE SPONSOR
Gordon Dewald Lecture
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2013-2014 Board of Directors
President
Mervat Ayad, BS, EMBA, CG(ASCP)CM, CCS
Quest Diagnostics at Nichols Institute
San Juan Capistrano, Calif.
[email protected]
President-Elect
Patricia K. Dowling, PhD
Pathline Labs
Suffern, N.Y.
[email protected]
Secretary-Treasurer
Denise Juroske Short, MSFS, MB(ASCP)CM
University of Texas MD Anderson Cancer Center
Houston, Texas
[email protected]
Public Relations Director
Jun Gu, MD, CG(ASCP)CM
Houston, Texas
[email protected]
Education Director
Sally J. Kochmar, MS, CG(ASCP)CM
Magee-Women’s Hospital-Pittsburgh Cytogenetics Lab
Pittsburgh, Pa.
[email protected]
2014 Annual Meeting Director
Jason Yuhas, BS, CG(ASCP)CM
Mayo Clinic
Rochester, Minn.
[email protected]
2014 Annual Meeting Co-Director
Adam Sbeiti, MT(ASCP)CGCM, DLMCM
Quest Diagnostics at Nichols Institute
San Juan Capistrano, Calif.
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2013-2014 Council of Representatives
Representative to CCCLW
Kathryn Sudduth, BA, CG(ASCP)CMDLMCM
Charlottesville, Va.
[email protected]
Representative to NAACLS
Peter C. Hu, PhD, MS, MLS(ASCP)CM, CGCM, MBCM
Houston, Texas
[email protected]
Representative to CAP/ACMG
Jonathan P. Park, PhD, CG(ASCP)CM
Dartmouth-Hitchcock Medical Center
Lebanon, N.H.
[email protected]
Representatives to the Board of Certification
Helen A. Bixenman, MBA/HCM, CHC, CG(ASCP)CMDLMCM, QLC
Phoenix Children’s Hospital
Phoenix, Ariz.
[email protected]
Amy R. Groszbach, BS, MB(ASCP)CM, MEd
Mayo Clinic
Rochester, Minn.
[email protected]
Representative to Foundation for Genetic Technology (FGT)
Patricia LeMay, MT(ASCP), CG(ASCP)CM
Monmouth Medical Center
Long Branch, N.J.
[email protected]
FGT Board of Trustees President
Robin Vandergon, CG(ASCP)CM, DLM(ASCP)
LabCorp
Fresno, Calif.
[email protected]
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Exhibitor Information
Exhibit Hall Floor Plan
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Abbott Molecular
1350 E. Touhy Ave.
Des Plaines, IL 60018
P: 224-361-7913
E: [email protected]
www.abbottmolecular.com
Booth # 9P
Abbott Molecular provides physicians with critical information based on the detection of
pathogens and subtle changes in patients’ genes and chromosomes, allowing for earlier
diagnosis, selection of appropriate therapies and monitoring disease progression. The
business includes instruments and reagents used to conduct sophisticated analysis of
a patient’s DNA and RNA. Our commitment can be seen in our broad portfolio of
products, focused on four critical areas: oncology, infectious diseases, pre- and postnatal testing and organ transplantation.
Affymetrix
3420 Central Expressway
Santa Clara, CA 95051
P: 408-731-5200
www.affymetrix.com
Booth # 8P
Affymetrix provides clinical research tools for whole-genome cytogenetic analysis. The
CytoScan® HD Solution provides the broadest coverage for high resolution detection
of copy number changes and the most SNP probes that genotype with >99% accuracy
for both constitutional and cancer cytogenetic applications.
Agilent Technologies
2850 Centerille Road
Wilmington, DE 19808
P: 800-227-9770
F: 302-636-8944
www.agilent.com/genomics
Booth # 16P
Agilent Technologies’ market-leading Genomics Solutions Division provides applicationfocused solutions. Perform gDNA sample QC with the TapeStation system, simplify the
sequencing of clinical research samples from custom design to mutation report using
the HaloPlex NGS target enrichment workflow and examine chromosomal aberrations
with CGH+SNP Arrays and SureFISH Probes.
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Allied Search Partners
1298 Minnesota Ave., Ste. E
Winter Park, FL 32789
P: 888-388-7571
F: 888-388-7572
www.alliedsearchpartners.com
Booth # 20
Allied Search Partners specializes in filling your permanent Anatomic and Clinical
Pathology Laboratory Job Openings. Our specialization is the driving force behind our
success and one of the reasons we are able to present candidates not found elsewhere
by other search firms or other added resources.
Applied Spectral Imaging, Inc.
5315 Avenida Encinas, Ste. 150
Carlsbad, CA 92008
P: 760-929-2840
F: 760-929-2842
www.spectral-imaging.com
Booth # 19P
Applied Spectral Imaging makes patient care better through advanced biomedical
imaging. ASI offers cytogeneticists and pathologists accurate analysis by providing
state-of-the-art diagnostic aids. ASI has over 2,500 systems deployed worldwide,
offices in the U.S., Europe and Asia as well as a global network of over 50 distributors.
Association of Genetic Technologists (AGT)
P.O. Box 19193
Lenexa, KS 66285
P: 913-895-4605
F: 913-895-4652
www.agt-info.org
Booth # 32P
The Association of Genetic Technologists, founded in 1975, is a non-profit professional
organization established to promote cooperation and the exchange of information
among those engaged in classical cytogenetics, molecular and biochemical genetics
and to stimulate interest in genetics as a career. Membership is open to all who have an
interest or are employed in the broad field of genetics. Stop by the AGT booth to pick
up a membership application and meet an AGT representative.
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BioDiscovery, Inc.
5155 Rosecrans Ave
Hawthorne, CA 90250
P: 310-414-8100
F: 310-414-8111
www.biodiscovery.com
Booth #1P
BioDot Inc.
2852 Alton Parkway
Irvine, CA 92606
P: 949-440-3685
F: 949-440-3694
www.biodot.com
Booth # 21P
BRONZE SPONSOR
BioDot is the leading supplier of systems for the research, development and
manufacturing of diagnostic tests. Its Mission is to enable, inspire and educate
scientists to commercialize their R&D ideas through to manufactured product. Using
its core competencies in low volume non-contact and contact dispensing, BioDot has
developed a range of equipment for the research and development, and manufacture
of rapid tests.
Biological Industries
83 Maple Ave.
Windsor, CT 06095
P: 860-298-8382
F: 860-298-8586
www.rainbowscientific.com
Booth # 12P
Biological Industries (BI) has been providing optimal and innovative solutions for cell
culture practice for 30 years. Their cytogenetic cell culture media are optimized for the
analysis of amniotic fluid cells, chorionic villus samples, peripheral blood lymphocytes,
primary bone marrow cells and hematopoietic cells. Visit booth 12P to learn about free
evaluation samples and high resolution banding reagents.
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SILVER SPONSOR
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BioView USA Inc.
44 Manning Road
Billerica, MA 01821
P: 978-670-4741
F: 978-670-4740
www.bioview.co.il
Booth # 25P
BioView develops and markets innovative automated cell diagnostic systems via
fluorescence in-situ hybridization (FISH) for clinical and research laboratories. The
Duet™ and ALLEGRO scanning workstations provide automated detection, analysis and
reporting of cells of interest, under fluorescence and brightfield microscopy. Bladder
Cancer FISH (UroVysion) is among the FDA cleared applications.
Caris Life Sciences
6655 N. MacArthur Blvd.
Irving, TX 75039
P: 214-294-5610
F: 214-294-5643
www.carislifesciences.com
Booth # 5
Caris Life Sciences is a progressive biosciences company specializing in molecular
profiling and blood-based diagnostic services which is leading the way in diagnostic,
prognostic and theranostic medicine. Caris combines the rigor of an academic medical
institution with the innovative spirit of a technology company. We believe that innovative,
high-quality testing and information can lead to more effective treatment selection and
ultimately to better outcomes for patients with cancer and other complex diseases.
Cytocell, an OGT company
520 White Plains Road, Suite 500
White Plains, NY 10591
P: (860) 298-8382
F: (860) 298-8586
www.cytocell.com
Booth # 6P
Cytocell celebrates more than 23 years as a leading provider of innovative DNA
screening solutions for the accurate detection of human genetic diseases. Cytocell
manufactures complete ranges of DNA FISH probes for use in clinical cytogenetics.
Please review our updated list of FISH probes for hematological malignancies including
our new full line of Aquarius® Hematopathology FISH probes. Check out www.
myprobes.com regarding our custom FISH probe-making services.
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CytoVision by Leica Biosystems
1360 Park Center Dr.
Vista, CA 92081
P: 800-248-0213
F: 760-539-1116
www.leicabiosystems.com
Booth # 24P
Enzo Life Sciences
10 Executive Blvd
Farmingdale, NY 11735
P: 631-694-7070
F: 610-941-9252
[email protected]
www.enzolifesciences.com
Booth #18P
Enzo Life Sciences is a leader in technologies for genomics, cellular analysis,
immunoassays, assay development, proteostasis, epigenetics, immunohistochemistry &
small molecule chemistry. Our expertise in labeling & detection includes fluorescent
labels/probes, ELISA and enzyme activity assays, biochemicals, antibodies, and proteins,
to serve life sciences research, drug development & clinical research.
Foundation for Genetic Technology
P.O. Box 19193
Lenexa, KS 66285
P: 559-392-0512
F: 559-432-5487
Booth # 30P/31P
The Foundation for Genetic Technology is a not-for-profit corporation with the taxexempt purpose of promoting education in genetic technology. The foundation, through
its contributors, funds the Outstanding Technologist Grant and the following Awards:
Best Poster, Best Platform Presentation, Best Exhibitor, EXCEL, Genome, Joseph Waurin
Excellence in Education and New Horizons. The Foundation also funds the AGT Student
Research Award and the Barbara J. Kaplan Scholarship. Additionally, the Foundation
promotes professional opportunities through new publications, regional workshops
and grants to educational institutions. Please stop by the booth to inquire about these
programs and to bid on silent auction items.
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BRONZE SPONSOR
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Genial Genetics
83 Maple Ave.
Windsor, CT 06095
P: 860-298-8382
F: 860-298-8586
www.rainbowscientific.com
Booth # 11
Genial Genetics offers the robotic MultiPrep Genie and Cell Sprint Harvesting systems
for both surface culture and suspension culture harvesting. Our ProCell cytogenetic
reagents yield high quality cytogenetic preparations. Our suite of genetic database
software products including Shire, iGene and iPassport QMS, offer integrated multidiscipline genetic patient management with excellent auditing and document/process
control capabilities.
Irvine Scientific
1830 E. Warner Ave.
Santa Ana, CA 92705
P: 949-261-7800
www.irvinesci.com
Booth # 15P
Irvine Scientific® is a leading supplier of Cytogenetics media for the Genetic and Cancer
Testing industry and a Fetal-Lung-Maturity assessment kit for the Prenatal/Diagnostic
industry. Chang Medium® is our brand of cytogenetics culture media for constitutional
and cancer genetic testing. AmnioStat-FLM®-PG is our unique, rapid, STAT test kit
that detects Phosphatidylglycerol (PG) for the assessment of fetal lung maturity during
prenatal care. Irvine Scientific is located in Santa Ana, California.
Kreatech
1821 Hillandale Road, Ste. 1B-388
Durham, NC 27705
P: 866-572-1432
F: 919-471-2366
www.kreatech.com
Booth # 23
Kreatech Inc. offers POSEIDON™ FISH probes with REPEAT-FREE™ technology.
Kreatech has an extensive portfolio of FISH probes and custom design capability.
Kreatech’s labeling technology, ULS™ – the Universal Linkage System – allows nonenzymatic, accurate and fast labeling of DNA, RNA and proteins; one universal labeling
system compatible with all biological samples.
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MatTek Corporation
200 Homer Ave.
Ashland, MA 01721
P: 508-881-6771
F: 508-879-1532
www.glass-bottom-dishes.com
Booth # 22G
MatTek’s Glass Bottom Dishes and Coverslip Kits combine the convenience of standard
size plastic petri dishes with the optics of glass, providing researchers with superior
quality high resolution microscopic images. These dishes and kits are routinely used in
confocal and fluorescence imaging techniques, amniocentesis, FISH, CVS and other
cytogenetic techniques.
MetaSystems
70 Bridge St., Ste. 100
Newton, MA 02458
P: 617-924-9950
F: 617-924-9954
www.metasystems.org
Booth # 17
MetaSystems provides fast, easy-to-use genetic imaging and high-throughput slide
scanning systems: ikaros for automatic karyotyping, isis for FISH imaging, CGH, mFISH,
high resolution color banding analysis, metafer for fully automatic slide analysis, spot
counting, rare cell detection, metaphase search, array analysis and XCyte DNA probes.
Please visit our website www.metasystems.org.
26
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ANNUAL MEETING
Oxford Gene Technology
Begbroke Business and Science Park
Sandy Lane
Yarnton, Kidlington, Oxfordshire OX5 1PF
United Kingdom
P: +44 (0)1865 856826
F: +44 (0)1865 848684
www.ogt.com
Booth # 4P
Oxford Gene Technology (OGT) is committed to providing researchers with innovative
products and services to accurately detect and analyze genetic disease, including cancer
and constitutional disorders. Our portfolio incorporates NGS cancer panel and exome
services plus a range of disease-specific microarray products. For further information
contact us at [email protected] or visit www.ogt.com.
Percival Scientific, Inc.
505 Research Drive
Perry, IA 50220
P: 515-465-9363
F: 515-465-9464
www.percival-scientific.com
Booth # 26
Promega
2800 Woodshollow Road
Madison, WI 53711
P: 608-274-4330
www.promega.com
Booth # 7P
Promega has over 30 years of expertise delivering reagents to life science and clinical
laboratories. We offer GPR- and GPLE-labeled products, including instruments and
reagents for DNA and RNA extraction, PCR, STR analysis, genetic analysis and mutation
detection.
27
AGT 39th
ANNUAL MEETING
SciGene
470F Lakeside Drive
Sunnyvale, CA 94085
P: 408-733-7337
F: 408-733-7336
www.scigene.com
Booth # 14
SciGene develops instruments and reagents to automate and enhance FISH and CMA
workflows used in research and clinical diagnostic laboratories. SciGene automation
boosts productivity, lowers costs, reduces re-test rates and standardizes tests for more
reliable results.
Staff Icons, LLC
115 Franklin Turnpike, Ste. 158
Mahwah, NJ 07430
P: 201-986-7888
F: 201-986-7444
www.stafficons.com
Booth # 13P
Staff Icons specializes in matching top talented professional candidates with companies
nationwide. We do full cycle recruiting in the biotech/pharmaceutical/health care industry
and service direct hire, short- and long-term or project-based staffing requirements.
We represent both clients and candidates and we recruit for the following disciplines:
cytogenetics, Histology, Molecular, Flow Cytometry, Microbiology and Engineering. We
have an exceptional team of recruiters ready to serve you.
STEMCELL Technologies, Inc.
570 West 7th Ave, Suite 400
Vancouver, BC Candada V5Z1B3
P: 800-667-0322
F: 800-567-2899
[email protected]
www.stemcell.com
Booth #3P
STEMCELL Technologies offers cell isolation products to enhance assay sensitivity
for HLA/chimerism, as well as multiple myeloma, CLL and other hematological
malignancies. RoboSep™, the automated cell separation instrument, offers true walkaway automation of cell isolation from whole blood or bone marrow while minimizing
sample handling and hands-on time. www.robosep.com
28
AGT 39th
ANNUAL MEETING
Tethis S.p.A.
Via Russoli 3
Milano, Italy 20143
P: +39 02 3656 8349
F: +39 02 3656 9183
www.tethis-lab.com
Booth #33G
Tethis has developed microFIND® a microfluidic device, used as alternative to
microscope glass slide. autoFIND F coupled to microFIND®, is an automated slide
staining system for the processing of the majority steps of the FISH (Fluorescent In Situ
Hybridization) assays on cytological samples both in interphase and metaphase.
Transgenomic, Inc.
12325 Emmet St.
Omaha, NE 68164
P: 888-813-7253
F: 402-452-5401
www.transgenomic.com
Booth # 27G
Transgenomic develops and commercializes biomarkers and personalized diagnostics
with the goal of improving medical diagnoses and patient outcomes. Transgenomic
leverages proprietary technology and molecular genetics expertise to provide a fully
integrated molecular diagnostic solution through our three integrated divisions —
Biomarker Identification, Genetic Assays and Platforms and Patient Testing.
UCLA Health
10920 Wilshire Blvd., Ste. 400
Los Angeles, CA 90095
P: 310-794-0506
F: 310-794-0620
www.uclahealthcareers.org
Booth # 10P
UCLA Health Systems defines greatness by the quality of the patient experience we are
able to deliver. Each and every time. To every single patient. If that’s where your ambitions
lie, UCLA is where you belong. We offer unequalled challenges and opportunities to
further your education, training and career.
29
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ANNUAL MEETING
30
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ANNUAL MEETING
Thursday, June 12, 2014
Pre-Conference Workshops
TIME
EVENT
LOCATION
7:00 a.m. –
3:00 p.m. &
6:30 p.m. –
8:00 p.m.
Registration Desk Open
Marriott Ballroom
Foyer
8:00 a.m. –
12:00 p.m.
FGT Board of Trustees Meeting
Filly
8:00 a.m. –
11:00 a.m.
Workshop 1: Introduction to Forensic
Science – Denise Juroske Short, MS, MB(ASCP)CM;
Crystal Simien, BS, MB(ASCP)CM
Bluegrass
8:00 a.m. –
10:00 a.m.
Workshop 2: Overview of HER2 FISH
Testing: Intricacies of Process & Challenges
in Interpretation – Reid G. Meyer, CG(ASCP)CM;
Jason Yuhas, BS, CG(ASCP)CM
Rose
8:00 a.m. –
10:00 a.m.
Workshop 3: Quality Assurance in Genetics
– Peggy Stupca, CG(ASCP)CM, DLMCM; Helen M.
Jenks, MT(ASCP)CGCM
Thoroughbred
10:30 a.m. – Workshop 4: FISH Analysis – Beyond
12:30 p.m.
Counting Dots – Shirong Wang, MS, CG(ASCP)CM;
Xiaojing Yang, MLT(ASCP)CGCM
Sponsored by Quest Diagnostics
Rose
10:30 a.m. – Workshop 5: Array CGH & SNP Array
12:30 p.m.
Validation, Data Interpretation & Quality
Control – Ming Zhao, CG(ASCP)CM, MB(ASCP)CM
Thoroughbred
1:00 p.m. –
5:00 p.m.
Poster Set-Up
Marriott I-V
2:00 p.m. –
4:00 p.m.
Workshop 6: FISH Testing Reimbursement
Challenges & Efficiency Opportunities –
Philip N. Mowrey, PhD, MS, FACMG, CG(ASCP)CM;
Fatih Boyar, MD, FACMG; Deborah W. Heritage,
MS, CG(ASCP)CM; Adam Sbeiti, MT(ASCP)CGCM,
DLMCM
Sponsored by Quest Diagnostics
Bluegrass
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AGT 39th
ANNUAL MEETING
TIME
EVENT
2:00 p.m. –
4:00 p.m.
Workshop 7: From BACS/OLIGO Array to
SNP Array – New Development in Perinatal
& Cancer Applications – Jun Gu, MD, PhD,
CG(ASCP)CM
Rose
2:00 p.m. –
4:00 p.m.
Workshop 8: Clinical Utility & Synergy of
Molecular Genetic Technologies –
Douglas Blake, CG(ASCP)CM
4-Hour FISH Workflow for FFPE, Blood &
Bone Marrow Specimens –
Sharon Alsobrook, CG(ASCP)CM, MLS(ASCP)CM
Sponsored by Agilent Technologies
Thoroughbred
4:00 p.m. –
6:00 p.m.
Genetic Educators Meeting
Filly
7:00 p.m. –
9:00 p.m.
Marriott Ballroom
Welcome Reception in the Exhibit Hall
Poster Viewing, FGT Silent Auction Opening I-V
32
LOCATION
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ANNUAL MEETING
Friday, June 13, 2014
Scientific Sessions
TIME
EVENT
LOCATION
7:00 a.m. –
4:00 p.m.
(closed 12:15 – 1:15 p.m.)
Marriott Ballroom
Foyer
7:00 a.m. –
8:00 a.m.
Continental Breakfast
Marriott Ballroom
Foyer
SCIENTIFIC SESSIONS
Marriott Ballroom VI
8:00 a.m. –
8:10 a.m.
Opening Remarks
8:10 a.m. –
9:00 a.m.
Keynote Address – Lee H. Hilborne, MD, MPH, DLM(ASCP)CM, FASCP
9:00 a.m. –
9:50 a.m.
Clinical Genome Sequencing: What’s Next in NextGeneration Sequencing? – Matthew J. Ferber, PhD, FACMG
9:50 a.m. –
10:20 a.m.
Break in the Exhibit Hall
Poster Viewing/Silent Auction Items Available
10:20 a.m. – Diagnostic Testing for HER2 Amplification in Breast Cancer:
11:10 a.m.
The New ASCO/CAP Guidelines – Gail H. Vance, MD
11:10 a.m. –
12:00 p.m.
Non-Invasive Pre-Natal Diagnosis – Charles “Buck” Strom, MD,
PhD, FAAP, HCLD
12:00 p.m. – Lunch on Your Own
1:30 p.m.
SCIENTIFIC SESSIONS
1:40 p.m. –
2:30 p.m.
Clinical Significance of the Leukemia Cell Karyotype
in Children with Acute Myeloid Leukemia (AML) –
Susana C. Raimondi, PhD
2:30 p.m. –
3:20 p.m.
Genetic Counseling in the Era of Microarrays –
Leslie Ross, MS, CGC
3:20 p.m. –
4:00 p.m.
4:00 p.m. –
4:30 p.m.
Personalized Medicine & Me – Peter C. Hu, PhD, MS, MLS(ASCP)CM,
CGCMMBCM
4:30 p.m. –
5:20 p.m.
Health & Safety – Mervat S. Ayad, BS, EMBA, CG(ASCP)CM, DLMCM,
CCS
33
AGT 39th
ANNUAL MEETING
Special Event
TIME
5:30 p.m. –
7:30 p.m.
EVENT
Job Fair
Companies participating as of May 7:
• Allied Search Partners
• NeoGenomics
• Staff Icons
34
LOCATION
Marriott Ballroom
Foyer
AGT 39th
ANNUAL MEETING
Scientific Sessions – Marriott Ballroom VI
8:10 a.m. – 9:00 a.m.
KEYNOTE ADDRESS
Lee H. Hilborne, MD, MPH, DLM(ASCP)CM , FASCP,
Professor of Pathology and Laboratory Medicine, David Geffen
School of Medicine, University of California Los Angeles;
Quest Diagnostics, Los Angeles, Calif.
NOTES
35
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NOTES
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ANNUAL MEETING
9:00 a.m. – 9:50 a.m.
Clinical Genome Sequencing:
What’s Next in Next-Generation Sequencing?
Matthew J. Ferber, PhD, FACMG, Director, Clinical Genome
Sequencing Laboratory, Mayo Clinic, Rochester, Minn.
With the advent of inexpensive genome sequencing, the amount of information
that can be inexpensively obtained is staggering. The presenter will review the
technologies and how they are changing clinical molecular diagnostics.
NOTES
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NOTES
38
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ANNUAL MEETING
10:20 a.m. – 11:10 a.m.
Diagnostic Testing for HER2 Amplification in
Breast Cancer: The New ASCO/CAP Guidelines
Gail H. Vance, MD, Professor and Sutphin Professor of Cancer
Genetics, Indiana University, Indianapolis, Ind.
The ASCO/CAP guidelines for HER2 testing in breast cancer were first published
in 2007. In 2013, an updated guideline document was published. The presenter
will crosswalk the differences between the two documents focusing on in situ
hybridization methodology, principally fluorescence in situ hybridization (FISH).
Comparative data from one institution will be highlighted.
NOTES
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ANNUAL MEETING
NOTES
40
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ANNUAL MEETING
11:10 a.m. – 12:00 p.m.
Non-Invasive Pre-Natal Diagnosis
Charles “Buck” Strom, MD, PhD, FACMG, FAAP, HCLD,
Senior Medical Director, Genetics, Quest Diagnostics at
Nichols Institute, San Juan Capistrano, Calif.
The presenter will highlight the scientific practices used for non-invasive
prenatal diagnosis. Dr. Strom will compare and contrast the currently available
methodologies and their strengths and weaknesses.
NOTES
41
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NOTES
42
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ANNUAL MEETING
1:40 p.m. – 2:30 p.m.
Clinical Significance of the Leukemia Cell Karyotype in
Children with Acute Myeloid Leukemia (AML)
Susan C. Raimondi, PhD, Director, Cytogenetics Laboratory,
St. Jude Children’s Research Hospital, Memphis, Tenn.
Acute leukemia is best managed through the use of risk-adapted therapy. The
karyotype of patients at diagnosis is an important factor in predicting response
to therapy. The distinct chromosomal abnormalities observed in AML will be
presented, and the practice used to stratify the children by Children Oncology
Group (COG) will be discussed.
NOTES
43
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ANNUAL MEETING
NOTES
44
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ANNUAL MEETING
2:30 p.m. – 3:20 p.m.
Genetic Counseling in the Era of Microarrays
Leslie Ross, MS, CGC, Genetic Counselor,
Quest Diagnostics, Denver, Colo.
New technology has made extensive genetic testing more accessible to a large
number of patients. However, difficult-to-interpret genetic test results have made
genetic counseling challenging.
NOTES
45
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ANNUAL MEETING
NOTES
46
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ANNUAL MEETING
4:00 p.m. – 4:30 p.m.
Personalized Medicine & Me
Peter C. Hu, PhD, MS, MLS(ASCP)CM , CGCMMBCM ,
Program Director, University of Texas, M.D. Anderson
Cancer Center, Houston, Texas
Dr. Hu will discuss his journey with personalized medicine. Can a normal person
understand this complex jargon? What does it all mean in the end?
NOTES
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NOTES
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ANNUAL MEETING
4:30 p.m. – 5:20 p.m.
Health & Safety
Mervat Ayad, BS, EMBA, CG(ASCP)CM , DLMCM , CCS,
Director, Laboratory Operations, Quest Diagnostics
at Nichols Institute, San Juan Capistrano, Calif.
Laboratory safety is an important part of our jobs every day, but what do the
regulations really mean? What steps can you take to better protect yourself from
the hazards you face? The presenter will answer these questions and more.
Attending this session will fulfill the ASCP safety training requirement.
NOTES
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NOTES
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ANNUAL MEETING
Saturday, June 14, 2014
Scientific Sessions
TIME
EVENT
LOCATION
7:00 a.m. –
6:00 p.m.
(closed from
12:30 p.m. –
1:30 p.m.)
Marriott Ballroom
Foyer
7:00 a.m. –
8:00 a.m.
Business Meeting Breakfast
SCIENTIFIC SESSIONS
8:00 a.m. –
8:10 a.m.
Opening Remarks
8:10 a.m. –
9:00 a.m.
Gordon W. Dewald Lecture: Clinical Laboratory Education
in Diagnostic Genetics – The Road to a Brighter Future in
Health Care – Vicki L. Hopwood, MS
Sponsored by Rainbow Scientific
9:00 a.m. –
10:40 a.m.
Abstract Platform Presentations & Student Abstract Award
Winner Presentation
10:40 a.m. –
11:20 a.m.
11:30 a.m. –
12:20 p.m.
Informed Consent for Whole Genome Sequencing – Katherine
S. Hunt, PhD, MS, CGC
12:20 p.m. –
1:45 p.m.
Lunch on Your Own
12:20 p.m. –
1:45 p.m.
UCONN Luncheon
SCIENTIFIC SESSIONS
Marriott Ballroom
I-V
1:45 p.m. –
2:35 p.m.
Miscarriages, Cell Morphology & Villi Formation: Detecting
High Abnormality Percentages in POCs – Philip J. Hardy, M. Clin
Cyto, B.Bus
2:35 p.m. –
3:25 p.m.
Targeted Therapies for Solid tumors & Companion
Diagnostics – Dianne Keen-Kim, PhD, FACMG
3:25 p.m. –
3:30 p.m.
Silent Auction Items Announced
3:30 p.m. –
3:45 p.m.
Break
51
AGT 39th
ANNUAL MEETING
TIME
EVENT
LOCATION
3:45 p.m. –
4:35 p.m.
The Integration of Clinical & Laboratory Elements in
Diagnosing & Managing Genetic Disorders – Kara Goodin, MD
4:35 p.m. –
5:25 p.m.
Overview of the New AMA Molecular Pathology CPT Codes –
V.M. Pratt, PhD, FACMG
5:25 p.m. –
5:30 p.m.
Closing Remarks
Special Events
TIME
EVENT
LOCATION
6:00 p.m. –
7:00 p.m.
Annual Awards Reception
Marriott Ballroom
Foyer
7:00 p.m. –
11:00 p.m.
Annual Awards Banquet & Dance
Marriott Ballroom V
52
AGT 39th
ANNUAL MEETING
Annual Business Meeting Agenda
Louisville Marriott Downtown
Louisville, Ky.
7:00 a.m. – 7:45 a.m.
1.Opening remarks by Mervat Ayad, AGT President
2.Introduction of the current AGT Board of Directors and Council of
Representatives
3.Approve the 38th Annual Business Meeting Minutes
4. Apprise the membership of highlights during the last year and initiatives
taken at the Board of Directors Meeting
5. Presentation of the approved 2014-2015 budget
6. Questions invited
7. Updates from Council of Representatives organizations:
• BOC
• FGT
• NAACLS
• CAP
• CCCLW
8.Introduction of the newly elected Board of Directors
9. Presentation of plaques to the outgoing Board/COR members and 2015
Annual Meeting Director
10. Meeting adjourned
53
AGT 39th
ANNUAL MEETING
38th Annual Business Meeting Minutes
The Cosmopolitan of Las Vegas
Las Vegas, Nevada
7:00 a.m. – 8:00 a.m.
AGT President Richard Pettersen called the 38th Annual Business Meeting to
order at 7:09 a.m. on Saturday, June 8.
1.President Richard Pettersen thanked members for their participation and
continued support. He also thanked the exhibitors and sponsors of the 38th
Annual Meeting.
2.The 2012-2013 AGT Board of Directors were introduced: President-Elect
– Mervat Ayad, Secretary-Treasurer – Denise Juroske-Short, Education
Director– Sally Kochmar, Public Relations Director – Jun Gu, Annual Meeting
Director – Denise Anamani, Annual Meeting Co-Director – Jason Yuhas.
3.Members present at the business meeting RESOLVED to approve the 37th
Annual Business Meeting Minutes.
4. President Richard Pettersen reported to the membership on the items completed in the past year and the initiatives taken at the Board of Directors
meeting.
5. Secretary/Treasurer, Denise Juroske-Short, presented a brief overview of the
current financial condition of the association and of the approved 20132014 budget.
6. Members were invited to ask questions about the Board initiatives, budget or
any related organizations. No questions were asked.
7. President Richard Pettersen introduced the following Council of Representatives members and asked them to provide brief reports:
• BOC – Helen Bixenman, Amy Groszbach
• FGT – Pat LeMay
• NAACLS – Peter Hu
• CAP – Jonathan Park (absent – report provided by Richard Pettersen)
• CCCLW – Kathy Sudduth
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ANNUAL MEETING
8.President Richard Pettersen introduced the newly elected and appointed
members of the Board of Directors and Council of Representatives for
2013-2014:
• Pat Dowling – President-Elect
• Adam Sbeiti – Annual Meeting Co-Director
9. The outgoing members of the Board of Directors and Council of Representatives were recognized for their service and presented with plaques. The
outgoing Board members were: Richard Pettersen – President and Denise
Anamani – Annual Meeting Director.
10. Jason Yuhas, 2014 Annual Meeting Director, was presented with the Annual
Meeting traveling plaque.
11. President Richard Pettersen passed the president’s gavel to President-Elect
Mervat Ayad.
12.President Richard Pettersen addressed the membership with some final
remarks.
There being no further business to come before the AGT membership, PresidentElect, Mervat Ayad, adjourned the 38th Annual Business Meeting at 8:00 a.m.
Pacific Time.
Respectfully Submitted,
Denise Juroske-Short
Secretary/Treasurer
55
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ANNUAL MEETING
Scientific Sessions – Marriott Ballroom VI
8:10 a.m. – 9:00 a.m.
Gordon W. Dewald Lecture: Clinical Laboratory
Education in Diagnostic Genetics –
The Road to a Brighter Future in Health Care
Vicki L. Hopwood, MS, Assistant Professor; Director,
Cytogenetic Technology Program, University of Texas,
M.D. Anderson Cancer Center, Houston, Texas
Sponsored by Rainbow Scientific
The presenter will provide an overview of genetic clinical laboratory education
from the perspective of a practitioner in the field for 30 years. This same speaker
is a patient who has survived radiation and chemotherapy to combat Stage IV
brain cancer. Through her presentation the presenter will also relate her personal
journey through diagnosis, standard treatment and clinical trials that are studying
targeting genetic biomarkers for improved therapy.
NOTES
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NOTES
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ANNUAL MEETING
9:00 a.m. – 10:40 a.m.
Abstract Platform Presentations
9:00 a.m. – 9:15 a.m.
COMPLEX CHROMOSOMAL REARRANGEMENTS IN B-CELL
LYMPHOMA: EVIDENCE OF CHROMOTHRIPSIS?
Veronica Ortega, BA, CG(ASCP)CM; Christina Mendiola, BS, CG(ASCP);
William Ehman, Jr., BS, CG(ASCP); Kumari Vadlamudi, MT(ASCP); Vijay Tonk,
PhD; Gopalrao Velagaleti, PhD
9:15 a.m. – 9:30 a.m.
MULTIPLE MYELOMA: THE TESTING, VALIDATION AND
IMPLEMENTATION OF CELL SEPARATION TECHNOLOGY FOR
IMPROVED PATIENT CARE
Elizabeth Harper Allen, CG(ASCP); Binh Vo, CG(ASCP); Joey Pena, CG(ASCP);
Soo Ha Cheong, CG(ASCP); Denise Lovshe, CG(ASCP); Dr. Gary Lu;
Dr. Xinyan Lu
9:30 a.m. – 9:45 a.m.
IMPLICATIONS OF DELAYED LEUKOCYTE ISOLATION AND
REPEATED CYCLES OF FREEZE-THAW ON THE ACTIVITY OF ELEVEN
LYSOSOMAL ENZYMES
Teresa Thompson, BS, MB(ASCP)CM; Tim Wood, PhD, FACMG; Laura Pollard,
PhD, FACMG
9:45 a.m. – 10:00 a.m.
CHROMOSOME SHATTERING: A LOOK AT CONSTITUTIONAL
CHROMOTHRIPSIS IN PATIENTS EXHIBITING MULTIPLE
COPY NUMBER CHANGES AND COMPLEX STRUCTURAL
REARRANGEMENTS REVEALED BY SNP MICROARRAY
April N. Harris; Rachel D. Burnside PhD, FACMG
10:00 a.m. – 10:15 a.m.
DETECTING LOW LEVEL MOSAICISM OF TRISOMY 9 WITH
MICROARRAY ANALYSIS
Jennifer Crawford, CG(ASCP)CM; Shamsa Naqvi, CG(ASCP)CM; Amanda Fortier,
PhD; Debra Rita, MD; Jillene Kogan, MD, PhD
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ANNUAL MEETING
10:15 a.m. – 10:30 a.m.
QUANTITATIVE ANALYSIS OF SNRPN GENE METHYLATION BY
PYROSEQUENCING: AN ACCURATE METHOD FOR IDENTIFYING
MOSAICISM
Crystal J. Symsick Propes; Kevin M. Babson, BS; Julie R. Jones, PhD, FACMG
10:30 a.m. – 10:40 a.m.
Student Abstract Award Winner
A COMBINATION OF ZYFLAMEND & VEMURAFENIB (PLX4032)
MAY HAVE A SYNERGISTIC EFFECT AGAINST MELANOMA CELL
PROLIFERATION
Jacob Yo; Peter C. Hu, PhD, MS, MLS(ASCP)CM, CGCMMBCM; Elizabeth A Grimm,
PhD
60
AGT 39th
ANNUAL MEETING
Platform Presentations
Please note: These abstracts have not been edited for grammar or spelling.
9:00 a.m. – 9:15 a.m.
COMPLEX CHROMOSOMAL REARRANGEMENTS IN B-CELL
LYMPHOMA: EVIDENCE OF CHROMOTHRIPSIS?
Veronica Ortega, BA, CG(ASCP); Christina Mendiola, BS, CG(ASCP); William
Ehman Jr., BS, CG(ASCP); Kumari Vadlamudi, MT(ASCP); Vijay Tonk, PhD;
Gopalrao Velagaleti, PhD
Genomic instability is a well-known hallmark of cancer. Thus, uncovering
pathways describing acceleration of such instability is not surprising. Recent
genome sequencing studies have led to identification of a novel phenomenon
called chromothripsis in which complex genomic rearrangements are thought to
be derived from a single catastrophic event rather than by several incremental
steps. Previously, genomic instability is thought to arise through a gradual multistep process.
Chromothripsis suggests an evolutionary modality for cancer cells to circumvent
individual mutational events with one simultaneous shattering of chromosomes
resulting in the random reassembling of segmented genetic material to form
complex derivative chromosomes. While chromothripsis is well documented in
solid tumors and leukemias, chromothripsis in lymphoma is rarely reported. We
report a case of possible chromothripsis in a patient presenting with a thyroid
mass suspicious for lymphoma/carcinoma of the thyroid.
Histology and immunostaining revealed that the mass contained characteristic
pattern of diffuse large B-cell lymphoma. Chromosome analysis from the
biopsy showed a complex karyotype with multiple numerical and structural
rearrangements including a translocation of chromosomes 3 and 7 involving
the BCL6 gene region, with the derivative chromosome further rearranging
with chromosomes 14, 7 and 22 with involvement of the IGH gene region. In
addition, an unbalanced structural rearrangement involving chromosomes 8 and
18 leading to 8p deletion and duplication of 18q, including the BCL2 gene
region and other structural rearrangements were observed.
The karyotype was interpreted as 51~56,XX,+X,+2,t(3;7)(q29;p11.2),der(7)t(3;7)
t(14;7;22)(q32;p11.2;q12),+der(7)t(14;7;22), der(8)t(8;18)(p12;q21),+der(9)t(5;9)
(q13;q22),+13,der(14)t(14;7;22),+21,+1~4r[cp20]. FISH analysis with B-cell
lymphoma probe panel confirmed the BCL6 gene rearrangement in 36.5% of
the nuclei, IGH gene rearrangement or 4-5 copies of IGH in 41.2% of the nuclei
and 3 copies of BCL2 in 7%. To further characterize this complex rearrangement,
array comparative genomic hybridization studies were performed with Cytochip
60K custom oligo array.
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The results showed multiple complex copy number variations including a
previously unidentified chromosome 12 abnormality, the complexity of which
appears to confirm the phenomenon of chromothripsis. However, array analysis
did not reveal any imbalance involving the BCL6, BCL2 or IGH gene regions
whose rearrangements were observed by FISH, thus suggesting that these
rearrangements are balanced in nature. Although the criteria used to identify
possible chromothripsis remains unclear, this pathway is said to occur more
often than expected. Our patients genomic abnormalities show characteristics
suggestive of chromothripsis and provides initial evidence that chromothripsis
is not confined to solid tumors, but can also be seen in B-cell lymphomas with
well characterized one or two-step lymphomagenesis. Our case further illustrates
that lymphomagenesis can be complex and may arise from a catastrophic event
resulting in multiple complex chromosome rearrangements.
9:15 a.m. – 9:30 a.m.
MULTIPLE MYELOMA: THE TESTING, VALIDATION AND
IMPLEMENTATION OF CELL SEPARATION TECHNOLOGY FOR
IMPROVED PATIENT CARE
Elizabeth Harper Allen, CG(ASCP); Binh Vo, CG(ASCP); Joey Pena, CG(ASCP);
Soo Ha Cheong, CG(ASCP); Denise Lovshe, CG(ASCP); Dr. Gary Lu; Dr.
Xinyan Lu
Plasma Cell Myeloma is the primary disease of all plasma cell neoplasms, resulting
from the expansion of clonal differentiated B-cells or plasma cells. Plasma
Cell, and Multiple Myeloma represent the second most prevalent hematologic
neoplasm worldwide. Approximately 15,000 new cases occur each year in the
United States of America.
Beginning in October 2012 until April 2013, the clinical cytogenetic lab tested
and validated a cell isolation instrument produced by StemCell known as the
RoboSep. Starting May 2013 to present, the lab has implemented this instrument
as a primary tool in the processing of clinical patient samples for Multiple Myeloma
and Plasma Cell Myeloma FISH testing. This technology, over time has improved
the detection rate of specific abnormalities through the use of Fluorescent In
Situ Hybridization (FISH), thus providing our clinicians with more sensitive and
accurate results for patient treatment determination. The implementation and
incorporation of the RoboSep into lab procedure for use in processing Myeloma
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samples has resulted in the ability to provide more relevant data to an increasing
patient population, and has helped to define more accurately the actual disease
status of the patient. The methods employed by traditional culturing and harvesting
of Myeloma samples have historically yielded diploid or normal results in most
cases. However, through the utilization of the RoboSep, positive results have been
reported in a majority of patient cases with significant plasma cell percentages.
Testing of the new technology was accomplished by comparing the outcome of 30
samples processed by conventional culture/harvest methods to those processed
using the RoboSep. Following the harvest of both sample types, slides were made
and FISH was performed using our Myeloma FISH panel, which includes the
probes: TP53 (17p13.1), RB-1 (13q14), CDKN2C/CKS1B (1p32.2/1q21), and
IGH@/CCND1/MYEOV-XT (14q32;11q13). Dramatic increases in abnormality
detection rate were documented, which has led to the refinement of the process
and development of algorithms based upon plasma cell percentages in the bone
marrow for all Myeloma samples which arrive to the lab. Isolation of plasma
cells in our Myeloma patient population has been achieved through the use
of gene expression profiling (malignant cells differ from normal plasma cells
by 120 genes), and plasma cell enrichment (using RoboSep), allowing normal
and abnormal CD138+ cells to be targeted and extracted through the use of
magnetic micro beads.
Data which compares the two methods of conventional culture/harvest vs. RoboSep
will be presented in the body of this poster to illustrate the successful results of
increased FISH sensitivity and positivity that have led to the implementation of this
technology into our clinical workflow, and consequently has become a standard
for the treatment of our Myeloma patients. This project not only highlights the
testing and implementation of a new technology for our clinical lab which has
led to improved patient care, but it also represents the importance of the QI/
QC process, which should always be an integral part of all clinical lab settings.
The success of this project has also resulted in the creation of a specialized
RoboSep processing and harvesting team in the lab, as well as the purchase and
implementation of an additional RoboSep instrument.
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9:30 a.m. – 9:45 a.m.
IMPLICATIONS OF DELAYED LEUKOCYTE ISOLATION AND
REPEATED CYCLES OF FREEZE-THAW ON THE ACTIVITY OF
ELEVEN LYSOSOMAL ENZYMES
Teresa Thompson, BS, MB(ASCP)CM; Tim Wood, PhD, FACMG; Laura Pollard,
PhD, FACMG
BiochemicalMeasurement of enzyme activity serves as the gold standard for the
diagnosis of a lysosomal storage disorder. Cultured fibroblasts are considered
the optimal sample type but the invasive nature and longer turn-around-time
(TAT) associated with this sample type can be problematic for physicians and
patients. Enzyme activity measurement in peripheral blood leukocytes (PBLs),
when possible, offers rapid TAT (typically one week) and a less invasive sample.
However, delayed shipment or other unanticipated environmental factors during
transport can impact the quality of peripheral blood samples, and ultimately
enzyme activity. Additionally, once the sample is stored in the laboratory, it is
recognized that repeated cycles of freeze-thaw can impact certain enzymes.
To further investigate these issues, we collected peripheral blood samples in
sodium heparinized tubes from five normal adult volunteers. PBLs were isolated
at blood draw (fresh) and 24, 48, 72 or 96 hours post-draw using a standard
dextran-gradient centrifugation method. The protein concentration and the activity
of each of eleven lysosomal enzymes (Arylsulfatase B, N-acetyl-galactosamine-6
sulfatase, N-acetyl-galactosaminidase, N-acetyl-glucosamine-6-sulfatase, Betahexosaminidase, Beta-mannosidase, Beta-glucuronidase, Beta-galactosidase,
Alpha-mannosidase, Alpha-iduronidase and Alpha-fucosidase) was measured in
each sample and compared to that from the fresh pellet. Enzyme measurements
were performed using 4 methylumbellierone (4MU) substrates with the exception
of ASRB which used a para nitrocatachol substrate. The total protein in each
lysate decreased 48 hours post-draw (60% reduction) and by 96 hours was only
10% of that in the fresh sample. Lysosomal enzyme activities also decreased after
48 hours (0-65%), even when the enzyme activity was corrected by the reduced
total amount of protein. By 96 hours post-draw the activity of several enzymes
was undetectable which could lead to a false positive result. Multiple cycles (5) of
freeze-thaw did not significantly impact enzyme activity values (0-35% reduction)
and, when limited to 5 cycles, would not appear to lead to a false positive enzyme
result.
Our data suggest that delayed isolation of PBLs results in a significant decrease
in total protein and can impact the measurement of a large number of lysosomal
enzymes. Diagnostic laboratories should continue to monitor the length of time
between blood draw and leukocyte isolation and, either reject delayed samples
or place caveats in reports describing the potential impact on enzyme activity.
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9:45 a.m. – 10:00 a.m.
CHROMOSOME SHATTERING: A LOOK AT CONSTITUTIONAL
CHROMOTHRIPSIS IN PATIENTS EXHIBITING MULTIPLE
COPY NUMBER CHANGES AND COMPLEX STRUCTURAL
REARRANGEMENTS REVEALED BY SNP MICROARRAY
April N. Harris; Rachel D. Burnside, PhD, FACMG
Chromothripsis is a recently described phenomenon whereby a single catastrophic
event shatters one or more chromosomes, resulting in multiple copy number
changes and/or complex structural rearrangements on one chromosome or
between multiple chromosomes. Non-homologous end joining of double strand
breaks is hypothesized as a mechanism for the repair of multiple breaks, although
the mechanism(s) which initiate these breaks is not clear. Originally described in
cancer, these highly complex chromosome rearrangements have recently been
demonstrated to also contribute to congenital anomalies constitutionally. The
phenomenon of constitutional chromothripsis is not often appreciated by routine
chromosome analysis, as copy number changes are often submicroscopic and
rearranged segments may not resemble known banding patterns.
Here we describe a series of individuals referred for microarray analysis
for clarification of results after structural rearrangements were observed by
chromosome analysis, each demonstrating constitutional chromothripsis by array.
The sizes of the copy number changes observed in these individuals ranged from
268 Kb to 16.31 Mb and a variety of structural rearrangements were observed,
including rings, insertions, and unbalanced translocations. While many of the
deletions and duplications include OMIM annotated genes which likely directly
affect the phenotype of each patient, it is also possible that position effects of
the rearranged segments may contribute to clinical presentations. Most complex
rearrangements occur de novo; however, follow up parental testing is needed to
confirm the origin and assess recurrence risk for these families.
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10:00 a.m. – 10:15 a.m.
DETECTING LOW LEVEL MOSAICISM OF TRISOMY 9 WITH
MICROARRAY ANALYSIS
Jennifer Crawford, CG(ASCP)CM; Shamsa Naqvi, CG(ASCP)CM; Amanda Fortier,
PhD; Debra Rita, MD; Jillene Kogan, MD, PhD
The application of SNP-based whole genome microarray testing is utilized for
detection of abnormal chromosomal copy number variations which include
micro-deletions and duplications, aneuploidy, unbalanced translocations, low
level mosaicism and long contiguous stretches of homozygosity. Some of these
pathogenic abnormalities are impossible to detect via conventional cytogenetics
and may be missed due to their small size, cryptic nature or whole chromosome
loss as a result of cultural artifact.
One such abnormality that may often be overlooked involves low grade
mosaicism in which the patient has multiple genetically distinct cell lines within
their genome. Here we discuss the prevalence of low level mosaicism and the
successes associated with detecting these additional cell lines using microarray
technology. Our laboratory has recently reported on three patient cases of mosaic
trisomy 9, which were not detected using conventional cytogenetics, utilizing
the AffymetrixÂ® CytoScanâ„¢ HD Microarray Assay. The patients presented
with a variety of physical characteristics and medical conditions which included:
developmental delay, dysmorphic facial features, poor feeding and abnormal
cry. All three cases had different levels of mosaicism. Only one case, which was
estimated at around 36% mosaic for trisomy 9, was prevalent enough for the
software settings to identify and flag the abnormality.
Thus, we were challenged with developing a method for the detection and
reporting of lower level mosaicism. Our laboratory employs the smooth signal
function, which identifies copy number variations, to detect low level mosaicism.
With the use of the heat map modifier function within the smooth signal track on
Affymetrix ChAS software, we have been able to detect mosaicism as low as 14%,
as reported in one of these cases. The heat map specifically uses varying color
schemes for optimum visualization of copy number variation, which allows for the
identification of very subtle changes in copy number. Our laboratory had success
using FISH on a direct and stimulated peripheral blood sample for verification of
a mosaic cell line to confirm the findings for all three patients.
The use of microarray technology for sensitive characterization of the human
genome has allowed for patients even with low level mosaicism to receive precise
diagnoses. These results benefit the patients as further diagnostic testing is
unnecessary; a treatment plan can be tailored to the diagnosis and the family
can be directed to the appropriate support group. These three cases reflect the
importance of proper analysis and optimization of the Affymetrix software tools
for identifying low level mosaicism.
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10:15 a.m. – 10:30 a.m.
QUANTITATIVE ANALYSIS OF SNRPN GENE METHYLATION BY
PYROSEQUENCING: AN ACCURATE METHOD FOR IDENTIFYING
MOSAICISM
Crystal J. Symsick Propes; Kevin M. Babson, BS; Julie R. Jones, PhD, FACMG
Angelman syndrome (AS) and Prader-Willi syndrome (PWS) are neurogenetic
disorders that result from defects in the q11-q13 imprinted region of chromosome
15. AS arises when only the paternal genes in this region are expressed due to
either a deletion involving the maternal chromosome 15 or to paternal uniparental
disomy (UPD) of chromosome 15. PWS occurs when only the maternal 15q11q13 region is inherited resulting from either a paternally derived deletion or
maternal UPD15. Methylation analysis of the SNRPN locus within this imprinted
region can be used to diagnostically test for and distinguish between these two
disorders. Previous studies in our laboratory have found pyrosequencing to be
a more sensitive and accurate method compared to methylation-specific PCR.
In the five years since the SNRPN pyrosequencing assay was validated and
implemented as a diagnostic test in our laboratory, 935 patient samples have
been analyzed. Of these, 50 were positive for PWS, 46 were AS positive, and 839
were normal. Methylation of the SNRPN locus by pyrosequencing is interpreted
as follows: 1-4% methylation is diagnostic for AS, 43-49% is considered normal,
and 94-99% is consistent with a diagnosis of PWS. Values outside of these ranges
may be indicative of mosaicism.
Patients who are mosaic for AS or PWS will often have a clinical presentation
that is very different from non-mosaic cases. Our analyses have identified two
mosaic PWS patients and eight patients mosaic for AS. Since methylation-specific
PCR is at best semi-quantitative, cases involving mosaicism may be missed by
this method. Pyrosequencing, however, is a truly quantitative technique which
allows for the accurate determination of mosaic PWS and AS patients. In addition
to diagnosing these mosaic cases, using a mathematical formula, we can also
determine the percent of mosaicism present in a patient based on the percent
methylation detected at the SNRPN locus. This information can be useful for
clinicians in understanding the atypical phenotype that may be present in their
patient.
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Student Research Award Winner
10:30 a.m. – 10:40 a.m.
A COMBINATION OF ZYFLAMEND AND VEMURAFENIB (PLX4032)
MAY HAVE A SYNERGISTIC EFFECT AGAINST MELANOMA CELL
PROLIFERATION
Jacob Yo; Peter C. Hu, PhD, MS, MLS(ASCP)CM, CGCMMBCM; Elizabeth A.
Grimm, PhD
Cutaneous melanoma is one of the most aggressive forms of skin cancer. In the
United States, its incidence rate increased on average 2.7% annually between
1985 and 2010.1 Targeted molecular chemotherapy drugs, such as BRAF and
MEK inhibitors, significantly improve survival and quality of life among late-stage
patients; but oftentimes de novo or acquired resistance renders these therapies
ineffective.2 Therefore, ongoing search for novel therapies for advanced
melanoma is imperative.
In laboratory studies, we found that Zyflamend (New Chapter, Inc., Brattleboro,
VT), a promising anticancer multi-herbal extract, inhibits melanoma proliferation
by regulating the autophagy-apoptosis switch.3 Based on our preliminary
findings, we speculated that a combination of Zyflamend and vemurafenib, a
mutant BRAF inhibitor, may synergistically inhibit melanoma proliferation. To test
this hypothesis, we prepared 3 concentrations of Zyflamend (low [LZ], 0.001
µL/mL; medium [MZ], 0.025 µL/mL; high [HZ], 0.050 µL/mL) and vemurafenib
(low [LV], 0.1 µM; medium [MV], 1.0 µM; high [HV], 5.0 µM) each. We
prepared 4 combinations of the agents—LZLV, LZMV, MZMV, MZLV. HZ and HV
concentrations were not used in combination treatments due to their individual
extensive toxicity. A375 (BRAF mutant) and MeWo (BRAF wild type) melanoma
cells and normal BJ fibroblasts were diluted to 50,000 cells/mL, seeded in 16-well
plates, and incubated with the treatments for 48 hours. We included controls of
50,000 cells/mL, media with DMSO (HV concentration), and media with DMSO
and olive oil (HZ concentration) in each experiment.
All media solutions were prepared with Dulbecco’s modified Eagle’s medium
supplemented with 5% fetal bovine serum. The Cellometer Auto T4 (Nexcelom
Bioscience, Lawrence, MA) and trypan blue was used for cell counting. Each
preparation was done in duplicate, and each experiment was conducted in
duplicate at separate times. We observed that the MZLV combination had the
greatest synergistic effect against our melanoma cell lines: this combination
resulted in a 54% reduction in A375 cells compared with a predicted reduction of
22%, while having less than 20% reduction in BJ fibroblasts. The proliferation of
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BJ fibroblasts incubated with HZ (60%) was dramatically lower than that incubated
with LZ (92%); but surprisingly, vemurafenib appeared to rescue partially this
inhibition. We also observed this phenomenon with MeWo Cells.
These findings with MeWo cells are in agreement with BRAF wild-type studies
showing BRAF inhibitor resistance via RAF-independent ERK signaling or RAF
dimerization.4, 5 In our study, we found that a combination of Zyflamend and
vemurafenib may be a novel therapy for late-stage cutaneous melanoma. Further
studies should focus on elucidating the mechanism behind this dosage-dependent
synergistic effect on BRAF-mutant cells and the partial rescue of Zyflamend’s toxic
effect on normal human fibroblasts.
1. Howlader N, Noone AM, Krapcho M, et al., eds. SEER Cancer Statistics Review,
1975-2010, National Cancer Institute. Bethesda, MD, http://seer.cancer.gov/
csr/1975_2010/, based on November 2012 SEER data submission, posted to
the SEER web site, April 2013.
2. Villanueva J, Vultur A, and Herlyn M. Resistance to BRAF Inhibitors: Unraveling
Mechanisms and Future Treatment Options. Cancer Res. 2011(71):
7137-7140.
3. Ekmekcioglu S, Chattopadhyay C, Akar U, Gabisi A, Newman Jr RA, Grimm
EA. Zyflamend mediates therapeutic induction of autophagy to apoptosis in
melanoma cells. Nutrition and Cancer. 2011; 63(6): 940-949.
4. Johannessen CM, Boehm JS, Kim SY, et al. COT drives resistance to RAF
inhibition through MAP kinase pathway reactivation. Nature. 2010: 468:
968-974.
5. Poulikakos PI, Zhang C, Bollag G, Shokat K, and Rosen N. RAF inhibitors
transactivate RAF dimers and ERK signalling in cells with wild-type BRAF.
Nature. 2010: 464: 427-430.
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11:30 a.m. – 12:20 p.m.
Informed Consent for Whole Genome Sequencing
Katherine S. Hunt, PhD, MS, CGC, Assistant Professor in Medicine,
Genetic Counselor, Mayo Clinic, Scottsdale, Ariz.
Whole genome sequencing is an exciting new technology available to patients.
Along with the promises of the technology, come multiple complexities and
uncertainties. One of the greatest challenges of genome sequencing is how to
properly consent patients prior to sequencing. The presenter will describe the
consenting challenges for whole genome sequencing and offer several solutions
for how to tackle these challenges and more effectively consent patients.
NOTES
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NOTES
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1:45 p.m. – 2:35 p.m.
Miscarriages, Cell Morphology & Villi Formation:
Detecting High Abnormality Percentages in POCs
Philip J. Hardy, M. Clin Cyto, B.Bus, Laboratory Manager,
Cyto Labs Pty. Ltd., Perth, Western Australia
With many laboratories struggling to achieve success and high abnormalities
in POCs, the pressure from new technologies presents an argument for
supplementing and replacing conventional cytogenetics. However, with refined
techniques and better knowledge it is possible to resolve these issues without the
cost.
NOTES
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2:35 p.m. – 3:25 p.m.
Targeted Therapies for Solid Tumors
& Companion Diagnostics
Dianne Keen-Kim, PhD, FACMG, Executive Director,
Cytogenetics,Genoptix Medical Laboratory, Carlsbad, Calif.
Genetic alterations within cellular proliferation or survival pathways are the
most common cause of cancer. New therapeutic interventions specifically target
these cellular pathways making them less toxic, better tolerated and more
effective than traditional cytotoxic chemotherapies. These targeted therapies
were first developed for cancers of the blood and bone marrow due to the easy
accessibility of neoplastic cells. However, tumors of the solid tissues are both
the most frequently diagnosed and deadly neoplasms. The presenter will discuss
the development of targeted therapies, companion molecular diagnostics and
efficient assay designs.
NOTES
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3:45 p.m. – 4:35 p.m.
The Integration of Clinical & Laboratory Elements in
Diagnosing & Managing Genetic Disorders
Kara Goodin, MD, Clinical Geneticist, Assistant Professor of Pediatrics,
University of Louisville School of Medicine, Louisville, Ky.
The presenter will review clinical cases that demonstrate the value of both
clinical and laboratory aspects of genetic disorders. The cases will highlight the
importance of both factors, particularly the interaction between the clinical and
laboratory facets, in diagnosing and treating affected individuals. Combining the
components allows for a more complete view of the phenotypes as well as an
evolution and expansion of our understanding of the disorders.
NOTES
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4:35 p.m. – 5:25 p.m.
Overview of the New AMA Molecular Pathology CPT Codes
V.M. Pratt, PhD, FACMG, Director, Pharmacogenetics Laboratory,
Indiana University School of Medicine, Indianapolis, Ind.
With the completion of the Human Genome Project and increased understanding
of the genes involved in human disease and cancer biology, clinical molecular
testing has grown by leaps and bounds. New biomarkers were brought to market
and largely reimbursed by using a molecular current procedural terminology
(CPT) code stacking system where each step of the process had its own CPT
code. At the request of payers, the American Medical Association (AMA)
developed a new CPT coding system based on a white paper proposed by the
Association of Molecular Pathology (AMP). Since there are well over a thousand
different molecular pathology tests, a tier system was created. Tier 1, where each
individual test gets a unique CPT code, was created to encompass the more
commonly ordered category 1 tests throughout the country. Tier 2, where there
are nine levels of complexity, was created to address the more rarely ordered
category 1 tests. This session will review the new CPT coding system.
NOTES
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Poster Abstract Presentations
Friday, June 13, 2014
9:50 a.m. – 10:20 a.m. (Even Poster Numbers)
3:20 p.m. – 4:00 p.m. (Odd Poster Numbers)
Presenters who are assigned even numbers are requested to stand by
their posters on Friday, June 13, from 9:50 a.m. – 10:20 a.m., and
presenters who are assigned odd numbers are requested to stand
by their posters on Friday, June 13, from 3:20 p.m. – 4:00 p.m. to
respond to attendee questions or for further discussion.
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Poster Abstracts
1
CHROMOSOMAL ABNORMALITIES IN GREAT APES REFLECT THEIR
COMMON EVOLUTIONTARY ORIGINS WITH HUMANS
Marlys L. Houck
Abnormalities in great apes similar to those observed in humansCytogenetic
studies of rare and endangered species can contribute to a better understanding
of the taxonomic and evolutionary relationships among species and provide
information crucial for enhancing captive breeding success. Documentation of
chromosomal abnormalities including aneuploidy and structural rearrangements
or deletions can identify individuals which should be excluded from captive
breeding programs. The great apes share a common ancestor with the lesser
apes (gibbons and siamangs). These two lineages diverged approximately 20
million years ago. Since the divergence, the lineages leading to modern humans,
chimpanzees, bonobos and orangutans have evolved. All species of great
apes have 2n=48, except, of course, humans (2n=46). Risk of specific types of
chromosomal rearrangements has been inherited from common ancestors in
these lineages. Thus, as trisomy 21 and 18 are notable anomalies in humans,
trisomy for the homologous elements in bonobos and other great apes has also
been observed.
Our lab has analyzed the karyotypes of over 200 great apes with several
significant findings including trisomy 18 in a newborn bonobo, premature sister
chromatid separation in cells of a young gorilla indicating possible Cornelia
de Lange Syndrome, and a de novo deletion in a male gorilla with postnatal
growth retardation and dysmorphic features. In addition, karyotype analyses on a
family of gorillas with a history of multiple spontaneous abortions and congenital
malformations indicated chromosome anomalies in some individuals in the
pedigree. Future investigations in great apes may focus on genomic disease risk,
such as occur as a result of tandem duplications and copy number variation as
has been observed in humans.
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4
A NOVEL VARIANT T(1;22) TRANSLOCATION - INS(22;1)
(Q13;P13P31) - IN A CHILD WITH ACUTE MEGAKARYOBLASTIC
LEUKEMIA
Ilham Atir; Svetlana Kleyman; May Wong; Ana Revelo-Sanchez; Julia T. Geyer;
Alexander Aledo; Susan Mathew
Acute megakaryoblastic leukemia (AMKL) is a de novo acute myeloid leukemia (AML)
where at least 50% of the blasts are of megakaryocytic lineage. AMKL is associated
with the t(1;22) translocation involving MKL1 and RBM15 genes. AMKL is most
commonly seen in infants during the first six months of life and young children below
the ages of three years with a female preponderance. Most of the patients present
with hepatosplenomegaly, anemia, bone marrow fibrosis, and moderately elevated
white blood cells. About 40 cases of AML with a t(1;22) translocation have been
described to date. We present a three month old female with AMKL with a variant
t(1;22) translocation. The patient presented with two weeks history of progressive
abdominal distension, intermittent vomiting and fever. Clinical examination showed
distended abdomen, massive hepatosplenomegaly and ascites. Initial CBC showed
HGB 5.7 g/dL, WBC 35.9 K/uL, and platelet count 86 K/uL. A differential diagnosis
of hemophagocytic lymphohistiocyosis was made. Bone marrow biopsy was very
small, normocellular, and showed maturing trilineage hematopoiesis without an
increase in blasts. Flow cytometry and immunohistochemistry studies showed no
evidence of leukemia/lymphoma. Cytogenetic analysis on the bone marrow showed
a variant t(1;22) translocation in six of 20 metaphase cells. Fluorescence in situ
hybridization (FISH) using the whole chromosome painting probes for chromosomes
1 and 22 showed an insertion of p13p31 bands of chromosome 1 onto the long
arm of chromosome 22, resulting in a variant t(1;22) translocation. The karyotype
of the patient was established as 46,XX,ins(22;1)(q13;p13p31)[6]/46,XX[14]. FISH
studies to establish the involvement of MKL1 and RBM15 genes are in progress.
Liver biopsy showed 90% fibrosis and the presence of numerous aggregates of
large, markedly atypical megakaryocytes. These cells expressed CD45, CD42b,
CD61, and CD117. A diagnosis of acute megakaryoblastic leukemia with extensive
involvement was made. Subsequently another bone marrow biopsy was performed
and showed marked bone marrow fibrosis and numerous megakaryoblasts. The
patient was treated with modified cytarabine/daunorubicin/etoposide (ADE)
chemotherapy regimen and she also received defibrotide. Following first month
of treatment, repeat biopsies of bone marrow and liver were both negative for
evidence of residual leukemia. The patient remains in complete remission. This case
report clearly emphasizes the importance of cytogenetic studies in the diagnosis,
prognosis, and treatment options in patients with acute leukemia, since the bone
marrow biopsy was non-diagnostic. The diagnosis of AMKL was suspected because
of the cytogenetic results and then confirmed by the liver biopsy. In some cases,
AMKL may present initially with <20% blasts in the bone marrow, thus cytogenetics
is required to make the correct diagnosis.
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5
IDENTIFICATION OF METHYLATED INK4A CO-EXISTING COPY
NUMBER VARIATIONS AND LOSS OF HETEROZYGOSITY IN
CIRCULATING CELL-FREE TUMOR DNA FROM HEPATOCELLULAR
CARCINOMA PATIENTS
Karam Hadidi; Gengming Huang; Peter Hu; Jianli Dong
BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most prevalent
and lethal cancers worldwide. When HCC is diagnosed at an early stage, the
patient can be cured by surgical resection, liver transplantation, or percutaneous
radiofrequency ablation. However, larger and advanced-stage tumors have poor
prognosis. Therefore, the need to improve early diagnosis of HCC is urgent.
Imaging scan and alpha-fetoprotein (AFP) are currently used to screen and
diagnose HCC, but new genetic and epigenetic markers are being discovered
to improve sensitivity and specificity. Studies have shown that the inhibitor of
cyclin-dependent kinase (CDK) 4 gene (INK4A) is inactivated by methylation in
70-80% of HCC liver tissues, and at various frequencies in the circulating cellfree DNA (cfDNA) of HCC patients. The present study aimed to assess whole
genome copy number variants (CNV) and copy-neutral loss of heterozygosity
(LOH) using circulating cfDNA, and identify recurrent genomic changes that
may modify the activity of INK4A methylation and serve as diagnostic markers
of HCC. METHODS: Serum and peripheral blood lymphocytes (PBLs) from cell
pellet were collected from 106 subjects with an AFP value of at least 100 Âµg/L.
Circulating cfDNA was purified from all serum samples and underwent bisulfite
conversion, PCR of the INK4A promoter, and pyrosequencing. Samples with >15%
methylation of the INK4A promoter were selected for SNP chromosome microarray
using CytoScan (Affymetrix). Results of INK4A methylation, CNV and LOH changes
in serum and corresponding PBL will be analyzed and compared. RESULTS:
Pyrosequencing of the corresponding PLB samples has revealed that all have have
INK4A promoter methylation below 5%. So far, 7 serum samples from 5 specimens
have been identified with >15% INK4A methylation in cfDNA. Several CNVs and
LOH have been identified by whole genome chromosome microarray analysis.
DISCUSSION: Once the microarray data is generated for all remaining serum and
PLB samples, HCC specific DNA aberrations will be identified by comparison of
the cfDNA results to the PLB DNA results. CNVs that occur in the cfDNA should
be characteristic of both the HCC tumor and the normal genome, while the blood
pellet should only represent genomic DNA, due to the concentration of tumor DNA
in the blood pellet samples being below the microarray threshold of sensitivity. In
the future, these identified CNVs may be used to non-invasively diagnose earlystage HCC, as opposed to waiting for a liver nodule to reach a certain size and
possibly metastasize.
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7
INVESTIGATION OF A RARE LIVEBORN TRIPLOID
April N. Harris, BS; Howell Dobbins, BS; Tatjana Gibson, BS; Rachel D.
Burnside, PhD, FACMG; Peter Papenhausen, PhD, FACMG
Triploidy is a rare condition where an extra haploid set of chromosomes is either
paternally or maternally inherited. The extra haploid dosage gain results in cells
having 69 chromosomes, and the majority of triploid pregnancies result in fetal
demise. The small percentage of instances in which a baby is born alive, they
usually only survive for a few hours. Among these, infants present with IUGR,
neural tube defects, heart defects, relative macrocephaly, and other congenital
anomalies. Most liveborn infants show some percentage of mosaicism for a diploid
cell line, which helps explain survival beyond the second trimester of gestation. It is
extremely rare that a non-mosaic triploid newborn is born alive. Here we present a
neonate who was born prematurely at 32 weeks presenting with Tetralogy of Fallot,
IUGR, small mouth with retrognathia and other congenital anomalies. Cytogenetic
testing was ordered, including DiGeorge/VCF FISH, chromosome analysis, and
SNP microarray. While microarray and chromosome analysis from peripheral blood
showed non-mosaic triploidy, FISH results indicated possible mosaicism with a
diploid cell line. SNP array and FISH techniques are approximately equally sensitive
for single chromosome mosaicism, however for triploidy, array is not as sensitive
as FISH due to normalization of copy number in the array software. Furthermore,
there is evidence that triploid cells may proliferate faster than diploid cells in the
blood, but in other tissues like skin, triploid cells may be selected against. In order
to investigate the possibility that our patient is actually mosaic for diploidy, buccal
smear FISH analysis is underway. It is known that the imbalance in imprinting due
to an extra set of chromosomes differentially affects placental growth and fetal
development. Diandric triploid (extra paternal set) pregnancies are associated with
placental overgrowth/cystic villi with little fetal development while digynic triploid
(extra maternal set) are associated with better fetal development and placental
growth restriction. Therefore, most triploid pregnancies that persist to the third
trimester are digynic and most that result in a liveborn infant are mosaic.
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8
DETECTING t(3;5)(q25;q35) IN ACUTE MYELOID LEUKEMIA WITH
MYELOSYSPLASTIC CHANGES
Melinda A. Claydon, BA; E. Richardson, BS; A. W. Block, PhD; S. N. J, Sait,
PhD
We present 3 cases with a t(3;5)(q25;q35). The t(3;5)(q25;q35)is a rare
cytogenetic finding, reported in œacute myeloid leukemia with myelodysplasticrelated change as defined by the 2008 WHO Classification of Tumours of
Haematopoietic and Lymphoid Tissues. The t(3;5)(q25;q35) is associated with
multilineage dysplasia and a younger age at presentation. Although t(3;5)
(q25;q35) is most often reported as a sole abnormality, trisomy 8 has also been
reported as a secondary change. AML with inv(3)/t(3;3) also shows evidence of
multilineage dysplasia however, the breakpoint of chromosome 3 involves the EVI1
locus which is not rearranged in the t(3;5)(q25;q35). Acute myeloid leukemia with
myelodysplastic features is reported to have a poor prognosis and a lower rate of
complete remission than other AML subtypes. Fluorescence in situ hybridization
(FISH) studies and molecular studies have shown that the t(3;5)(q25;q35) results
in the fusion of the nucleophosmin (NPM) gene on chromosome 5 and the MDS/
myeloid leukemia factor 1 (MLF1) gene on chromosome 3. Using a combination
of conventional cytogenetic analysis and FISH, we identified t(3;5)(q25;q35) in
the bone marrow samples from 3 patients with a diagnosis of acute myeloid
leukemia with myelodysplasia-related changes between 2010 and 2013. All
patients are female with a median age of 38 years. FISH studies showed that the
NPM/MLF1 fusion was positive and that EVI1 was not rearranged. Conventional
karyotypes showed no other clonal abnormalities. One patient has the variant
constitutional inv(9)(p11.2q13). Because the t(3;5)(q25;q35) shows evidence of
multilineage dysplasia similar to the separately WHO defined 3q abnormality
entities, which have a poor prognosis, it is important to accurately define these
abnormalities so that appropriate treatment can be started in a timely manner.
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9
UNEXPECTED LACK OF A RUSSELL-SILVER PHENOTYPE IN A
FAMILY WITH DUPLICATION OF THE 11P15.5 IMPRINTED REGION
Howell Dobbins, BS; Shobana Kubendran, MBBS, MS,CGC; Brook Rush, MS,
CGC; Stuart Schwartz, PhD, FACMG; Rachel D. Burnside, PhD, FACMG
Genomic imprinting is a mechanism to control gene expression by methylation
of DNA that is heritable. The result is parent-of-origin silencing of genes and
monoallelic expression, which deviates from the typical biallelic expression.
There is a cluster of genes located at 11p15.5 which are imprinted,and defects
in imprinting resulting from sequence mutations or copy number changes in
the imprinted region can be associated with Beckwith-Wiedemann Syndrome
(BWS) or Russel-Silver Syndrome (RSS), depending on the parent-of-origin of the
defect. Features of BWS include overgrowth, abdominal wall defects including
omphalocele, and macroglossia, while RSS features include short stature,
cafÃ© au lait spots, and a triangular shaped face. We present a male newborn
who was referred for microarray analysis due to suspicion of BWS. Results
showed three non-contiguous duplications in 11p15.5->p15.4, specifically at
[hg19] 11p15.5(558,632-766,250), 11p15.5p15.4(1,291,555-2,820,200) and
11p15.4(3,103, 972-3,413,174). Methylation testing confirmed a diagnosis of
Beckwith-Wiedemann syndrome in the proband. Familial follow up analyses
showed that the father and paternal grandmother also carry the duplications.
Interestingly, the father does not present with the expected RSS phenotype,
nor does the grandmother present with a phenotype. Further pedigree review
indicates that a cousin of the proband also shows features of BWS, suggesting
that the cousin and paternal uncle may also be duplication carriers, although the
paternal uncle does not present with features of RSS either.
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10
A NOVEL T(2;14)(P11.2;Q32) TRANSLOCATION INVOLVING THE
REARRANGEMENTS OF IMMUNOGLOBULIN HEAVY CHAIN
(IGH@) AND IMMUNOGLOBULIN KAPPA LIGHT CHAIN (IGÎš) IN
B-CELL LYMPHOMA
Jennifer Hwang; Tatyana Shklovsky; Parmeswaran Ganeshan;
Govinda Rama Hancock; Patricia Massino; Joseph Gillis; Swarna Gogineni;
Susan Mathew, PhD, FACMG; Shivakumar Subramaniyam, PhD, FACMG
Chromosomal translocations associated with Immunoglobulin (Ig) loci (IGH@,
IGÎº, IGÎ») are commonly observed in B-cell lymphoma and mature B-cell
neoplasm. The most commonly rearranged genes with IGH@ include: BCL1,
BCL2, BCL6, and c-MYC. However, the simultaneous occurrence of Ig heavy chain
and Ig light chain gene rearrangements in the same patient is very uncommon.
We describe two cases of B-cell lymphoma with a t(2;14)(p11.2;q32) translocation
involving IGH@ and IGÎº gene rearrangements. Patient 1 is a 60 year old male
who presented with a history of fever, night sweats, and a right supraclavicular
lymphadenopathy in 2006. Immuno-histopathological studies on the excisional
biopsy of the lymph node established a diagnosis of follicular lymphoma grade
1-2. Cytogenetic analysis on the lymph node identified the following karyotype:
46,XY,t(2;14)(p11.2;q32),add(12)(q22),t(14;18) (q32;q21.3),ins(17;?)(q21;?)[16]/
46,XY[4]. Fluorescence in situ hybridization (FISH) analysis using the LSI IGH@BCL2 dual color dual fusion probes and LSI IGÎº (2p11.2) dual color break apart
probe showed rearrangement of IGH@ and IGÎº genes. Both IGH@ alleles
were rearranged; one with BCL2 (90%) resulting in the t(14;18) translocation and
the other allele with IGÎº (92%) resulting in the t(2;14) translocation. In 2010,
the patient was treated with six cycles of R-CHOP chemotherapy and achieved
remission in 2011. A follow up PET/CT scan in 2013 showed disease recurrence.
The patient was offered Rituximab therapy, but the patient declined. Patient 2 is
an 80 year old woman presented with a history of generalized weakness and
left anterior flank pain for one month. Initial CT and MRI scans identified left
renal mass. Studies on the left renal biopsy sample established a diagnosis of
diffuse large B cell lymphoma. Cytogenetic analysis on the bone marrow sample
showed the following karyotype: 48,XX,+X,der(2)t(2;14)(p11.2;q32),+add(3)(q12),
-4,del(6)(q15),der(14)add(14)(p13)t(2;14)(p11.2;q32), +18,-21,-22,+1~4mar[cp6]
/46,XX[14]. FISH assay showed both IGÎº and IGH@ gene rearrangements. The
patient declined chemotherapy and is being monitored. The t(2;14)(p11.2;q32)
translocation involving IGH@ and IGÎº gene rearrangements observed in
our patients appears to be a recurrent translocation in B-cell lymphoma. The
involvement of Ig heavy and Ig light chain genes in the same sample is very rare.
It is difficult to establish whether the t(2;14)(p11.2;q32) is a primary or secondary
event in the pathogenesis of B-cell lymphoma. Molecular studies to confirm the
fusion of IGH@ and IGÎº in these cases will be important to understand the
molecular mechanisms in the pathogenesis of B-cell lymphoma.
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11
MOSAIC 22Q13.3 DELETIONS: VALUE OF CONCURRENT
KARYOTYPE, FISH AND ARRAY CGH STUDIES TO PROVIDE THE
PROPER GENETIC DIAGNOSIS
Lisa Warren, BS, CG(ASCP); Julie Joyce, BA, MB(ASCP); Stephanie Fiedler, MS,
MB(ASCP); Trish Stanley, BA, CG(ASCP); Mike Tiller, BS, CG(ASCP);
Norwood Tosatto, CG(ASCP); Lei Zhang, PhD; Linda Cooley, MD, MBA;
Elena Repnikova, PhD
Mosaicism is defined as the presence of more than one cell line in an individual.
Mosaicism has been implicated as a cause of developmental delay, dysmorphic
features, and multiple congenital anomalies. Mosaicism may be missed
by conventional cytogenetics analysis if the abnormality is not present in T
lymphocyte blood cells, which are stimulated by the commonly used mitogen,
Phytohaemagglutinin (PHA). Additionally, the mosaic cell line may fail to respond
to the mitogen and be masked by a high percentage of cells with a normal
karyotype. The routine chromosomal analysis in a constitutional chromosomal
study is 5 cells, which increases the probability of overlooking a mosaic structural
chromosomal abnormality in the analyzed cells. Fluorescent in situ hybridization
(FISH) analysis, another widely used clinical cytogenetic technique that allows
detection of significantly smaller imbalances may be performed when mosaicism
is suspected, since it confers the ability to scan hundreds of non-cultured and
cultured blood cells. However, the diagnosis must be suspected, to choose the
proper probe. Microarray-based comparative genomic hybridization (aCGH)
from whole blood or tissue-specific samples provides a significantly higher
resolution analysis of the genome, thus is an alternative to standard chromosome
analysis. Multiple studies have shown that aCGH may be a better technique
for detection of low-level mosaic structural chromosomal changes with negative
conventional chromosome studies. However, the lower limit for mosaicism
detection by various platforms is generally not less than 30%. Our experience
suggests that for certain cases, use of all three techniques optimizes the detection
of low-level structural chromosomal changes. We studied three unrelated
clinical cases with various clinical presentations for which karyotype analysis,
FISH or array CGH were ordered in attempt to find the etiology of congenital
malformations. Karyotype analysis included standard GTG-banding of PHAstimulated blood lymphocytes. FISH analysis is performed by scoring metaphase
and interphase cells from unstimulated or PHA-stimulated T-cells. Microarray
analysis is performed using Oxford Gene Technologys (OGT) whole genome
oligonucleotide 4x180k ISCA v2 microarray chip (Oxfordshire, UK) manufactured
by Agilent Technologies (Santa Clara, CA) with four individual sections each
containing ~180,000 oligonucleotide probes and backbone probe density of
25 kb (23.8 kb backbone, 4.44 kb disease region, 19.97Â±10 kb genes with an
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average of 6 probes per gene). Data analysis was performed with Cytogenomics
software (Agilent Technologies) using a log 2 ratio threshold of Â±0.25 and a
4 probe minimum. The first patient was a 2 year old male who presented with
global developmental delay and features of fetal alcohol syndrome. Chromosome
analysis initially yielded a normal 46,XY karyotype, while microarray analysis
revealed an approximate 7.3 Mb mosaic terminal deletion within chromosome
bands 22q13.2q13.33. The deleted region contained 50 genes including
SHANK3. Careful re-examination of this patients chromosomes revealed 35%
of the cells contained a deletion of 22q13.3. FISH using a 22q telomere probe
confirmed a single copy of chromosome 22q telomere in 39.5% of nuclei and
45% of metaphase cells. FISH using a N85A3 (22q13.3) probe showed a single
copy of N85A3 in 51% of nuclei and 40% of metaphase cells, thus indicating a
terminal 22q deletion. The second male patient presented at 4 days of age with
dysmorphic facial features and seizures. Microarray analysis was negative for
DNA copy number variants. Chromosome analysis showed a low-level mosaic
deletion of 22q13.3 region in 6% (2/30) of examined metaphase cells. FISH
using a 22q telomere probe showed a normal signal pattern in 200 nuclei
and 100 metaphase cells. FISH using a N85A3 (22q13.3) probe showed a
single copy of N85A3 in 16.5% of nuclei and 10% of metaphase cells, thus
suggesting an interstitial deletion in this patient. The third patient was a 33 6/7
week gestation female infant with prenatally diagnosed hydrops and tetralogy
of Fallot pulmonary atresia. The infant had pleural effusion drained at birth
and then developed pneumothoraces. FISH analysis, ordered on fetal pleural
fluid for 22q11.2 microdeletion syndrome revealed a normal result, however,
a concurrently used control probe (N85A3) for 22q13.3 showed 16% to 20%
mosaicism for a single copy of 22q13.3. Chromosome and FISH analyses
performed at birth confirmed the above finding and revealed the deletion in 29%
of metaphase cells and 32.5% of nuclei. Microarray analysis performed on whole
blood did not confirm the deletion identified by FISH and chromosome studies.
All three described cases were determined to have a well documented diagnosis
of 22q13.3 deletion syndrome, also known as Phelan-McDermid syndrome.
Major phenotypic features of patients with non-mosaic 22q13.3 deletion include
neonatal hypotonia, global developmental delay, absent to severely delayed
speech, normal to accelerated growth, large fleshy hands, dysplastic toenails, and
decreased perspiration that results in a tendency to overheat. Phelan-McDermid
syndrome can result from a de novo or inherited chromosome abnormality, which
can be determined by parental chromosome analysis. Parental studies were not
performed in any of the examined cases to determine the inheritance pattern of
the 22q13.3 deletion. Patients with mosaic 22q13.3 deletion are not frequently
described in the literature. The patients examined in our study revealed some but
not all major phenotypic features of the 22q13.3 deletion syndrome. Deletion of
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the 22q13.3 region with haploinsufficiency of the SHANK3 gene is sufficient to
cause the described phenotypes including the neurological symptoms (as seen in
patients one and two). The level of mosaicism detected in the blood may not reflect
the level of mosaicism in other tissues. The age of the patient at diagnosis and
the level of mosaicism in various tissues likely influence the phenotypic features
of Phelan-McDermid syndrome. These factors make the clinical diagnosis of
mosaic Phelan-McDermid syndrome challenging, and thus laboratory diagnosis
all the more important. Our study illustrates that the combination of traditional
karyotyping, FISH and aCGH can significantly improve the detection of low-level
mosaicism for chromosomal aberrations. Without careful and attentive analysis of
the chromosomes, FISH and array results, mosaicism may be missed with the use
of just a single technique, e.g., aCGH. Our work also emphasizes the importance
of using more than one technique to refine the aberration and provide a more
precise definition of such on the molecular level.
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15
SIMULTANEOUS OCCURRENCE OF t(9;22)(q34;q11.2)
AND JAK2V617F MUTATION IN TWO PATIENTS WITH
MYELOPROLIFERATIVE NEOPLASMS
Hin Ting V. Wong; Carline Joseph; Linda Dean; Sheryl Asplund, MD;
Linda M. Pasztor, PhD, FACMG
The 2008 revision of the WHO classified MPNs into two categories, BCR/ABL1
positive CML and Ph-negative MPNs. The latter includes polycythemia vera,
essential thrombocythemia, and myelofibrosis. JAK2V617F mutation is specific
to Ph-negative MPNs, occurring in more than 95% of polycythemia vera and
approximately 50% of essential thrombocythemia and primary myelofibrosis
cases. Very few cases have been reported with the coexistence of BCL/ABL1
fusion and JAK2V617F mutation. Among these reports, the majority of patients
either had a preexisting BCR/ABL1-positive CML and developed JAK2V617F
mutation while undergoing treatment or vice versa. By contrast, a small number
of patients showed the apparently simultaneous occurrence of both JAK2V617F
and the t(9;22) rearrangement exhibiting both phenotypes by morphology.
We present two rare MPN cases that were concurrently positive for both the
JAK2V617 mutation and t(9;22). The first case was an 80-year-old female with
polycythemia vera and CML showing two independent clones “ trisomy 8 and
t(9;22) by conventional cytogenetic analysis. The second case was an 84-year-old
female with myelofibrosis and CML showing two unrelated clones “ an interstitial
deletion in the long arm of chromosome 13 and t(9;22). Both showed BCR/ABL
fusion by FISH. Our studies underscore the importance of correlating the clinical,
histopathology, molecular, FISH and conventional cytogenetic analyses for this
exceptional entity.
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16
UTILITY OF SNP MICRO ARRAY IN DELINEATING CLONAL
EVOLUTION
Tatjana Gibson; Peter Papenhausen; Rachel D. Burnside; Lynn Moscinski
SMyelodysplastic Syndrome (MDS) often takes a turn for the worse and begins
to transform into acute myeloid leukemia (AML) in conjunction with karyotypic
progression. An 82yo female patient suffering from low grade MDS that had
advanced to intermediate grade, with 6% blast cells, had a chromosome analysis
and MDS panel Fluorescence in situ hybridization (FISH) performed from a bone
marrow sample. The chromosome analysis showed the common macrocytic
anemia related deletion of 5q and an apparently balanced insertion of 2p21p13
into 15q22 in all cells. Half of the metaphases contained two copies of the
derivative 15 without a normal 15. Interestingly, the two derivative 15s showed
different satellites, but were otherwise identical. This suggests that the subclone
evolved through copy-neutral loss of heterozygosity (CN-LOH), a mechanism
for most mutation driven acquired CN-LOH. Since this evidence of mitotic
recombination can be detected by microarray analysis along with potential gene
involvement of the duplicated insertion, the SNP microarray was pursued. This
analysis revealed the anticipated CN-LOH which was initiated from a 15q13
site and included about 26% of the DNA while the 5q deletion was present in
about 90%. The smaller ratio of the subclone dosage in relation to the original
clone, as compared to the 50:50 chromosome analysis ratio, indicates that the
subclone is dividing faster, as can be expected. The 2p segment that was inserted
into 15q showed unexpected deletions on both sides of the segment inserted
into 15, as well as the gain of the region due to the doubled derivative. On the
distal side of the duplicated segment the ASXL2 gene was truncated, suggesting
oncogenic involvement in fusion with a 15q gene. Unfortunately, lacking a 15q
dosage change, there was no clue to the identity of the reciprocal gene. Since
the PML gene locus is very close to the insertion site, targeted FISH was run, but
failed to split the signal. Whatever the identity of the reciprocal gene, the fusion
appears to have offered significant selective advantage to the subclone. ASXL
family members (1,2 and 3) are involved in transcriptional regulation. Mutations
of ASXL2 occur in prostate cancer, pancreatic cancer and breast cancer. EPC1ASXL2 gene fusion occurs in adult T-cell leukaemia/lymphoma. The prognosis of
myeloid malignancies with misregulating truncation mutations of ASXL1 is poor,
but less is known about ASXL2. The subclone is then likely to be associated with
high risk of transforming into Acute Myeloid Leukemia (AML) and did rise with
the patient blast cell population. Further FISH analysis or sequencing would be
needed to identify the partner gene to fully understand the pathology.
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18
HOMOZYGOUS DELETION OF TEL (ETV6) IN CHILDHOOD ACUTE
LYMPHOBLASTIC LEUKEMIA (ALL): PROGNOSTIC IMPLICATIONS
Christina Mendiola, BS, CG(ASCP); Veronica Ortega, BA, CG(ASCP);
Juana Rodriguez, BS, CG(ASCP); Gopalrao Velagaleti, PhD
The fusion of TEL (ETV6) and AML1 (RUNX1) gene regions resulting from the
translocation of chromosomes 12 and 21, t(12;21), is the most common genetic
abnormality observed in childhood ALL occurring in about 25% of patients.
Studies showed that the neoplasm in these patients persists not by the initial
fusion event involving these gene regions, but in most cases by a secondary
or subsequent leukemogenic event involving deletion of the TEL gene region
on the non-rearranged TEL allele. As these rearrangements usually remain
undetected by conventional cytogenetics, it is readily observed by fluorescence
in situ hybridization (FISH) studies. Although other variant patterns have been
reported by FISH including extra copies of AML1 gene region, extra fusions
of TEL/AML1 and rearrangements of TEL, deletion of the second TEL allele is
the most common and can have significant prognostic implications. In these
cases, persistence of the original pre-leukemic clone may indicate proliferative
advantage and lead to phases of prolonged remission followed by periods of
relapse as the residual secondary clones emerge. We report a case on a 26 year
old male with ALL harboring homozygous deletion of the TEL gene region. To our
knowledge, this is the first reported case of a homozygous TEL deletion without
a concomitant TEL/AML1 fusion. His initial presentation to our laboratory was in
2003 status post bone marrow transplant and showing normal result by routine
chromosome analysis (RCA). Nine years later (07/2012) the patient returned with
relapse. Chromosome analysis showed a complex karyotype, 46~48,XY,add(1)
(p22),add(7)(q11.2),del(7)(p13p15),add(9)(p22),-10,del(12)(p12),add(12)(p13),13,add(14)(q22),del(15)(q22q24),add(16)(p13.1),del(18)(q21.1q21.3),+i(21)(q10)
x2,+mar1,+mar2[cp18]/46,XY[2]. FISH analysis with ALL panel showed 3 copies
of cMYC gene region in 14.5% of the nuclei, deletion of p16 gene region in 36.5%
of the nuclei and homozygous deletion of TEL gene region with 6 copies of AML1
in 81% of the nuclei. Follow-up chromosome and FISH studies five (12/2012), and
nine months later (04/2013), were all normal. At present, the patient continues
to be in remission with the most recent chromosome analysis (01/2014) showing
normal results. Homozygous deletion of the TEL gene region in our patient may
suggest that absence of TEL gene region on both alleles behaves similarly to cases
with the classic TEL deletion of only one allele. Furthermore, the manifestation of
relapse following remission clearly supports previous reports of the TEL deletion
being the principle causative factor for subsequent and late-onset relapses by its
ability to evade complete elimination during therapy. Since our patient presented
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to us following BMT, we cannot ascertain if the homozygous TEL deletion is the
second hit following the initial TEL/AML1 fusion, or homozygous TEL deletion,
which has never been reported, is by itself the initial causative agent. Given the
preponderance of evidence, we hypothesize that our patient may have the initial
TEL/AML1 fusion with deletion of one TEL allele at diagnosis and as confirmed by
other studies, the loss of one TEL allele may have resulted in the loss of second
TEL allele similar to loss of heterozygosity in other cancers.
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19
A NOVEL AUTOMATED METHOD FOR FLUORESCENCE IN
SITU HYBRIDIZATION PRE-TREATMENT IN FORMALIN-FIXED,
PARAFFIN-EMBEDDED TISSUES
Jessica A. Roseberry Baker; Joel D. Cook, BS; Andrew E. Schade, MD, PhD
One of the most important steps in a fluorescence in situ hybridization (FISH)
assay is the pretreatment of formalin-fixed, paraffin embedded (FFPE) tissue. The
pretreatment allows access to the nuclei and in turn the cellular DNA for the
probes to bind to. Formalin fixing tissues aids in the preservation of DNA by
cross-linking, however this can be challenging to reverse. Archival tissues are
often used to develop assay conditions and to complete validation studies. Due to
their age as well as variable fixation times, the cross-linking is stronger and more
difficult to reverse. Most labs follow a version of commercially available assays
that involve sodium isothiocyanate (NaSCN) at 80-85ËšC or 2-[N-morpholino
]ethanesulphonic acid (MES) at 95-99ËšC and incubation with a protease at
37-40ËšC. Use of these protocols with archival tissue gives less than optimal
results in regards to signal strength and cell morphology. Another limitation is
the instrumentation available to automate FISH. Currently, the main automation
platform uses 500 mL of reagents regardless of slide number required and has
a temperature deviation of +/- 5. This may be a costly use of reagents, as well
as less specific temperatures for critical steps. Three main FISH methods were
tested to improve cell morphology and probe signal strength on archival NSCLC
and gastric carcinomas using a ThermoBrite Elite (TBE). These methods included
initial pretreatment with NaSCN at 80ËšC, MES at 95ËšC or DIVA at 95ËšC
followed by incubation with protease at 37ËšC. The pretreatment with NaSCN
at 80ËšC followed by incubation with 0.05 mg/mL pepsin/0.01N HCl was less
than optimal. The target cells were digested away leaving connective tissue and
no signal. The pretreatment with MES at 95ËšC followed by incubation with
pepsin was improved from the NaSCN pretreatment. The cells are intact with
signal, however the signal intensity was low due to the presence of stroma and
background noise. The pretreatment with DIVA at 95ËšC followed by incubation
with 1 mg/mL pepsin/0.01N HCl was superior to the other methods. The cell
morphology remained intact with optimal clearing and the signal strength was
very strong and punctate. This was consistent in NSCLC and gastric carcinomas
with varying age and fixation times. Also, utilizing the TBE wasted less reagents and
had more accurate temperatures (+/- 1 degree). In conclusion, we demonstrated
that the novel use of DIVA as a pretreatment at 95ËšC followed by incubation
with 1 mg/mL pepsin/0.01N HCl on the TBE gave a higher quality FISH signal in
archival tissues than other widely used methods.
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20
CLINICAL IMPLEMENTATION OF AN EFFICIENT, AUTOMATED
SOLUTION FOR CELL AND FISH PROBE APPLICATION
Shannon Dingivan; Niccole Cox; Kristi Wolfe; Dianne Keen-Kim, PhD, FACMG
Facing reduced reimbursement for medical laboratory tests and higher reagent
costs, clinical laboratories are increasing the use of automation to decrease the
cost and the time required to perform each test. Given the high-volume of FISHtesting found in our hematology-focused laboratory, we sought instrumentation
to reduce labor and the amount of FISH probe needed. Additionally, we wanted
automation that could be utilized for multiple activities throughout the lab (e.g.,
chromosome and FISH testing), while also reducing the potential for specimen
and FISH probe application error. To this end, we found and implemented
the CellWriterâ„¢ 480 workstation (BioDot, Irvine CA). We present data and
methodology for the validation and implementation of this fully-automated
solution for cell application to FISH slides and applying both FISH probe and
DAPI counterstain. In our in-lab pre-implementation assessment, we compared
probe cost per test, time per test, as well as accuracy and quality in both cell
and probe application, to demonstrate the efficient and cost-effective use of
automation in the clinical laboratory. Using the CellWriter workstation, we first
designed protocols for applying cultured cell pellet, FISH probes, and DAPI onto
slides for downstream FISH analysis. In our lab, specimens for hematologic
evaluation have an average of >4 probes analyzed per patient. Therefore, we
found the addition of 8-well slides into our workflow to be most efficient, in terms
of sample, glass use, and storage. The CellWriter uses <10% of patient sample
volume (15ul for 16 wells) when compared to manual application, thus increasing
the number of evaluations available per patient specimen. This is particularly
important for low-yield procedures such as plasma-cell enrichment and dry taps.
Less probe volume is also required per test when using the CellWriter. By using
a nanoliter dispenser within the CellWriter, we use only 0.7ul of FISH probe per
test, compared to the probe manufacturer recommended 10ul of FISH probe
per test volume. However, due to a dead volume of 6ul per probe, more than
0.7ul/test must be calculated in the cost savings and the efficiency increases with
the total number of applications of the same probe. When a single test (probe)
is batched on the CellWriter, we realize a >10-fold cost savings. To determine
time savings of probe application using the automated method, we compared
a typical manual hybridization to a typical automated hybridization. At â‰¥20
probe applications, the CellWriter is faster than the manual method. With larger
batch sizes, probe application time is halved on the CellWriter. Additional
time savings is realized through a Teflon gasket barrier that defines the wells
in our new 8-well slides and eliminates the need for individual cover slips and
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rubber cement. Qualitative evaluation also demonstrated less damage to the
specimen from human handling, such as scratches produced on the slides due
to the elimination of rubber cement in the hybridization step. Finally, we have
developed a proprietary IT solution, which transfers test information for each
patient directly from our Laboratory Information System to the CellWriter and
eliminates the possibility of specimen or probe mix-up. In conclusion, we have
reduced our reagent and labor costs and the potential for error through the
implementation of an automated cell, FISH probe and DAPI application system.
The biggest draw-back to our implementation experience was the amount of
time required in development and validation of the system, and the training of
personnel in our lab. Additional testing, including the confirmation of scoring
concordance of the manual method and automated method are ongoing. When
compared to the manual method, the benefits of the automation provided by
the CellWriter include a decrease in the required volume of the patient sample
and the FISH probe while also reducing the labor required. In addition, the use
of the CellWriter decreased the potential for error by improving consistency and
reducing variability in probe application.
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21
A NON-INVASIVE, REAL-TIME APPROACH TO AID IN THE
PROFILING OF RARE CELLS IN CANCER
Natalee Bales; Florence Lee, PhD; Dena Marrinucci, PhD
Circulating tumor cells (CTCs) are cells located in the bloodstream that have been
shed from primary tumors and play a critical role in the initiation of the metastatic
spread of human cancers. CTCs are traditionally defined as cells with large, intact
nuclei which are CD45 negative and epithelial cell adhesion molecule (EpCAM)
and/or cytokeratin (CK) positive. The majority of CTC analysis platforms focus
on CTC enumeration by selecting cells based on specific criteria including size,
shape, and/or cell surface marker expression. While these methods achieve cell
isolation, atypical CTC candidates (e.g. small cells, EpCAM negative cells, etc.)
are often missed because they do not fit the preset criteria. In addition, significant
manipulations of fragile CTCs in these methods can compromise cellular integrity,
thereby limiting the possibility of downstream molecular characterization. Our
approach aims to identify and molecularly characterize all CTCs, including the
atypical CTC candidates, without using physical selection techniques. With our
platform, nucleated blood cells are first plated onto glass microscope slides and
immunofluorescently stained with a cocktail of antibodies (CK and CD45). The
slides are then scanned with our proprietary scanner, and the captured images
are analyzed by multi-parametric analysis algorithms to identify and locate each
CTC or CTC candidate. The tumor cells can then be further characterized by
DNA fluorescent in situ hybridization (DNA FISH) using locus-specific fluorescently
labeled probe sequences to assess their genomic status. Our mission is to employ
our platform to aid in the improvement of patient lives by enabling precision
medicine through non-invasive, real-time tests that profile rare cells in cancer.
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22
13Q DELETION SYNDROME. CASE REPORT AND LITERATURE
REVIEW
Viviana Gomez, MSC, MD; Marisol Ibarra, MD; Gloria Garcia, CG(ASCP);
Carmen Quezada, CG(ASCP); Laura Martinez, MD; Daniel Campos, MD
CYTOGENETICSIntroduction. 13q deletion syndrome is a rare condition
described by Allderdice et al. in 1969. Clinical features include growth retardation,
brain malformations, mental retardation, facial dysmorphism, and urogenital,
gastrointestinal, and musculoskeletal malformations. In the literature, 180 cases
have been reported with the vast majority being de novo deletions. We present
the case of a patient with an interstitial deletion of the long arm of chromosome
13 karyotype GTG 46, XY, del(13) (q14.1q32.1) with multiple malformations. A
review of the literature was performed. Case report. The patient is a newborn
male, obtained by cesarean section at 36 weeks of gestation with a history of
IUGR, duodenal atresia and polyhydramnios. On physical examination: weight
2010 g (p10) length 42 cm (< P3) CP 33 (p50 ), aplasia cutis, areas of alopecia
with scarce thin hair, wide anterior fontanelle communicating with the posterior
fontanelle, broad forehead, arched eyebrows, straight eyelid openings of 2.5 cm,
telecanthus, broad nasal bridge, anteverted nostrils, square tip, low-set auricles
with posterior rotation, small thick helix, nuchal redundancy, a holosystolic murmur
grade III/VI on chest exam, reducible umbilical hernia, bilateral cryptorchidism,
phimotic penis of 1.5 cm, central hypotonia, incomplete Moro reflex, weak
suck, brachydactyly and clinodactyly of the 5th finger with bilateral hypoplastic
nails, aberrant folds, and bilateral proximal implantation of the 3rd toe. Brain
MRI showing dilatation of both ventricles, communicating hydrocephalus, and
cortical atrophy; ECG: patent ductus arteriosus and interatrial communication.
EEG, abdominal US, and ophthalmologic evaluation are normal. The patient
died at 6 months of age from complications of a hospital infection. Cytogenetic
analysis. GTG karyotype formula 46, XY,del(13)(q14.1q32.1). Karyotype of both
parents with normal chromosomal formula. Since the area of rupture involves
the locus 13q14, a FISH analysis was performed using the probe 13q14 LS1
13 Green aneuvysion spectrum, which reported 46,XY.ish del(13)(q14q14)
(LSI 13-) that shows the absence of the signal in the chromosome that has the
deletion at 20 metaphases analyzed. Discussion. The deletion syndrome 13q,
is classified according to Brown 1993 into 3 groups according to the deleted
regions 13q32. This is considered a critical region and is associated with major
CNS malformations. This region appears to be conserved in our patient and is
consistent with the failure to present severe SNC malformations. Even though
no aCGH was performed to accurately define the breakpoints, we were able to
define that it is an interstitial deletion and have a greater accuracy of the region
involved.
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23
DOUBLE INV(3)(Q21Q26.2) IN ACUTE MYELOID LEUKEMIA
Bing Bai, MD, CG(ASCP); Jun Gu, MD, PhD, CG(ASCP); Guilin Tang, MD,
PhD;
Denise Lovshe, BS, CG(ASCP); Xinyan Lu, MD, FACMG
Inv(3)(q21q26.2) is one of the rare recurrent cytogenetic abnormalities found
in acute myeloid leukemia (AML). Latest World Health Organization (WHO)
classification system has defined AML with inv(3) or t(3;3) and associated RPN1/
EVI1 fusion as a distinct subgroup with poor clinical outcomes. Double inv(3)
(q21q26.2), a paracentric inversion occurred at 3q21 and 3q26.2 on both
chromosome 3, is even harder to find. Only ten such cases have been reported
so far. Three AML cases with double inv(3)(q21q26.2) were presented here. They
were 57 year-old female AML patient with minimal differentiation (M0), 65 year-old
female patient with AML-M4 transformed from CMML (deceased), and 73 yearold male AML-M6A patient (deceased). Bone marrow differentials, morphology,
flow cytometirc immunophenotyping, molecular analysis, treatment, and survival
data were collected. EVI1 rearrangement was confirmed by fluorescence in situ
hybridization (FISH). This study added new cases to limited literature which will
facilitate the better characterization of this unique WHO subgroup.
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24
ENHANCED DIAGNOSTIC YIELD FOR MULTIPLE MYELOMA BY
CHROMOSOME GENOMIC ARRAY TESTING OF CD138-ENRICHED
PLASMA CELLS
Lena Glaskova; Lucy Fei; Sarah Schroeder; Scott McElhone; Min Fang, MD,
PhD, FACMG
BACKGROUND: Chromosome genomic array testing (CGAT) of multiple
myeloma (MM) samples is a great tool to reveal new insight of this disease. It can
detect submicroscopic losses and gains not seen by conventional cytogenetics or
FISH panel that ascertains poor prognostic markers in MM. In addition, CGAT
incorporating SNP probes can also detect copy-neutral loss-of-heterozygosity
(cnLOH) important for cancer biology. However, this technology does not detect
minimal residual disease among patients post-treatment. As a tertiary care
center, most of our MM patients are post-treatment with low levels of disease.
To enhance the detection rate of CGAT in MM, we employed cell separation
technique to enrich CD138 plasma cells prior to CGAT. The purpose of the study
is to compare the CGAT findings with FISH and Cytogenetic results performed on
the same patients. METHOD: We evaluated 23 MM patients with the abnormal
plasma cells in the bone marrow ranging between <0.1~60% by flow cytometry
analysis. To increase sensitivity, samples with abnormal plasma cells between
<0.1~ 40% were separated. Of 20 samples in this category, 14 were separated.
CD138+ cells were enriched using autoMACS Pro-Separator, DNA extracted
by the Qiagen kit, and analyzed using the CytoScan HD platform. A total of
22 samples had concurrent FISH study, 20 of which also had cytogenetics
performed. In three cases, CGAT and FISH were performed on different tubes.
RESULTS: Of all 23 MM patients tested by CGAT, 19 (83%) were abnormal.
Seventeen (90%) showed additional abnormalities not detected by FISH and
cytogenetics. Of the 3 samples with 40~60% plasma cells and not separated,
all were positive by CGAT and, with the exception of one with no FISH ordered,
were consistent with FISH and cytogenetics findings. All of these studies showed
additional abnormalities not seen by G-banding. One study did not correlate
with FISH probably due to different tubes used in these studies. Six of the 9
samples with 6~40% plasma cells were separated and were 89% concordant
between array and FISH. One discrepancy was due to different tubes used for
CGAT and FISH. Cytogenetics was normal in 3 of these studies with flow results
between 20~36%. All 9 patients showed additional abnormalities not seen by
G-banding. All 5 samples with 1~6% plasma cells were separated and split
between CGAT and FISH and showed 100% concordance. Cytogenetics was
normal in 2 of these cases with flow between 3~6%. Three of the 5 patients
showed additional abnormalities not seen by G-banding. All 5 samples in the
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0.1~1% plasma cell category were also separated. All were abnormal by FISH,
but only one CGAT study (20%) correlated with FISH findings. Interestingly, a
sample with 0.06% abnormality by flow yielded robust array (30% abnormal) and
FISH (39% abnormal) data after CD138 enrichment even though cytogenetics
was normal. CONCLUSIONS: Our data showed that enrichment of MM samples
prior to CGAT could enhance diagnostic yield, even on samples with very low
plasma cells. Sample variation may contribute to some false-negative results.
Separation of samples with <15% plasma cells shows the most benefit. Array
analysis is an invaluable addition to the genetic evaluation of MM patients. It not
only confirms FISH and cytogenetics findings, but also uncovered a multitude of
cryptic abnormalities making the whole picture of this disease better. These new
markers could be developed into new prognostic FISH probes for more accurate
post-treatment follow-up.
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25
FLUORESCENCE IN SITU HYBRIDIZATION (FISH) TEMPERATUREDEPENDENT EQUIPMENT COMPARISONS: TEMPERATURE
GRADIENT EFFECTS AND ANALYTICAL OUTCOME
Mary Lowery Nordberg; Roderick Jackson; Kathleen Kelly
OBJECTIVE: Differences in hybridization platforms used in fluorescence in situ
hybridization (FISH) analysis experiments can lead to significant differences in
hybridization results. To address some of the challenges involved in genomic
hybridization technology as a clinical tool, variations in performance characteristics
of hybridization platforms were evaluated (HYBrite and ThermoBrite (Abbott
Molecular), CytoBrite (SciGene)). We proposed that based on the thermoelectric
technology (i.e. Peltier technology) for rapid heating and cooling, the performance
of the CytoBrite would allow for improved and more accurate probe denaturation
and hybridization.
BACKGROUND: Historically, our laboratory has experienced several FISH failures
due to inefficient probe hybridization. The primary reason for the failure of a
patient assay is the uncertainty of analytical results as a function of diffuse signal
patterns (i.e. œspatter) or lack of hybridization. This has been especially evident in
the evaluation of peripheral blood and bone marrow smears for straight-forward
assays such as BCR/ABL t(9;22) fusions. In a single month, a total of eight FISH
failures (requiring repeat preparation and processing) all involving hematology
FISH targets (BCR/ABL, PDGFRA, PDGFRB, PML/RARA). While FFPE tissues can
be problematic, fresh specimens should be highly accurate, analyzable, and
reproducible. Technologist notes in the analysis log trace back to diffuse, unable
to analyze signals. In the first 6 months of 2013, a total of 60 FISH failures
(requiring repeats) were logged. As with any other DNA hybridization assay,
the main factors influencing failures and/or background are: (1) the amount of
repetitive sequences of the probe, and the extent to which they are blocked;
(2) hybridization temperature (lowering it increases non-specific binding of the
repetitive sequences); (3) the balance between hybridization time and amount of
DNA probe; and (4) the stringency of the post-hybridization washes.
METHODS: Using expired probes from commercial vendors, comparative
studies were performed using the CytoBrite, HYBrite, and ThermoBrite. Slide
preparation, denaturation and hybridization conditions were performed
according to the probe manufacturers specifications and DeltaMDx standard
laboratory protocol. Post-hybridization wash solutions were performed using the
ready-to-use FISH Wash Buffer 1 (0.4%SSC/0.3% IGEPAL, pH7) and FISH Wash
Buffer 2 (2xSSC/0.1% IGEPAL, pH7) (SciGene). All samples were sealed with
CytoBond prior to denaturation and processing. Specimen types included both
FFPE tissues, cytogenetic preparations (pellets fixed in Carnoys), and peripheral
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blood/bone marrow smears. Triplicate slides were prepared on all specimens.
Positive controls (if available) were incorporated into the study as appropriate.
RESULTS: A total of 50 prepared slides from various specimen types and various
probes were compared. FISH slides were compared for hybridization efficiency,
probe signal intensity, and overall assay results. Based on the initial visual
interrogation, the CytoBrite hybridization platform appeared to produce less
spatter and more analyzable signals (regardless of the probe). Out of 25 fresh
specimens (bone marrow/peripheral blood smears) and 25 FFPE specimens, 20
(80%) and 25 (100%) fresh and FFPE, respectively showed improved analyzable
signals using the CytoBrite. Improved signal data included subjective and
objective parameters such as signal intensity/strength, background, and tissue
architecture (FFPE).
CONCLUSION: Peltier technology is a vast improvement over existing FISH
hybridization platforms. Used for many years in traditional polymerase chain
reaction (PCR) assay procedures, the rapid heating and cooling parameters
allows for efficient denaturation and hybridization in FISH assays. This efficiency
results in reproducible and successful assays for many distinctly different tissues
and FISH probe types.
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26
8q ABNORMLITIES IN CLL PATIENTS WITH RICHTERS SYNDROME
Andrew McFaddin; H. Breidenbach; E. Hertein; J. Jones; J. Bryd, MD;
N. Heerema, PhD
Richters transformation, or Richters syndrome (RS), is described as
clinicopathological transformation of chronic lymphocytic leukemia (CLL) into an
aggressive, fast growing diffuse large B-cell lymphoma (DLBCL). This condition
affects about 5-10% of patients with CLL and is associated a poor prognosis, with
median survival of about 10 months. Although specific risk factors have yet to be
identified, TP53 abnormalities, non-del(13q) cytogenetics, unmutated IGHV, and
CMYC abnormalities may predispose patients to RS.1 In the clinical Cytogenetics
laboratory at The Ohio State University Wexner Medical Center, between 1999
and 2012, 51 CLL patients with suspected Richters transformation were analyzed.
Sixteen of the samples were cultured for 72 hours and stimulated with pokeweed
and PMA, and 35 were cultured for 72 hours and stimulated with pokeweed,
PMA, and oligodeoxynucleotide (ODN). Analyses of these samples demonstrated
18 cases with apparent normal 8q by chromosome analysis. Of these 18, 11 had
normal FISH results for CMYC and 7 had no CMYC FISH performed. Of these
18, 13 cases had complex karyotypes involving other chromosome abnormalities
and 5 had simple abnormal karyotypes. Another set of samples revealed 9
cases of structural 8q abnormalities by chromosome analysis, with breakpoints
ranging from 8q11.2-q24. These samples all had complex karyotypes but normal
CMYC FISH results. Twenty-three additional cases showed abnormal copy
number of 8q along with abnormal copy number of CMYC by FISH. Twenty
of these demonstrated complex karyotypes, while three had simple abnormal
karyotypes. Finally, one case showed a structural abnormality of 8q24 as part of
a complex karyotype with corresponding partial deletion of CMYC by FISH. In
examining this set of patients with CLL and RS, it can be seen that abnormalities
involving 8q are often part of a larger complex karyotype. These abnormalities
include copy number variants as well as structural rearrangements along the
q-arm of chromosome 8, not always involving the CMYC gene. It should be
noted that stimulation of these samples with pokeweed, PMA, and ODN allow
for proliferation of abnormal cells for chromosome and FISH analyses which aid
in acquiring more information regarding the cytogenetic findings in these unique
patients with RS. 1Oncology. Vol.26 No.12
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27
ADVENTURES WITH ASS1: THREE COLOR STRATEGY PROVIDES
ADDITIONAL INFORMATION BEYOND THAT ANTICIPATED
Hong Huang, CG(ASCP); Sandi Gagneaux, CG(ASCP); Shelley Taylor;
Beth Barnett, CG(ASCP); Jason Ford-Green, PhD; Suzanne Hurley, CG(ASCP);
Brandice Nowell; Holly H. Hobart, PhD Dipl ABMG
The three-color Dual-fusion FISH probe cocktail was designed to address the
10-15% of cases that have the BCR/ABL1 gene fusion but have lost the reciprocal
fusion signal located on the derived #9 chromosome. This finding is clinically
significant because these 10-15% of patients do not respond to treatment as do
the 85-90% of patients who do not have the deletion. This approach works well
for this purpose. Also, when these loci (=FISH signals) are lost on the der #9,
distinguishing between low level residual disease and artifact is not possible in the
absence of the third color, because these patients have FISH patterns that do not
specifically demonstrate the BCR/ABL fusion on the Philadelphia chromosome.
Those two-color patterns are consistent with three things:
1. BCR/ABL1 fusion on the Philadelphia chromosome
2. BCR/ABL1 reciprocal fusion on the der 9
3. Artifact of cell flattening
Examples from four patients demonstrate the basic utility of the three-color probe
cocktail strategy as well as additional information gleaned:
1. Clonal evolution, addition of Philadelphia chromosome
2. Variable size deletions on der 9
3. Clonal changes following treatment
4. Clarification of G-band abnormalities
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28
UNEXPECTED FINDINGS ILLUSTRATE AN IMPORTANCE FOR
CONFIRMATORY TESTING FOLLOWING POSITIVE NON-INVASIVE
PRENATAL SCREENING
Kimberly Gobac, BS, CG(ASCP)CM; Anna Horne, BS; Heather Absher, BS,
CG(ASCP)CM;
Felicia Johnson, BS; Spring Brooks, BS, CG(ASCP)CM; Marc Delos Angeles, BS,
CG(ASCP)CM; Catherine Rehder, PhD, FACMG; Kristen Deak, PhD, FACMG
Non-invasive prenatal screening (NIPS) analyzes circulating cell-free fetal DNA
found within the maternal blood. Testing was developed as an early, non-invasive
option for detecting fetal aneuploidies and sex chromosome abnormalities.
With both false positive and negative results being possible, follow-up testing is
recommended for any positive result. In 2013, we received three blood samples
from infants for which NIPS was positive for trisomy 21; all patients declined
prenatal follow-up testing. In one case, chromosome analysis showed straight
forward free trisomy 21, but the other two cases produced unexpected findings.
In Case 1, NIPS was performed after an ultrasound showed increased nuchal
translucency. At birth, the baby had features of Down syndrome, and cord blood
chromosome analysis showed free trisomy 21. For Case 2, maternal serum screen
suggested an increased risk of trisomy 21 and subsequent NIPS was positive for
trisomy 21. At birth, the child showed multiple anomalies consistent with Down
syndrome. Microarray analysis of a peripheral blood specimen revealed a 12.69
Mb duplication of material from 21q22.11-q22.3, which contains the Down
syndrome critical region including the genes DYRK1A and RCAN1. Chromosome
analysis showed that this duplicated material from 21q was also located on 21p,
consistent with an unbalanced t(21;21). Analysis of maternal blood showed no
translocation. Lastly, in the Case 3, although NIPS was positive for trisomy 21,
no obvious features of Down syndrome were seen at birth. For this reason, FISH
analysis was performed, which showed no evidence for trisomy 21. Subsequent
chromosome analysis was also normal. The mother has a history of polycythemia
vera, which can show trisomy 21 as an acquired clonal abnormality, although
no record of maternal cytogenetic studies were available. Several reasons for
false-positive screening include confined placental mosaicism and maternal
malignancy. In summary, positive NIPS could reveal rearrangements other than
free trisomy 21, which may result in a substantial recurrence risk for a family.
These results also emphasize that NIPS is not a diagnostic test and that it is
highly important for patients to understand the limitations associated with this
new technology and seek genetic counseling.
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29
VARIANT TLX3/BCL11B REARRANGEMENT IN A 4-YEAR-OLD BOY
WITH T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA
Jennifer Otani-Rosa; Rhonda Mackendrick; Tri Tchen; Laura O’Leary; Nancy
Hsu; Christine Bryke; Zunyan Dai; Philip Mowrey
We describe an interesting case involving a 4-year-old boy referred for acute
lymphoblastic leukemia. A bone marrow aspirate was cultured for 24 and 48
hours. Chromosome analysis on G-banded metaphase cells revealed a complex
karyotype in 19 of 20 cells, with unidentifiable material on 2q, 5q, and 14q.
FISH results on interphase cells, using probes for BCR-ABL1, ETV6-RUNX1, MLL,
CEP4, D10Z1, D17Z1, TRA/D, and TCL1, were normal. Oligo-SNP Microarray
analysis detected a small gain [~1.7-MB] of 2p25.3, two deletions in the long
arm of chromosome 5 [~14-MB deletion at 5q11.2q12.3 and ~10-MB deletion
at 5q35.1q35.3] encompassing the 3rd end of TLX3, as well as biallelic deletion
of 9p21.3 [~1.4-MB and ~362-KB], which encompassed CDKN2A. Additional
FISH testing of CDKN2A was performed to confirm the biallelic deletion at
9p21.3. Metaphase FISH using the IGH breakapart probe demonstrated
that a small portion of 14q32.2 containing the proximal 3rd end of IGH was
inserted into 2q31 while the 5th end remained on the derivative chromosome
14. We concluded that the remaining segment of the 5q13q35.1 was inverted
and inserted into 14q32.2, with the following karyotype: 45,XY,ins(2;14)
(q31;q32.2q32.3),der(5;22)(p10;q10),ins(14;5)(q32.2;q35.1q13). Chromosome
analysis together with the microarray and FISH studies suggests that the
rearrangement involving chromosomes 5 and 14, is likely a variant of the t(5;14)
(q35.1;q32.2), which results in the TLX3/BCL11B gene rearrangement associated
with T-lymphoblastic leukemia.
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30
SUCCESSFUL FIBROBLAST CULTURES FROM BONE MARROW
SAMPLES TO RULE OUT CONSTITUTIONAL KARYOTYPES
Michael Babineau; Ann Thomas; Paola Dal Cin
Occasionally, cytogenetic analysis on bone marrow (BM) specimens will result
in an abnormal cytogenetic finding atypical for the presented disease state.
For example, a patient’s BM sample received with a questionable indication for
anemia exhibited a 45,XX,der(14;21)(q10;q10) karyotype in all 20 metaphases. In
the past, to determine whether this patient carried a constitutional chromosome
rearrangement, peripheral blood was requested, thus further stressing an already
sick individual. Currently, by culturing the fibroblasts present in the remaining
(originally submitted) BM sample, a possible constitutional karyotype can be
obtained. Procedure: incubate the remaining bone marrow sample with media
in a T-25 flask establishing a monolayer culture. Change media as needed once
growth has been established. When mitotic cells are observed: trypsinize to
cover slips, harvest, GTG-band and analyze as you would for routine fibroblast
cultures. In our specific example, we found an identical 45,XX,der(14;21)
(q10;q10) karyotype indicating this Robertsonian translocation was not
related to the disease status of the patient. Since 2008, our laboratory has
successfully cultured the remaining BM specimen from 32 patients to determine
constitutionality of reported non disease related chromosome abnormalities
and has found 9 balanced translocations, 3 Robertsonian translocations, and
3 sex chromosome abnormalities. The risk to develop hematological disorders
has been explored in the past, and in some cases, has been confirmed. The
best example of a constitutional abnormality resulting in hematological disease
is trisomy 21 individuals with Down syndrome developing acute lymphoblastic
leukemia. Constitutional Robertsonian translocations have been associated with
various cancers, although cases with de novo translocations have been previously
reported. Therefore, the successful fibroblast culture of BM samples to rule out
a possible constitutional chromosome abnormality has: a) reduced stress for the
patient, b) recognized the possibility of an increased risk of malignancy, and c)
with factoring in the patients age and health has added to the discussion of how
to proceed further with their standard of care.
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31
VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTOR AND
PLATELET DERIVED GROWTH FACTOR RECEPTOR ALPHA GENE
HETEROGENEITY IN GLIOBLASTOMA
Joel Cook; Jessica Baker, MS; Crystal Dotson-Roberts, BA; Erin Felke, BS;
Lisa Riegle, BS; Hiral Oza MS; Gerard Oakley III, MD; Andrew Schade MD,
PhD
The receptors for vascular endothelial growth factor (VEGF) and platelet derived
growth factor (PDGF) represent two closely related families of receptor tyrosine
kinases. Phylogenetic evidence suggests that these may have arisen from
duplication of an evolutionarily distant precursor. This is supported by the close
proximity of VEGFR2 and PDGFRa on chromosome 4q12. Both have also been
reported to be amplified in certain tumors including glioblastomas. The purpose
of this study was to determine the frequency and signal patterns of VEGFR2
and PDGFRa gene amplification in formalin fixed, paraffin embedded (FFPE)
glioblastoma (GBM) by fluorescence in situ hybridization (FISH) and compare
amplification with protein expression levels. 13 FFPE GBM’s were hybridized
with a three color custom probe set for VEGFR2, PDGFRa and centromere 4
to detect VEGFR2 and PDGFRa amplification. Four of 13 tissues were amplified
for VEGFR2 and PDGFRa. Of the four samples, focal PDGFRa amplification
ranged from 2.5-63% with 4-~50 copies, and combined amplification of both
genes spanned from 29.5-72.5% with 4-~50 copies. The PDGFRa and VEGFR2
copy number levels reported here are significantly higher than most published
instances of gene amplification. Interestingly, PDGFRa is often increased without
VEGFR2 despite their close proximity on 4q12. In fact, one of 13 tissues had focal
VEGFR2 amplification occurring in only 5% of the cells, whereas focal PDGFRa
amplification occurs in four of the 13 tissues in up to 63% of the cells. When
both genes are amplified (≥4 copies) they are not increased to the same extent,
possibly indicating multiple amplification events. PDGFRa gene amplification
correlates with PDGFRa immunohistochemistry (IHC) positivity, however this is not
true for VEGFR2 gene amplification and VEGFR2 IHC. This implies that in GBM,
VEGFR2 amplification does not increase receptor expression. The high copy
number, variation in copy number range, and low incidence of focal VEGFR2
amplification may offer opportunities for targeted therapies against PDGFRa and
VEGFR2 in glioblastoma, as well as a better understanding of the underlying
cytogenetic mechanisms.
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32
THE UTILITY OF CYTOGENOMIC SNP MICROARRAY TESTING IN
THE ANALYSIS OF PRODUCTS OF CONCEPTION SPECIMENS
Kristin S. Petras, CG(ASCP)CM; Qun Shi; Jennifer Keller-Ramey, PhD;
Carrie Fitzpatrick, PhD
Cytogenomic microarray (CMA) testing has been recommended for the analysis
of copy number variants in postnatal specimens. The benefits of this testing have
been widely described and include: higher resolution whole genome abnormality
detection, more objective assay, and success without active dividing cells. CMA
testing has also been more recently recommended for the analysis of copy
number variants in prenatal and products of conception (POC) specimens with
many of the same benefits. We validated the Affymetrix CytoScanÂ® HD SingleNucleotide Polymorphism (SNP) CMA platform for use in POC specimens in
April, 2013. Here we summarize our clinical experience since that time including
a few interesting cases. We have performed CMA testing on 13 POC specimens
to date in conjunction with routine chromosome analysis. Ten of these cases were
reported as normal by both chromosome analysis and CMA. One case was
a culture failure for chromosome analysis due to contamination of the culture
present at the time of collection, but was able to be reported by CMA as normal.
Interestingly enough, the remaining two samples were also culture failures for
chromosome analysis due to lack of cell attachment and growth, and both were
found to be monosomy 21 by CMA. This abnormality is very rarely seen in routine
chromosome analysis and could explain the lack of cell growth in these cultures.
Monosomy 21 could represent a cause of pregnancy loss that has previously
been underreported. One of the biggest advantages of performing CMA testing
on POC specimens is the fact that it can provide results that are unable to be
detected by routine chromosome analysis due to culture failure. Since validating
CMA in our laboratory we have been able to report CMA results in all instances
of culture failure which remains approximately 5-15% of all POC specimens.
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33
PRENATAL DIAGNOSIS OF DUPLICATION (21)(q22.13q22.2):
FINDINGS OF CHROMOSOME ANALYSIS, FISH AND
CHROMOSOMAL MICROARRAY FOLLOWING A NORMAL NIPT
RESULT
Deborah Heritage; Jane Kang; Jennifer Jahn; Jocelyn Dayanghirang; Tarryn
Quinlan; Anita Vishwakarma; Leslie Ross; Renius Owen; Mohamed Mohamed
El Naggar; Philip Mowrey; Steven Schonberg
We report a rare case involving dup(21)(q22.13q22.2) detected in amniotic fluid
at a gestational age of 17 weeks and 2 days. The initial noninvasive prenatal
testing (NIPT) results were normal. However, ultrasound findings revealed
increased nuchal translucency and echogenic intracardiac focus indicative of
increased risk for trisomy 21. The case was thus referred to us for chromosome
analysis, FISH prenatal aneuploidy screen, and chromosomal microarray (CMA).
A FISH aneuploidy screen on uncultured amniocytes suggested a male fetus
with three signals for 21q22.13-q22.2, consistent with trisomy 21. Analysis of 20
G-banded metaphase cells showed a male karyotype (46,XY) with two normal
appearing chromosomes 21. As this result was discrepant with interphase FISH
results, a FISH assay with the same probe for the Down syndrome critical region
was repeated on metaphase chromosomes. This analysis yielded a signal on
each chromosome 21, one of which appeared significantly enhanced. The CMA
analysis revealed a 3.5-Mb duplication within bands 21q22.13q22.2 extending
from nucleotide position 38,589,731 to 42,113,758. This case supports the use
of multiple genetic tests following negative results for NIPT when ultrasound
findings or other prenatal tests are abnormal. Molecular cytogenetics can
detect abnormalities below the resolution of conventional cytogenetic analysis.
G-banding and metaphase FISH can assist in determining the associated structural
rearrangement to aid in genetic counseling for this and future pregnancies.
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34
ROS1 AND RET ANEUPLOIDY IN NON-SMALL CELL LUNG CANCER
Maricar Fernando; J. Williams; A. Felder; S. Stuart; I. Gadi, PhD;
J. Tepperberg, PhD
The chromosomal rearrangement involving the anaplastic lymphoma kinase
(ALK) gene in non-small cell cancer (NSCLS) has stimulated interest in oncogenic
tyrosine kinase (TK) gene fusions as potential therapeutic targets. Recently, genetic
rearrangement in ROS1 (chr 6) and RET (chr 10) were identified as well with
NSCLC. Similar to ALK, genetic alteration in ROS1 and RET involves chromosomal
rearrangements that result in the formation of chimeric fusion kinases capable of
oncogenic transformation. These structural chromosome rearrangements have
received substantial attention in the field of translational research. However, the
role of whole chromosome aneuploidy, as seen by the gain of the control probe,
is much less well understood, although it is generally considered to be a driver
of tumor progression. It is unclear whether this progression is due to the over
expression of these genes or overall instability of the genome. The interpretation
of extra ROS1 and RET hybridization signals is limited by the absence of an internal
control site probe and other probes in the genome. The goal of this study was to
determine whether the extra ROS1 and RET hybridization signals detected by FISH
are due to additional copies of these genes or additional chromosome 6 and 10
homologues. Our data showed that of 478 cases, 58.9% of of ROS1 cases were
negative, 5.2% were positive for a ROS1 gene rearrangement and 35.7% showed
extra copies of ROS1. For 101 RET cases, 47. % were negative, 2.0% were positive
for a RET gene rearrangement and 48.5% of cases showed additional copies of
RET. In our relatively large cohort, the percent positive for ROS1 is slightly higher
than the ~2.0% positive cases cited in the literature. For RET, the 2.0% of samples
positive for RET is similar to what is cited in the literature.
A pilot study on NSCLC FFPE using samples containing extra copies of ROS1 and
RET were tested with pericentromeric FISH probes for chromosomes 6 and 10,
respectively. Our data consistently showed a similar number of extra copies of
pericenentric signals for chromosome 6 and ROS1 and chromosome 10 and RET.
This indicates that there are additional copies of of ROS1 and RET, secondary to
aneusomy for chromosomes 6 and 10. Molecular testing may help further define
the level of aneuploidy and gene content. While NSCLC patients with ROS1 and
RET gene rearrangements appear to benefit from targeted tyrosine kinase inhibitor
therapy, it is unclear whether patients with extra copies of these chromosomes
will benefit from this targeted treatment. Serial monitoring of patients with these
probes will be needed to determine whether aneuploidy for chromosomes 6
and 10 could possibly be indicators of TK targeted therapy in advanced NSCLC
patients. Comparison of tumors with polysomy and histology and/or molecular
microarray studies will be examined to determine whether these tumors can be
differentiated from their non-polysomic counterparts.
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35
ARRAY DETECTED GPC3 GENE DELETION ASSOCIATED
SIMPSON-GOLABI-BEHMEL SYNDROME
Tracy Hummel; M.Fernando; A. Felder; R.Pasion; I. Gadi, PhD;
J. Tepperberg, PhD
A 30 year old female was referred for amniocentesis at 18 weeks 3 days
gestational age (GA) with ultrasound findings of diaphragmatic hernia and fetal
size disproportionate to GA. FISH studies for chromosome 13, 18, 21, X, Y and
karyotype resulted in normal male. AF-AFP was also within normal range. Due to
the presence of ultrasound anomalies, Pallister Killian syndrome was suspected.
Additional FISH studies and microarray were performed to rule out the presence
of a supernumerary chromosome 12 usually associated with Pallister Killian
syndrome. FISH using a chromosome 12p13 region probe ETV6 showed normal
2 hybridization signals. A whole genome microarray analysis revealed 151 Kb
interstitial deletion of Xq26.2. This deletion encompasses GPC3 gene for X-linked
Simpson-Golabi-Behmel syndrome (SGS). Mutations in the GPC3 gene cause
Simpson-Golabi-Behmel Syndrome, a syndrome very similar in clinical phenotype
to Pallister Killian Syndrome. Prenatal ultrasound similarities include possible
diaphragmatic hernia and larger growth rate. SGS postnatal features include
coarse facial features, intellectual hardships. This X-linked disorder and generally
affects males whereas females are generally unaffected carriers. It has only
been clinically found in 130 people worldwide. Follow-up parental FISH studies
showed that the mother was a mosaic carrier of the deletion with a deletion of
Xq26.2. FISH studies on grandmother showed cells with one hybridization signal
due to age related loss of one X chromosome. Data to interpret these FISH results
will be presented. The result clearly illustrates the effectiveness and significance
of prenatal microarray analysis and indicates its utility as a primary cytogenetic
test for prenatal evaluation.
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36
PRENEOPLASTIC MOSAICISM DETECTED BY MICROARRAY
Mary Ann West, CG(ASCP); Sally Kochmar, MS, CG(ASCP); Maureen Sherer,
MS, CG(ASCP); Jie Hu, MD, PhD; Svetlana Yatsenko, MD; Urvashi Surti, PhD
We present the finding of cytogenetic abnormalities due to neoplastic process in
a newborn detected by microarray prior to clinical manifestation in the patient.
This male proband was born to a 33 y.o. mother at 36 wks gestation with a
prenatal history of IUGR. Patient was noted to have mild dysmorphic features,
microcephaly and gram-negative rod sepsis at birth. Chromosome analysis on
peripheral blood lymphocytes showed a 46,XY karyotype. Oligonucleotide CGH
microarray analysis revealed a mosaic 51.7 Mb deletion of 7p12.1->7pter and a
mosaic 61.3 Mb deletion of 7q21.3->7qter. At age 8 months, a repeat sample
for aCGH showed the 7p and 7q deletions identified by previous studies as well
as detecting a mosaic 19.41 Mb duplication of 1q21.1-q21.3. FISH confirmed
the mosaic 7p and 7q deletions and 1q duplication. At age 13 months the
patient presented to the ER with signs of acute leukemia which was subsequently
diagnosed as AML. Bone marrow chromosome analysis at this time revealed
a
46,XY,der(1)t(1;11)(p36.1;q13)[5],del(5)(q22q33)[20],r(7)(p13q21)[14],-7[6],
t(9;14)(q34;q24)[2][cp20] karyotype. FISH analyses using the probe D7S486
were performed on left and right cheek buccal smear samples and showed
normal results, indicating that the chromosome 7 deletions found on aCGH were
most likely acquired abnormalities. Repeated bone marrow analyses over the
next 6 months continued to show progression of chromosome abnormalities with
persistent mosaic monosomy 7. The patient was unresponsive to chemotherapy
and underwent allogenic cord blood stem cell transplant. He was also found to
have a dysplastic right kidney, hypertension, and developed cafÃ©-au-lait spots.
Mutation analysis for NF1 and SPRED1 showed normal results. Testing for the
FANCD1 (BRCA2) gene revealed compound heterozygous mutation, which is
considered to be an aggressive form of Fanconi anemia and present in about 7%
of Fanconi patients. DEB breakage studies confirmed the diagnosis of Fanconi
Anemia. Parental testing revealed heterozygous BRCA2 mutations in each parent
and an extensive history of multiple cancers on both sides of the family. At age
29 months the patient was diagnosed with a secondary malignancy, a stage
III clear cell renal carcinoma of the left kidney, and also developed a large
pericardial effusion. The left kidney was removed. A lesion developed on the
second kidney which could not be removed. The patient died at the age of 2.5
years. This unusual case illustrates the utility of microarray analysis in detecting
chromosomal abnormalities not seen by traditional cytogenetic analysis.
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37
BIALLELIC TP53 DELETION IN PLASMA CELL MYELOMA AND
ACUTE MYELOID LEUKEMIA
Linda Dean; Jackie Han; Rosa Belanger; Alicia Tamayo; Anthony Safo, DO;
Linda Pasztor, PhD, FACMG
p53 is a tumor suppressor protein which is encoded by the TP53 gene in
humans. TP53 is critical in multicellular organisms where it regulates the cell
cycle and thus functions as a tumor suppressor. p53 has many mechanisms of
anti-cancer function and plays an important role in apoptosis, genome stability,
and angiogenesis. If TP53 is mutated or deleted, tumor suppression is severely
compromised. Monoallelic TP53 loss occurs in lower frequency in hematological
malignancies than in solid tumors. Its importance in treatment resistance is welldocumented in CLL and plasma cell myeloma. However, TP53 alterations in
acute myeloid leukemia have not been widely examined. We present a case of
both monoallelic and biallelic TP53 deletion in acute myeloid leukemia. Case 1
was a 64 year old female with plasma cell myeloma which showed plasmablastic
features by morphology. Kappa-restricted plasma cells were observed in 36% of a
bone marrow aspirate. Interphase FISH revealed hyperdiploidy as well as monoand bi-allelic TP53 deletion. A MYC breakapart probe also showed that MYC
was involved in a translocation, a rare phenomenon in plasma cell myeloma.
Conventional cytogenetic analysis revealed a normal female karyotype. Case 2
was a 67 year old male with leukocytosis and extensive bone marrow involvement
(80-90% blast burden) by acute myeloid leukemia. The blasts displayed high
nuclear-cytoplasmic ratios with irregular indented/bilobed nuclei having
prominent to distinct nucleoli, finely granular cytoplasm, and no Auer rods.
Also present were a monocytic hyperplasia with shift toward immaturity to the
level of blast equivalents and prominent eosinophilic hyperplasia. Conventional
cytogenetic analysis revealed a 45,XY,der(9)t(9;17)(q34;q21),inv)16)(p13.1q22),17,add(17)(p12)[15]/46,XY[5] chromosome complement. This complex
hypodiploid karyotype predicted CBFB gene probe breakapart and biallelic TP53
by interphase FISH. Interphase FISH confirmed these hypotheses. Metaphase
FISH demonstrated the correct interpretation of the unbalanced rearrangement
between 9q and 17q. Biallelic TP53 deletion may confer an even greater adverse
effect than monoallelic deletion. However, although testing for TP53 deletion is
widely performed in CLL and plasma myeloma, its examination in myeloid disease
is not. Unlike the karyotype in this case, monoallelic/biallelic TP53 deletion may
not be readily appreciated by conventional cytogenetic analysis. Testing for p53
deletion by FISH will uncover the presence of TP53 loss important for prognosis
in myeloid neoplasia.
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39
8p11 MYELOPROLIFERATIVE SYNDROME: A RARE CASE OF
CONCURRENT MYELOID AND LYMPHOID NEOPLASMS IN
A 61-YEAR-OLD FEMALE RESULTING FROM FGFR1 GENE
REARRANGEMENT
Ernesto Taylor; Jagmohan Sidhu; Tracy Auster; Melissa Anderson; Violeta
Villani; Alma Ganezer; Daniel Di Bartolo; Steven Gersen
We present a patient with 8p11 myeloproliferative syndrome (EMS) - a rare,
atypical, biphenotypic hematologic disorder associated with FGFR1 gene
rearrangement. On initial examination, our patient, a sixty-one year old female,
presented with a rapidly growing lymphadenopathy, fevers, and night sweats.
Morphologic and flow cytometric analysis on a right inguinal lymph node
biopsy revealed immunophenotypic features consistent with T-lymphoblastic
lymphoma (T-LBL) and eosinophil hyperplasia. Upon analysis of the bone core
biopsy, however, a seemingly disparate picture emerged of excess eosinophil and
basophil precursors and dwarf megakaryocytes, which prompted a diagnosis of
myeloproliferative neoplasm with eosinophilia and basophilia. The patient was
subsequently administered an aggressive chemotherapeutic regimen (Linkers
regimen) to address her T-LBL. Shortly thereafter, she developed pancytopenia
and numerous blasts in the peripheral blood and bone marrow; the disease
had now transformed into an acute myeloid leukemia. Cytogenetic analysis of
a post-treatment bone marrow specimen produced the following karyotype:
50,X X,+8,t(8;13)(p12;q12),+13,+13,der(13)t(8;13)x2,+21[2]/51,idem,der(8)
t(8;13),+mar[18]. Fluorescence in situ hybridization (FISH) to metaphase cells
with a break-apart probe for FGFR1 demonstrated involvement of this gene in
the t(8;13). Interphase FISH for FGFR1, performed retrospectively on the original
lymph node biopsy, established that it was also disrupted at initial presentation,
thereby substantiating that this was a biphenotypic disease which must have
resulted from a genetic lesion involving FGFR1 in a pluripotent hematopoietic
stem cell. Herein we provide further detail on this rare case and discuss clinical
features, cytogenetic abnormalities, and the molecular mechanism underlying
EMS.
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40
CHROMOSOME MICROARRAY ANALYSIS (CMA) AS A DIAGNOSTIC
ROUTINE SERVICE IN SINGAPORE: ONE CENTRES JOURNEY
Yon Hui Yi; Tan Mui Li; Robin Roch; Maggie Brett; Tan Ene Choo; Lim Jiin Ying;
Breana Cham; Ivg Ng; Dr. Tan Ee Shien; Dr. Law Hai Yang; Dr. Angeline Lai
Genome-wide arrays are replacing conventional karyotyping in post-natal
diagnostics and it is recommended as a first-line test for the evaluation of
individuals with multiple congenital anomalies, developmental delay, intellectual
disability, or an autism spectrum disorder. Our embarkment on the CMA journey
started with a research grant, which analyzed more than 300 samples over a
period of six years. This research experience was then translated onto diagnostic
service. From June 2013 to December 2013, a pilot study was conducted with
53 post-natal samples which were referred from patients with a variety of clinical
phenotypes ranging from development delays, cognitive delays, craniofacial
dysmorphisms, musculoskeletal and neurological disorders. We performed the
analysis using Agilent Technologies 4x180K SurePrint G3 Human CGH+SNP
Platform and Cytogenomics software. Copy number variants (CNVs) ranging in
size from 104 kb to 36.4 Mb, were found in 20 out of 53 samples. In five patients,
pathogenic CNVs were found in known microdeletion and microduplication
syndrome regions including the Cat Eye syndrome, Cri du Chat syndrome,
Williams syndrome, Miller Decker syndrome and 3q29 microdeletion syndrome.
Fifteen samples (28%) had CNVs <3 Mb that might not have been detected
by traditional G-Banding analysis. CMA was shown to be useful in revealing
the imbalances which was not specifically identified by conventional cytogenetics
and Fluorescence in-situ Hybridization (FISH) for patients with variable clinical
features. The additional information will aid clinicians in diagnosing and
pinpointing cause of anomalies in patients. Therefore, CMA will be most helpful
as a routine diagnostic tool.
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41
CLINICAL IMPLEMENTATION OF CMA ANALYSIS IN A
DIAGNOSTIC LABORATORY: EXPERIENCE WITH OVER 1000
POSTNATAL CASES
Monika Thapa PhD; Patricia J. Mouchrani, MS; Margaret J. Barch, MS;
Kathryn Platky, MS; Joseph H. Hersh, MD
Background: Chromosomal microarray analysis (CMA) is a powerful and highly
sensitive technique for detecting copy number variations in the human genome
mainly for unexplained developmental delay/intellectual disability, multiple
congenital anomalies and autism spectrum disorders. Objective: To evaluate the
importance of CMA analysis in pediatric clinical practice. Method: Peripheral
blood specimens were collected for CMA testing. High-density oligonucleotide
array NimbleGen CGX-3 platform with 3x135K array was used for the CMA testing
and the results were validated using conventional karyotyping/FISH techniques.
Results: A total of 1,234 cases were received by the Frank F. Yen Cytogenetics
Laboratory at the Weisskopf Child Evaluation Center in the Department of
Pediatrics at the University of Louisville from March 2011 through June 2013.
Pathogenic CMA results were obtained in 17.9% (221/1,234) cases, of which
135 aberrations were smaller than 3 Mb, 15 between 3-5 Mb and 71 > 5
Mb. Approximately 10.9% (135/1,234) of clinically significant aberrations would
have remained undetected if only conventional karyotyping was performed on
these cases, although there were instances when CMA rather than chromosomal
analysis was performed to confirm a chromosomal syndrome that was strongly
suspected clinically and was diagnosable by chromosomal analysis. Conclusion:
Our experience provides support to previous claims that CMA is a first-line
diagnostic test for unexplained clinical phenotypes and for characterizing
submicroscopic genomic imbalances. In contrast, conventional cytogenetic
analysis is the diagnostic study of choice when a recognizable chromosomal
syndrome is thought to be present based on clinical findings.
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42
NEXT GENERATION SEQUENCING TECHNOLOGIES AND THE
DEVELOPMENT OF A PANEL FOR SKELETAL DYSPLASIAS
Jessica A. Cooley, BS, MB(ASCP)CM; Stephen McGee, MS; Katharine Kubiak,
MS; Kellie King, MS, CGC; Jamie Butler, BS; Jennifer A. Lee, PhD, FACMG;
Julie R. Jones, PhD, FACMG; Michael J. Friez, PhD, FACMG; Monica J.
Basehore, PhD, FACMG
Skeletal dysplasias make up a large and diverse group of over 450 disorders
affecting bone and cartilage formation, many including limb shortening,
lengthening, complete or partial limb absence, along with other abnormalities.
Analyzing genes one at a time by Sanger sequencing can be costly and timeconsuming and may even lead to a delay in diagnosis. In an effort to provide
testing in a more rapid and cost-effective manner, a skeletal dysplasia panel has
been developed for use with Next Generation Sequencing (NGS) that includes 10
genes (COL1A1, COL1A2, COL2A1, COMP, SLC26A2, FGFR3, FLNA, HSPG2,
SOX9, TRPV4) known to be associated with various skeletal dysplasias. About
90% of individuals with a skeletal dysplasia disorder are found to have a mutation
in at least one of these ten genes. RainDanceTM Enrichment and SOLiDTM
NGS 5500xL technologies are used to analyze these 10 genes. RainDanceTM
Enrichment uses micro-droplet based technology. Both primer library droplets
and genomic DNA template are added to a microfluidic chip, and an electric
field causes the two paired droplets to merge into one droplet so that they can be
amplified by PCR. The emulsion must then be broken to release the amplified PCR
products into solution so they can be used for NGS. During a SOLiD sequencing
run, five rounds of primers are used to sequence the template by ligation of
di-base labeled probes. When the probe containing the correct base pairs binds
to the template, a fluorescent signal is given off that the SOLiD can detect. For
the validation of the skeletal dysplasia panel, 16 patients were used, 7 with a
known molecular diagnosis, and 9 with an unknown diagnosis. The NGS results
found at least one mutation or variant of unknown significance for 15 of the 16
validation patients. A cohort of 42 patients, including the 9 unknown validation
patients, was analyzed and 19 (45.2%) patients had at least one pathogenic or
likely pathogenic alteration. Many other patients have been run since this original
data was collected. In addition, our lab has recently purchased an Illumina MiSeq
NGS platform. Sequencing by synthesis (paired-end) takes place as the machine
adds all 4 dNTPs simultaneously. Each dNTP has a specific fluorescent molecule
attached which will release a signal of differing wavelength for each of the 4
bases, allowing the instrument to analyze the data. The MiSeq has a shorter
sample preparation time and shorter instrument run time (12-24 hours compared
to the SOLiD run time of about 1 week) which will help results to be obtained
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quicker. Many of our skeletal dysplasia samples are prenatal samples, so a
quicker turnaround time for this test would be beneficial. Given the high detection
rate of alterations for this skeletal dysplasia panel (45.2%), the ability to barcode
and run multiple patients at once, the ability to analyze multiple genes at once,
and the short time period in which all this work can be accomplished, it can be
concluded that next generation sequencing technologies have many advantages
over Sanger sequencing.
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43
CLINICAL PRESENTATION OF A PATIENT WITH MOSAIC WHOLE
CHROMOSOME PUPD 11 RESULTING IN BECKWITH-WIEDEMANN
SYNDROME
Mark Rolla, BS, CG(ASCP)CM; Jillene Kogan, MD, PhD; Debra A. Rita, MD;
Aida Catic, MS, CG(ASCP)CM
Beckwith-Wiedemann syndrome (BWS) is a growth disorder resulting from an
altered expression of imprinted genes at 11p15. Differentially methylated imprinting
centers (IC) on chromosome 11 containing growth regulating genes are believed to
play a direct role in the observed BWS phenotype. Affected individuals may present
with macrosomia, visceromegaly, embryonal tumors, omphalocele, ear creases/
pits, renal abnormalities, and other phenotypic abnormalities. Several molecular
mechanisms can be responsible for the altered expression of these critical genes:
gain or loss of methylation at IC1 and IC2, respectively, mutation of the maternal
CDKN1C allele, paternal Uniparental Disomy(UPD) of 11p15, and various
cytogenetic aberrations involving 11p15 region. Multiple molecular and cytogenetic
testing techniques are available to identify the cause of BWS including whole
genome SNP array analysis. We present the case of a patient with a mosaic (~36%)
whole chromosome pUPD 11 karyotype resulting in a BWS diagnosis and document
our laboratorys experience in identifying the underlying molecular mechanism. The
patient was born prematurely at 24 3/7 weeks gestation and initially presented
with retinopathy of prematurity, chronic lung disease, anemia, apnea, and bilateral
inguinal hernias. The patient was later observed to have macroglossia and
abnormal facies. A differential diagnosis included Beckwith-Wiedemann syndrome,
hypothyroidism, chromosomal abnormality, and possible normal variant. Whole
genome SNP microarray analysis was completed and an abnormal allele peak track
consistent with mosaic UPD of the entire chromosome 11 was observed. In order
to determine parental origin of the UPD 11, methylation-sensitive multiplex ligation
dependent probe amplification (MLPA) was ordered and confirmed a paternal
origin. The patients final karyotype, arr[hg19] 11p15.5q25(198,510-134,942,626)
x2 mos hmz pat (~36%), supports a BWS diagnosis. After diagnosis, the patient
developed a dramatically rapidly growing mass within the abdomen which was
diagnosed as a hepatoblastoma, epithelial type. Commercially available platforms
for whole genome SNP microarray are increasingly helpful in providing an accurate
and rapid analysis where classical cytogenetics falls short. The determination of
zygosity patterns is critical to the care of patients suspected of conditions involving
genomic imprinted regions and recessive disease alleles. The mechanism resulting
in pUPD 11 most likely occurred when a gain/loss resulted in an abnormal cell line
which was then rescued. The timing of these two separate events is critical for the
development of mosaic cell lines giving rise to the entire fetus. The importance of
the SNP analysis in this case is that it revealed such an extremely rare mechanism.
Only one other case of whole chromosome pUPD 11 resulting in BWS has been
reported (see Dutly, et al., 1998, AM J Med Genet 79(5):347-53). The resulting
diagnosis of BWS has guided medical management of this patient.
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44
TRISOMY 13 IS A RARE PRESENTATION IN MYELODYSPLASTIC/
MYELOPROLIFERATIVE NEOPLASM, UNCLASSIFIABLE
Patricia LeMay; Noel Kowal, MD; Wendy Shertz, MD
Trisomy 13 as the sole cytogenetic aberration in patients with hematological
neoplasms is both exceedingly rare and a poor prognostic indicator. We present
a case of a 77 year old woman with a chronic myelodysplastic/myeloproliferative
neoplasm with a trisomy 13 in the absence of a BCR/ABL gene rearrangement.
The patient presented with symptoms of pneumonia including fever, chest pain,
and shortness of breath, and a peripheral blood differential showing an absolute
monocytosis at 17 000 (WBC 70 700), anemia, and thrombocytopenia. A bone
marrow biopsy showed hypercellular marrow with a 3:1 myeloid:erythroid ratio with
full range of maturation and unilineage granulocytic dysplasia; megakaryocytes
were increased in number. Mild fibrosis and scant stainable iron were observed
by reticulin and iron stains, respectively. Analysis by flow cytometry showed the
presence of 83% immature myeloid precursor cells with the following positive
(CD64, CD16, CD13, CD33, CD15, CD10, CD32, and myeloperoxidase) and
negative (HLA-DR, CD56, and CD117) immunophenotypic pattern, respectively.
Cytogenetic analysis of bone marrow aspirate revealed a 46, XX, +13 karyotype.
Florescent in-situ hybridization for BCR/ABL gene rearrangement utilizing Vysis
LSI BCR/ABL1/ASS Tri-Color Dual Fusion Probe showed no evidence for BCR/ABL
gene rearrangement. Trisomy 13 has been reported rarely in myelodysplastic/
myeloproliferative neoplasm, unclassified. In a Mayo Clinic study (1990-2006)
of 27 patients with trisomy 13 in hematologic malignancies only 4 patients had
myelodysplastic/myeloproliferative disorder1. The Mayo Clinic study concluded
trisomy 13 in hematological malignancies is usually associated with elderly males
and carries a poor prognosis.
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45
THE DIAGNOSIS OF MOSAIC TRISOMY 14 IN A FEMALE INFANT
Carol S. Deeg CG(ASCP); Don Roman CG(ASCP); Elizabeth Hamelberg
CG(ASCP); Sheri Hedricks CG(ASCP); Aimee McKinney CG(ASCP); Inga
Calloway CG(ASCP); Sarah Ramsey CG(ASCP); Devon Lamb Thrush, MS,
CGC; Sayaka Hashimoto, MS, CGC; Shalini Reshmi, PhD; Caroline Astbury,
PhD
Peripheral blood from a 2 day-old female was received for STAT high resolution
chromosome analysis. Her phenotypic findings included low-set ears, a small
chin, and a sacral dimple with a hair tuft. She had a normal echocardiogram,
renal untrasound and brain MRI, but an abnormal hearing test. The STAT
chromosomes were interpreted as 46,XX. Subsequent oligonucleotide array
indicated a pattern suggestive of trisomy 14 mosaicism. Additional analyis of
the peripheral blood specimen revealed that 4% (2/53) of metaphase cells
contained a third chromosome 14 (mos 47,XX, +14[2]/46,XX[51]. These findings
were also confirmed with FISH using the subtelomere probe specific for the
long arm of chromosome 14, which demonstrated three chromosomes 14 in
12% (6/50) of metaphase cells counted and three signals for the probe in 21%
(21/100) of interphase cells. Uniparental disomy 14 (UPD 14) testing was then
requested, to determine if UPD in the normal cell line may be contributing to
the patient’s phenotype. Normal biparental inheritance was reported (by an
outside laboratory). The patient had a normal methylation index, indicating that
both unmethylated (maternal) and methylated (paternal) alleles were present.
While full trisomy 14 is frequently observed in products of conception and is
generally considered to be incompatible with life, mosaicism for trisomy 14 has
been described. Approximately 20 patients have been reported with trisomy 14
mosaicism since its first description in 1975 by Rethore’ et al. Most, if not all,
cases are de novo. The syndrome presents with a distinct phenotype, including
growth retardation, low-set ears, asymmetrical growth, short stature, failure to
thrive, broad nose, brain anomalies, micrognathia, short neck, congenital heart
disease, and deafness. The phenotype appears to be more severe in those
individuals with a higher percentage of trisomic cells. This case again emphasizes
the benefits of utilizing combined cytogenetic methods for determining a clinical
diagnosis.
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46
THE UTILIZATION OF HIGH RESOLUTION CHROMOSOME
ANALYSIS, ARRAY CGH AND FISH IN DETECTING A COMPLEX
CHROMOSOME REARRANGEMENT IN A MALE PATIENT WITH
der(20)t(X;20)(q28;p13)
Olivia Thoele, BS, CG(ASCP); Leslie Willis, BS, MG(ASCP); Heather Workman,
MS, CGC; Rae Jean Spatz, BS, CG(ASCP); Sue Ann Berend, PhD, FACMG
We report a 12 month old male patient with global developmental delay,
hypotonia, microcephaly, low set ears and failure to thrive. Upon examination, it
was noted that patient had a petite mouth and mild maxillary hypoplasia and the
inability to sit independently; who was found to have a complex chromosomal
translocation involving chromosome 20 and the X chromosome, detected by
standard high resolution chromosome analysis and microarray comparative
genomic hybridization (aCGH). High resolution chromosome analysis performed
in 2013 showed additional material of unknown origin on the short arm of one
chromosome 20 at 20p12. Microarray analysis was performed and showed a
4 Mb duplication at band Xq28 (MECP2 gene), 312kb deletion in band 20p13,
1Mb duplication at band 20p13, and 384kb duplication at band 18p11.32.
The karyotype is arr Xq28(150,721,923-154,912,933)x2,18p11.32(330,610714,609)x3,20p13(13,039-325,929)x1, 20p13(369,370-1,315,670)x3. FISH
using the probe RP11-954J6 (Xq28), and XYpter subtelomere probe revealed a
derivative chromosome 20 resulting from an unbalanced translocation between
chromosome 20 and the X chromosome[ish der(20)t(X;20)(q28;p13)(RP1195J6+)]. The Xq28 duplication region contains approximately 90 OMIM genes
including MECP2 decipher gene. This MECP2 decipher gene region is typically
seen in patients with Rett Syndrome, clinically defined as neurodevelopment
disorder, a condition which affects mostly female patients and is characterized
by intellectual disability, delayed development, and seizures. The 20p13 deletion
region contains at least 11 OMIM genes including TRIB3, DEFB32, ZCCHC3,
NRSN2, and SOX12. The 20p13 duplication region includes at least 14 OMIM
genes which includes TCF15, SDCBP2, SCRT2, and SNPH. The 18p11.32
duplication region contains several genes. Parental microarray studies were
performed on both parents of the patient, which revealed that both parents were
negative for the Xq28 duplication, 18p11.32 duplication, 20p13 duplication and
the 20p13 deletion seen in this childs study-; therefore these were de novo findings.
De novo findings are more likely to impact the development of the patient. This
study demonstrates the usefulness of combining aCGH with cytogenetic analysis
and FISH for detecting complex genomic imbalances. Â Materials & Methods Â
Metaphase cells were prepared from PHA “stimulated peripheral blood culture
using standard methods. Slides were Giemsa-trypsin banded for routine analysis.
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Â Fluorescence in-situ hybridization was performed using probes for the XYpter
subtelomere (Rainbow Scientific Cytocell) as the internal control along with BAC
clone (Blue Gnome) RP11-954J6 specific to the Xq28 region. FISH was performed
according to the laboratory protocols. Image analysis of karyograms and FISH
pictures was performed on Cytovision image analysis systems. Â CGH microarray
testing was performed on the proband using the Agilent Technologies system
(ISCA 180K Oligo Array version 1.0, Cytochip platform). Microarray analysis
on both the probands parents was performed on the ISCA 180K Oligo Array,
version 1.0, Cytochip platform as well (using the UCSC 2006 hg 18 assembly).
Discussion We present here a male patient initially diagnosed with 46,XY,add(20)
(p12)-with additional material of unknown origin on the short arm of one
chromosome 20 at 20p12 at the age of 13 months old. FISH studies showed that
the material was derived from the Xq28 region; thus, the genomic imbalances
involving the X chromosome and chromosome 20 identified by the microarray are
likely the results of an unbalanced translocation between chromosome 20 and
the X chromosome, 46,XY,add(20)(p12).ish der(20)t(X;20)(q28;p12). Duplication
of the Xq28 region, specifically including the MECP2 decipher gene, is seen in
patients with Rett Syndrome, a condition characterized by intellectual disability,
delayed development, and seizures (primarily in females, as it is lethal to males).
However, in recent years the duplication in the MECP2 gene has been seen in
male patients. Typically these males present with a phenotype to that of Rett
Syndrome when there is an extra X-chromosome or a mosaic cell line for the
mutation. However, duplication of the MECP2 gene region in males in non-mosaic
form and in chromosomally normal males result in a severe neurodevelopment
disorder, including developmental delay and severe mental retardation, recurrent
infections, spasticity and seizures. Many of these duplications are inherited from
a carrier mother. Parental studies were negative both by the chromosome study
and by microarray (also negative for the 20p13 loss, 20p13 gain, and 18p11.32
loss which was also seen in the proband via microarray); thus, these findings
were de novo aberrations. De novo unbalanced chromosomal rearrangements
are more likely to impact the development of this patient. This case illustrates the
capability of utilizing high resolution chromosomes analysis with array CGH and
subsequent FISH testing to evaluate patients structural aberrations, and provide
a specific diagnosis.
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47
A CASE OF RELAPSED ACUTE MYELOID LEUKEMIA WITH A T(15;17)
NOT ASSOCIATED WITH ACUTE PROMYELOCYTIC LEUKEMIA
Laura Ross; B. Schneider; A. Safo, MD; W.L. Flejter, PhD
A bone marrow sample from a 46 year old male with a history of AML was
evaluated by morphology, flow cytometry and cytogenetics after receiving
four cycles of high-dose consolidation therapy. There was no morphologic or
immunophenotypic evidence of acute myeloid leukemia. Cytogenetic analysis was
normal. Seven months later, our laboratory received a follow-up bone marrow
for testing with morphology, flow cytometry, cytogenetics and fluorescence in
situ hybridization (FISH). Morphology revealed relapsed acute myeloid leukemia
with 10-15% blasts. The blasts displayed high nuclear-cytoplasmic ratios with
finely granular cytoplasm but no Auer rods. They lacked the typical bi-lobed
nuclei and hypergranular cytoplasm of classic-type leukemic promyelocytes
and no œfaggot cells were identified. The residual hematopoietic elements
showed striking trilineage dysplasia. Flow cytometry detected increased blasts
(9%) expressing CD34, CD117, and HLA-DR. Cytogenetics showed a complex
karyotype: 45,XY,add(5)(q31),-7,inv(9)(q11q13[2]/46,idem,+mar[3]/45-46,idem,
t(15;17)(q22;q;21),+mar[cp12]/46,XY[3]. The morphology of the t(15;17)
appeared identical to that typically observed in acute promyelocytic leukemia
(APL). An AML FISH Profile including probes for PML/RARA was performed.
No PML/RARA fusion was seen in any of the 200 interphase nuclei examined.
However, 81/200 cells showed an additional copy of RARA. FISH analysis of
metaphase cells showed a third RARA signal on the long arm of chromosome 5.
These results indicate that the t(15;17) identified by cytogenetics likely involved a
rearrangement of genes other than PML and RARA. Further, the breakpoint on
chromosome 5 is not consistent with the variant APL t(5;17). Although the t(15;17)
is most often seen in APL, it may also be seen in treatment-related MDS or AML.
Due to therapeutic implications, this study illustrates the importance of FISH as a
tool for the characterization of a t(15;17) observed in patients with features not
typical of APL.
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48
COMPLEX KARYOTYPE INCLUDING A PARACENTRIC INVERSION
AND AN APPARENTLY BALANCED THREE-WAY TRANSLOCATION:
46,XX,INV(3)(Q23Q28),T(6;17;15)(P21.1;P11.2;Q26.1)
Jeffrey Wobser; Elizabeth Hamelberg, CG(ASCP); Carol Deeg, CG(ASCP);
Inga Calloway, CG(ASCP); Aimee McKinney, CG(ASCP); Devon Lamb-Thrush,
MS, LGC; Sayaka Hashimoto, MS, LGC; Robert Pyatt, PhD; Shalini Reshmi,
PhD, FACMG; Caroline Astbury, PhD, FACMG
A 1 day old female with dysmorphic features, hypoplastic genitalia, sandal gap
toes, and hypertelorism was referred to our laboratory for chromosomal analysis
of peripheral blood. High resolution chromosome analysis revealed a modal
number of 46 chromosomes including two X chromosomes. However, each
cell had one chromosome 3 with a paracentric inversion in the long arm, with
breakpoints estimated at bands q23 and q28. Each cell also had a complex
translocation involving one chromosome 6, one chromosome 15, and one
chromosome 17. The short arm of chromosome 6 (bands 6p21.1 to the terminus)
was attached to chromosome 17 at band 17p11.2. The short arm of chromosome
17 (bands 17p11.2 to the terminus) was attached to chromosome 15 at band
15q26.1. The long arm of chromosome 15 (band 15q26.1 to the terminus) was
attached to chromosome 6 at band 6p21.1. Fluorescence in situ hybridization
(FISH) using subtelomeric probes for the involved chromosomes (Tel 3q, Tel
6p, Tel 15q, Tel 17p; Abbott Molecular) confirmed that the chromosome 3q
terminus was intact and the arms of the three-way translocation were exchanged
as described above. By G-banding, these chromosome abnormalities did not
suggest a gain or loss of material at the breakpoints and suggested an apparently
balanced three-way translocation and apparently balanced inversion. Microarray
was ordered to extend these findings. Chromosomal microarray (Nimble Gen
135K oligonucleotide array) suggested that these abnormalities were more
complex including regions of loss not detectable by G-banding. The microarray
analysis revealed a 1.84 Mb loss within chromosome bands 3q27.3 through
3q28 containing 3 genes and 4 non-coding RNAs. Additionally, there is a 1.41
Mb loss within chromosome bands 6p21.2 through 6p21.1 containing 20 genes
and 1 micro RNA. A likely benign copy number change was also identified at
16q23.1. This case reiterates the clinical utility of follow up microarray analysis of
an apparently balanced, complex chromosomal rearrangement in a patient with
phenotypic findings.
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49
THREE CASE STUDIES OF MOSAIC DELETION OF 22Q13.3
Lisa Warren; Elena Repenikova; Julie Joyce
We describe three separate blood cases with different levels of mosaicism for
deletion of 22q13.3, also known as Phelan-McDermid syndrome. Mosaicism
should be seen in routine chromosomes, FISH and microarray (aCGH). PHA
stimulated blood cultures were set on all patients. FISH probes N85A3 probes
were used. Microarrays were run on all three patients. Deletion of 22q13.3 was
seen in 35% of the metaphase cells in case one, in 6% of metaphase cells in
case number two and 29% of metaphase cells in case number three. FISH using
probe N85A3 for 22q13.3 show a single copy of N85A3 in 51% of nuclei in case
number one, in 16.5% of nuclei of case number two, and in 32.5% of nuclei
in case number three. Copy number oligonucleotide microarray analysis shows
an approximate 7.3 Mb mosaic terminal deletion within chromosome bands
22q13.2q13.33 that contains 50 genes including SHANK3 for case number
one. Copy number oligonucleotide microarray analysis was negative for DNA
copy number variants in case number two and three. Phenotypic characteristics
of mosaic deletion 22q13.3 individuals may vary greatly depending on the
distribution of contributing cell populations. Ultimately aCGH could not be used
as a stand-alone test to fully elucidate the patients chromosome mosaicism, thus
demonstrating the necessity of routine chromosome analysis with aCGH and
FISH.
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50
EARLY CONFIRMATION OF SUSPECTED CYTOGENETIC
ABNOMALITIES USING WHOLE BLOOD LYMPHOCYTE ISOLATION
Kimberly Machowski, CG(ASCP), MB(ASCP); Kirsten Casavant, CG(ASCP);
Solveig Pflueger, PhD, MD, FACMGE
Introduction: Early confirmation of suspected cytogenetic abnormalities in
newborn babies is an important service in the cytogenetic laboratory. It is a
useful tool for the physician not only for diagnosis but also for giving resolution to
the parents and family of the newborn. With traditional karyotyping, a preliminary
result is usually not available for 48-72 hours from time of specimen receipt.
Using the Stemcell EasySep Whole Blood Lymphoid Positive Selection kit and
FISH technology, preliminary results may be available within 24-48 hours from
time of specimen receipt. Here, the results are reported detailing the validation
that used a parallel study performed on 14 peripheral blood cases analyzed
both cytogenetically as well as with FISH on isolated whole blood lymphocytes.
Materials and Methods: 0.2-1.0ml of peripheral blood was taken and isolated
using the EasySep whole blood lymphoid selection kit protocol. FISH was
performed on the patient slide and a normal control using one of three probe
sets depending on the indication for the case: numerical, microdeletion, or sex
confirmation. Two technologists scored 100 cells each of patient and control
slide. An Excel spreadsheet was created to show a side-by-side comparison of
the results. Results: There was 100% concordance between FISH and cytogenetic
findings on the 14 cases involved in this validation. Conclusion: This validation
showed that EasySep technology can enable the cytogenetic laboratory to
provide physicians with preliminary results on newborns earlier than traditional
karyotyping when a known cytogenetic abnormality is suspected.
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51
ATYPICAL CLINICAL PRESENTATION OF AN INFANT
HEMATOPOEITIC NEOPLASM WITH AN MLL-SEPT6
REARRANGEMENT
Sudabeh Balakhani; Sudabeh Balakhani; Billie Carstens; Lynne Meltesen;
Xiayuan Liang; Kelly Maloney; Veronica McDaniel; Charla Spies; Lynn Werhane;
Diane Minka; Karen Swisshelm
We present an interesting case of a five month old infant who presented to
Childrens Hospital Colorado with absolute neutropenia, atypical monocytosis,
and moderate thrombocytopenia. The bone marrow aspirate demonstrated
19% abnormal monocytes and promonocytes, with flow cytometric analysis
also revealing a population of abnormal monocytes. The initial differential
included myelodysplasia versus acute myeloid leukemia (AML). Cytogenetic
studies were submitted with a FISH panel that included MLL (KMT2A). Interphase
molecular cytogenetics (FISH) studies were positive for an MLL rearrangement
in 31% of cells scored. Since the blast percentage in the aspirate was below
20%, it was classified as an acute monocytic neoplasm. A standard cytogenetic
culture revealed an ins(X;11)(q24;q23.3q13.1) with sequential metaphase FISH
demonstrating the 5th portion of MLL inserted into the long arm of chromosome
X and the 3rd portion of the probe remaining on the deleted chromosome 11.
This is unlike the more common reciprocal translocations in which the active 5th
centromeric portion of the probe remains on the derivative chromosome 11.
This altered juxtaposition may be due to the opposite transcriptional direction of
SEPT6 on Xq24, requiring more complex and sometimes cryptic rearrangement
mechanisms such as inversion/insertion and recombination events to produce
the MLL-SEPT6 chimeric protein. Nearly half of the reported cases of MLL-SEPT6
have presented with monocytic differentiation and almost all were less than 2
years old presenting as AML. The (X;11) MLL-SEPT6 rearrangement is observed
infrequently, making up about 1.9% of all AML patients and less than 0.01% of all
acute leukemias (C. Meyer et al., Leukemia 27: 2165“2176, 2013).
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Student Poster Presentations
10:40 a.m. – 11:20 a.m.
Student Poster Presenters are requested to stand by their posters on
Saturday, June 14, from 10:40 a.m. – 11:20 a.m. to respond to attendee
questions or for further discussion.
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Student Poster Abstracts
S1
IDENTIFICATION OF METHYLATED INK4A CO-EXISTING COPY
NUMBER VARIATIONS AND LOSS OF HETEROZYGOSITY IN
CIRCULATING CELL-FREE TUMOR DNA FROM HEPATOCELLULAR
CARCINOMA PATIENTS (UPDATED RESULTS)
Karam Hadidi; Gengming Huang, PhD; Peter Hu, PhD; Jianli Dong, MD, PhD
BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most prevalent
and lethal cancers worldwide. When HCC is diagnosed at an early stage, the
patient can be cured by surgical resection, liver transplantation, or percutaneous
radiofrequency ablation. However, larger and advanced-stage tumors have poor
prognosis. Therefore, the need to improve early diagnosis of HCC is urgent.
Imaging scan and alpha-fetoprotein (AFP) are currently used to screen and
diagnose HCC, but new genetic and epigenetic markers are being discovered to
improve sensitivity and specificity. Studies have shown that the inhibitor of cyclindependent kinase (CDK) 4 gene (INK4A) is inactivated by methylation in 70-80%
of HCC liver tissues, and at various frequencies in the circulating cell-free DNA
(cfDNA) of HCC patients. The present study aimed to assess whole genome
copy number variants (CNV) and copy-neutral loss of heterozygosity (LOH) using
circulating cfDNA, and identify recurrent chromosomal changes that may serve
as diagnostic markers of HCC. We hypothesize that microarray analysis on serum
samples with high INK4A methylation will reveal the CNV and LOH present in the
patients genomic DNA as well as the HCC tumor. HCC-specific aberrations will
be elucidated by comparison of the serum and whole blood microarray results.
METHODS: Serum and peripheral blood lymphocytes (PBLs) from cell pellet were
collected from 149 subjects with an AFP value of at least 100 µg/L. Circulating
cfDNA was purified from all serum samples and underwent bisulfite conversion,
PCR of the INK4A promoter, and pyrosequencing. Samples with >15% methylation
of the INK4A promoter, as well as one HCC tissue sample from liver biopsy, were
selected for SNP chromosome microarray using CytoScan (Affymetrix). Results of
INK4A methylation, CNV, and LOH changes in serum and corresponding PBL will
be analyzed and compared. RESULTS: Pyrosequencing of the serum samples has
revealed 7 samples from 5 specimen with >15% INK4A methylation in cfDNA.
Pyrosequencing of the corresponding PLB samples has revealed that each has an
INK4A promoter methylation below 5%. Whole genome chromosome microarray
analysis has been performed on three serum samples and the HCC tissue sample
thus far. Many recurrent CNV’s have been identified in the tested samples,
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including copy number losses on the q arm of chromosome 4 and the p arm of
chromosome 17, as well as copy number gains on the q arms of chromosome
8, 14, 19, and 20. DISCUSSION: Once the microarray data is generated for
all remaining serum and PLB samples, HCC specific DNA aberrations will be
identified by comparison of the cfDNA results to the PLB DNA results. CNVs that
occur in the cfDNA should be characteristic of both the HCC tumor and the
normal genome, due to the high concentration of INK4A methylated DNA. DNA
from the blood pellet should only represent the patient’s hereditary genome,
because the concentration of INK4A methylated DNA is below the microarray
threshold of sensitivity. In the future, these identified chromosomal changes may
be used to non-invasively diagnose early-stage HCC, as opposed to waiting for
a liver nodule to reach a certain size and possibly metastasize.
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S2
A SYSTEMATIC REVIEW OF LRRK2 GENE VARIANTS IN
PARKINSON’S DISEASE PATIENTS AMONGST ETHNIC GROUPS
Jeremiah Sherwood; Heather Aldis; Megan Rist; Chandler Culbreath; Shawn
Miller
Parkinson’s Disease (PD) is the second most common neurodegenerative disease
next to Alzheimer’s. Recent studies have shown that genetics play a larger role
than previously thought, and a majority of the genetic alterations implicated
in the pathogenesis of PD include variants of the LRRK2 gene. Products of the
LRRK2 gene disrupt MAP kinase pathways, increasing neuronal toxicity, and
modulate protein-protein interactions, leading to increased aggregation of
α-synuclein and tau proteins. We conducted a systematic review to assess the
worldwide distribution of LRRK2 G2019S, A419V, and Q1111H SNPs in order
to determine their relative frequencies among ethnic groups. The results of nine
studies examining 39,055 individuals, obtained from PubMed, Summons, and
SCOPUS were summarized in a systematic review. The papers included in this
review measured LRRK2 variants using multiple platforms including SNP arrays
(3/9), and MLPA (1/9), direct sequencing (2/9), and PCR sequencing methods
(3/9). Four studies show combined frequencies of 26.04% and 30.43% for the
mutant G2019S allele in Ashkenazi Jew and Arab populations, respectively, and
two show an overall frequency of 2.45% for the A419V mutant allele in Asians.
Although multiple studies indicate that Q1111H mutations most frequently occur
in Latin American populations, sufficient frequency data was not available for
analysis. Knowing the frequency of LRRK2 variants among different populations
allows for better appropriation of resources for genetic testing, and provides
a framework for future endeavors focused on disease prevention. More testing
should be conducted to assess the significance of these and other regional
hotspots in Parkinson’s Disease.
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S3
PPC MITOGEN COCKTAIL INCREASES MITOTIC INDEX IN MATURE
B-CELL NEOPLASMS OTHER THAN CLL
Ching-Hua Liu; Soo Ha Cheong; Binh Tan Vo; Elizabeth Harper Allen; Peter C.
Hu; XinYan Lu
Background: Success of mature B-cell cultures is often hindered by low mitotic
index. Previous study has shown the enhanced detection of chromosome
abnormalities in chronic lymphocytic Leukemia (CLL) by conventional cytogenetics
using PPC cocktail [1]. The PPC mitogen cocktail, consists of Pokeweed (PKW),
phorbol-12-myristate-13-acetate (PMA), and CpG oligonucleotides (CpGODN), has been proved to give consistent karyotype and has since been
adapted by many laboratories for the stimulation of CLL cultures [2]. However,
PPC mitogen cocktail in other mature B-cell neoplasms culturing hasn’t been well
studied. Hypothesis: We hypothesize that PPC cocktail works as a new mitogen
for mature B-cell neoplasms other than CLL, resulting in higher mitotic index and/
or higher clonal chromosomal aberration detection rate. Methods: Bone marrow
aspirations were obtained from ten patients and 72 hours parallel culturing was
set up, using lipopolysaccharide (LPS) and PPC. The cultures were harvested and
suspensions were dropped using standard protocol. The slides from both cultures
were g-banded using trypsin and stained with Leishman(GTL). Mitotic index was
calculated, averaging counts from three random areas on the slide using the
following equation: number of metaphase/(number of metaphase+number of
interphase)x100. Results: Higher mitotic index in PPC mitogen cocktail stimulated
bone marrow culture was observed in seven out of ten samples tested, in comparison
with standard LPS culture. Conclusion: The result supports our hypothesis that
PPC yields good mitotic index in mature B-cell neoplasms, although our sample
size is limited. PPC mitogen cocktail might be used to produce a better mitotic
index, and eventually help increase the chromosomal aberration detection rate.
References: 1.Muthusamy, N., Breidenbach, H., Andritsos, L., Flynn, J., & Jones,
J. (2011, February). Enhanced detection of chromosomal abnormalities in chronic
lymphocytic leukemia by conventional cytogenetics using CpG oligonucleotide
in combination with pokeweed mitogen and phorbol myristate acetate. Cancer
Genetics, 204(2), 77-83. 2. Heerema, N., Byrd, J., Dal Cin, P., Dell’ Aquila, M.,
Koduru, P., Aviram, A., & Smoley, S. (2010, December). Stimulation of chronic
lymphocytic leukemia cells with CpG oligodeoxynucleotide gives consistent
karyotypic results among laboratories: a CLL Research Consortium (CRC) Study.
Cancer Genetics and Cytogenetics, 203(2), 134-140.
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S4
GENETIC ALTERATIONS OF ACUTE LYMPHOBLASTC LEUKEMIA
DETECTED BY ARRAY CGH: META-ANALYSIS OF 13 STUDIES AND
1428 SUBJECTS
Hiroko Sato; My Nguyen; Mina Wei; Sam Beriji; Tracy Garza; Vicki L.
Hopwood; Ming Zhao; Jun Gu
Current advanced cytogenetic technique, array comparative genomic
hybridization (aCGH), has enabled more accurate detection of cryptic genetic
aberrations in patients with Acute Lymphoblastic Leukemia (ALL), aiding the
process of disease classification and prognostic evaluation. The purpose of this
study was to review new genetic aberration detected by aCGH in ALL patients.
We hypothesized that aCGH could reveal new genetic aberrations in addition
to BCR/ABL1 rearrangement for ALL. A systematic review and meta-analysis
were conducted to evaluate DNA copy number aberrations detected by aCGH
in all types of ALL patients (n=1428). Thirteen studies (1428 patients) published
between 2009 and 2014 were identified through SUMMONS and PubMed
using searching keywords of “aCGH” and “Acute Lymphoblastic Leukemia”.
After combining all the data, the most frequent loci with aberrations were on
9p21-pter (7%), 12q21.2-q24.1(3%), 14q31-q32.33(3%), suggesting CDKN2,
NAP1L1, and IgH genes aberrations might be important to ALL tumorogenesis.
An interesting gain in 1q21-qter(2%) was noted which is popular region for
translocations of BCL9 and ARNT. aCGH could reveal new genetic aberrations
that were previously unknown. The significance of the new aberrations needs to
be further investigated.
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S5
GENETIC HOTSPOTS OF AUTISM DETECTED BY ARRAY CGH: A
SYSTEMATIC REVIEW
Juan Raudales; Erica Neel; Shannon Gonzales; Sui. F. Lee; Meagan Denos;
Vicki L. Hopwood, MS, CG(ASCP), MB(ASCP); Ming Zhao, MD, CG(ASCP),
MB(ASCP); Jun Gu, MD, PhD, CG(ASCP), MB(ASCP)
Autism Spectrum Disorder (ASD) is characterized as a developmental disorder
that includes communication and social impairment. Array comparative genomic
hybridization has been used to detect copy number variants (CNVs) within the ASD
community. The purpose of this study was to compile previous research together
to determine the most common CNVs in ASD patients. PUBMED, SCOPUS, Ovid,
and Medline were extensively searched for relevant articles. Five relevant studies
were identified. Two out of the five studies used Agilent platforms for detection
of CNVs while the other three studies used custom-designed arrays. Out of the
five studies selected, 16p11.2 (34.7%), 15q11-q13 (14.8%), and 22q11.2 (2.9%),
were the most frequently involved loci by CNVs in ASD patients. Duplication was
observed more frequent than deletion, with frequencies of 100% for 15q11-q13,
44.2% for 16p11.2, and 77.3% for 22q11.2, respectively. Our data suggests that
more research is needed to confirm the frequency of these CNVs in ASD patients.
A FISH probe panel could be developed based upon the results of this analysis as
a quick screening method for an initial diagnosis of ASD.
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S6
MUTATION SCREENING IN PATIENTS WITH PULMONARY
ARTERIAL HYPERTENSION
Katie Hay; RuiRui Ji, MD, PhD; Peter Hu, PhD, MS, MLS(ASCP)CMCGCM, MBCM;
Yuxin Fan, MD, PhD
BACKGROUND: Hereditary Pulmonary Arterial Hypertension (HPAH) is
characterized as an autosomal dominant disease with ~20% penetrance,
variable expressivity, and genetic anticipation. The majority of mutations in
patients presenting with HPAH have been found in the Bone Morphogenetic
Protein Receptor (BMPR2) gene. This gene plays an important role in the
expansive transforming growth factor beta (TGF-beta) pathway of receptors
that appear to be related to other vascular pulmonary diseases like hereditary
hemorrhagic telangiectasia (HHT). BMBR2, ALK1, SMAD4, SMAD8, and ENG,
have all been shown to be associated with HPAH and are the most commonly
investigated genes for patients with PAH. 70% of patients with HPAH have
mutations in the BMPR2 gene, and it is the first gene examined when looking for
genetic causes of PAH. These mutations often cause a haploinsufficiency or a
deleterious protein product creating an imbalance in apoptosis and proliferation
signaling of vascular endothelial cells. Endogenous variables such as the type of
mutation found and exogenous variables such as drug exposures are thought to
contribute to the initiation and severity of the disease. The disease onset of PAH
has been documented to occur at any age, and many die or undergo a lung
transplant within 1-5 years. Genetic testing for patients and their family can help
determine course of treatment, possibly prolong a patient’s life, and determine
which family members need closer monitoring for possible onset of the disease.
METHODS: This study examines 21 patient samples, of known age, gender, and
family history, for genetic mutations in genes including BMBR2, ALK1, SMAD4,
SMAD8, and ENG known to be associated with HPAH. Bidirectional PCR-based
sequencing and capillary electrophoresis is used to detect the mutations in the
exons and splicing junction regions of each gene. Any changes are recorded and
determined to be polymorphisms or disease-causing mutations. RESULTS: Data is
being analyzed for this project and will be included in the finished poster.
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S7
CONVENTIONAL FISH VERUS CIG-FISH: META-ANALYSIS
COMPARING DETECTION RATES IN PATIENTS WITH MULTIPLE
MYELOMA
Huy Huynh; Emeric Munyantore; Samar Abumahaimeed; Xue Gu; Jacqueline
Pac; Vicki L. Hopwood, MS, CG(ASCP), MB(ASCP); Ming Zhao, MS,
CG(ASCP), MB(ASCP);
Jun Gu, MD, PhD, CG(ASCP)
Objective – It has been reported that CIG FISH has produced accurate diagnoses
much more than conventional FISH for the detection of Multiple Myeloma (MM).
This study systematically analyzes the risks versus the rewards of using CIG FISH
for the diagnosis of MM against conventional FISH. Methods - SUMMON,
PubMED, Google Scholar, and MEDLINE were searched for papers that used
both CIG FISH and a conventional FISH technique to diagnose MM. Only papers
that specifically compared I-FISH and CIG-FISH probe panels on diagnosed
MM patients were included. Probes selected for analysis must be used in at least
2 or more of the studies. The percentage of cases that each FISH technique
(CIG/conventional) diagnosed correctly was recorded and compared. Results –
Overall the conventional FISH methods had a positive detection rare of 54.4%
while the CIG FISH method had a detection rate of 85.2%. The individual probes
all showed increases in detection ranging from 1.4% (MAF probe) to 31.4% (IGH
probe) except for one probe that had the exact same detection rate for both
FISH detection techniques (CEP3 probe). Three probes produced statistically
significant differences in detection rate (IGH (p=4,43E-11), TP53 (p=4.55E-02),
Rb1 (p=4.23E-05)) while two probes did not (FGFR3 and MAF). Conclusion –
Since the overall data showed an elevation in positive detection rate, the benefits
to employing CIG for the detection of MM outweigh the negative risks, including
time and cost. Further investigation, such as the need for multiple studies using a
FISH probe panel that is exactly the same, is needed to confirm our preliminary
analysis.
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AGT 39th
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S11
SYNTHETIC CONTROL OF MUTATION RATES IN SACCHAROMYCES
CEREVISIAE
Andrew Frink; Gábor Balázsi, MD
BACKGROUND: Mutator phenotypes are often observed in microbial populations
and human cancers and are implicated in oncogenesis and the evolution of drug
resistance. Mutator phenotypes can be caused by mutations in polymerase or
mismatch repair (MMR) pathways. Different endogenous polymerases confer
different mutation rates. Translesion polymerases, which have increased
expression during cellular stress, have particularly high mutation rates when
replicating normal DNA and could contribute to stress-induced mutagenesis.
Whether transient mutator effects are present due to expression level variation of
error-prone polymerases is unclear. In this work we utilized a previously described
tetR/doxycyline based ”linearizer” synthetic gene circuit to linearly and uniformly
control the expression of an error-prone variant of polymerase delta, pol3-01, in
replicating Saccharomyces cerevisiae. We hypothesize that the resulting change to
the mutation rate will be correlated with the imposed expression of pol3-01, but will
be non-linear. Testing this hypothesis will shed light on the underlying properties
of polymerase proofreading and mismatch repair. METHODS: We constructed
three integrative plasmids by placing the genes tetR::yEGFP, pol3-01, and wildtype POL3 downstream of identical tetR repressible promoters. tetR::yEGFP and
pol3-01 were integrated at the GAL1 locus to create the p3m controllable mutator
strain and tetR::yEGFP and POL3 were integrated to created the p3a control
strain. Linear expression dose-response to doxycycline was confirmed by assessing
tetR-yEGFP fluorescence by flow cytometry Mutation rates were estimated by
fluctuation assay for canavanine resistance (Canr) for p3m at 0,1,2,3,4, and 5
μg/ml doxycycline and p3a at 0 and 5 μg/ml doxycycline. RESULTS: Doxycycline
control of tetR-yEGFP expression was highly linear and uniform for both p3m
and p3a (R2 = 0.99 and R2 = 0.96, respectively). Expression of the polymerase
from an adjacent identical promoter was assumed to be correlated to tetR-yEGFP
expression, based on earlier characterization of the linearizer gene circuit. The
rates of Canr per division for p3a were 6.0 x 10-7 and 3.9 x 10-7 for 0 and 5 μg/ml
doxycycline, respectively. The mutation rates of Canr per division for p3m were 8.9
x 10-7 and 24.1 x 10-7 for 0 and 5 μg/ml doxycycline, respectively. Mutation rates
were significantly correlated (R2 = 0.48; p=) with pol3-01 expression. Currently,
we are improving the accuracy of fluctuation assays to measure mutation rates
more precisely and develop mathematical models of the relationship between
polymerase expression and mutation rates. DISCUSSION: Utilizing a synthetic
gene circuit, we achieved non-environmental control of mutation rates in isogenic
populations. Expression of an error-prone polymerase was shown to increase the
mutation rate and could serve as a possible mechanism for transient mutagenesis.
Similar circuits could be constructed for components of the MMR pathway. In
future work this and other gene circuits could be used to further explore the
dynamics of mutator subpopulations in drug resistance and cancer.
142
AGT 39th
ANNUAL MEETING
S12
DETECTION OF CMYC AMPLIFICATION BY FISH ANALYSIS
Anusha Tejomurtula; Ruby Liu; Peter C. Hu; Carlos Bueso-Ramos; Awdhesh
Kalia; Jun Gu; Zhao Ming
Background: Fluorescence in situ hybridization (FISH) is a valuable adjunct
technique for identifying genetic abnormalities that affect diagnosis, risk, and
prognosis. Many studies have investigated the capability of FISH to produce
results from a variety of samples, including cytopreps, tissue imprints, and
20-year-old archived bone marrow smears [1-3]. For example, one retrospective
study investigated using FISH analysis of 12-year-old archived bone marrow
smears to detect MYC-IGH translocations in lymphoma patients [1]; another
study investigated the using FISH analysis of bone marrow samples to detect
C-MYC amplifications in patients with mantle cell lymphoma [4]. However, no
study has investigated the use of FISH analysis of archived bone marrow smears
to detect both translocations and amplifications involving C-MYC in patients
with Burkitt lymphoma. C-MYC is the most frequently altered and amplified
oncogene in B-cell lymphomas [7] and can be used as a specific genetic marker
with FISH to diagnose Burkitt lymphomas within 24 hours. Aim: Our aim was
to use FISH analysis of 6-year-old bone marrow smears to study translocations
and amplifications involving C-MYC in patients with Burkitt lymphoma.
Methods: C-MYC amplification was assessed using the dual-color break-apart
rearrangement probe. BM smears were pre-fixed using a 3:1 methanol: glacial
acetic acid solution, pretreated at a high temperature and were subjected to
enzymatic treatment. The slides then underwent the standard FISH protocol.
Two different cell counters equipped with an Olympus fluorescence microscope
(Olympus America, Center Valley, PA) were each used to count 100 interphase
cells (200 interphase cells total). Result: Hybridization was successful in all BM
smears. All smears had normal cells with two fusions and no amplification of
C-MYC. Conclusion: FISH analysis of archived bone marrow smears rapidly
produces reliable, reproducible results that can be used for retrospective studies.
The use of methanol and glacial acetic acid at a 3:1 ratio enabled the lysis and
washing away of erythrocytes from the bone marrow smears. The pretreatment
followed by protease treatment enabled the proper digestion of the cell membrane
and facilitated the binding of the probe to target DNA.
143
AGT 39TH
ANNUAL MEETING
Save the Date!
AGT 40th Annual Meeting
June 4-6, 2015 Savannah, Ga.
144

39th 39th - Association of Genetic Technologists

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