Scientific Program

Transcription

Scientific Program

IX. Congress
September 10 – 12, 2015
Berlin
Scientific Program
including Abstracts
Imprint
Publisher
Agency KONSENS GmbH
Stockumer Straße 30
59368 Werne
Germany
Phone: +49 23 89 / 52 75 0
Homepage: www.agentur-konsens.de
Editor
Dr. rer. nat. Michael Fleischhacker
Abteilung Pneumologie, Molekularbiologisches Labor
Universitätsklinik und Poliklinik für Innere Medizin I
Universitätsklinikum Halle/Saale
Ernst-Grube-Str. 40
06120 Halle/Saale
Germany
Priv.-Doz. Dr. med. habil. Bernd Schmidt
Leiter Pneumologie
Universitätsklinik und Poliklinik für Innere Medizin I
Ernst-Grube-Str. 40
06120 Halle/Saale
Germany
Design & Layout
Agency KONSENS GmbH
Print
CEWE Print GmbH
Meerweg 30-32
26133 Oldenburg
Germany
2
Contents
Welcome Messages ................................................................................................................................................ 4
Congress Organization . .......................................................................................................................................... 6
Sponsors / Exhibition . ............................................................................................................................................. 8
Scientific Program ................................................................................................................................................. 10
Welcome reception at the Palais ........................................................................................................................... 18
List of Oral Presentations (OP) ............................................................................................................................. 20
List of Poster Presentations (P) . ........................................................................................................................... 26
Abstracts of Keynote Lectures (KL) . ..................................................................................................................... 34
Abstracts of Introduction Lectures (IL) .................................................................................................................. 38
Abstracts of Oral Presentations (OP) .................................................................................................................... 41
Abstracts of Poster Presentations (P) ................................................................................................................... 65
Abstracts of Industrial Session ............................................................................................................................ 100
Author Index ........................................................................................................................................................ 103
3
Welcome messages
Dear Colleagues,
On behalf of the organizers we would like to welcome you to this years‘ CNAPS IX‘s destination: Berlin.
When our forerunners and field-founders Philippe Anker and Maurice Stroun organized the first event in 1999
in Menthon, France, none of us attendees could have imagined that Circulating Nucleic Acids would be a game
changer.
A short 15 years later the development of methods for the isolation and characterization of circulating fetal nucleic
acids found in the blood of pregnant women has turned into commercially available and almost non-invasive tests
for an early detection of fetal disease. In addition, physicians and researchers use cell-free nucleic acids to develop
methods for the examination and monitoring of cancer patients. Soon, this knowledge will be useful when making
treatment decisions concerning patients with benign diseases.
We are confident that the application of methods and tests developed in the field of humans will spread across
species to be advantageous in animal research as well.
We will do our very best to cover all important aspects of CNAPS and make this meeting as successful as its
predecessors, and we look forward to welcoming you to Berlin.
Michael Fleischhacker
4
Bernd Schmidt
Dear participants of the CNAPS IX meeting,
The times of the sole embedment of nucleic acids into life science by the classical paradigm of molecular biology
are history. Nowadays we know, for example, that RNA does not only come in messenger, ribosomal and transfer
flavors. There are several additional species that control translation, modulate transcription or even exert a sponge function on other nucleic acids. But there is even more - nucleic acids have become extracellular.
Maurice Stroun, Philippe Anker and Peter Gahan were the first to explore the field of extracellular nucleic acids.
Their work included the examination of effects of bacterial RNA synthesis on the induction of crown gall tumors
in tomato cells. Additionally, they demonstrated that a direct DNA uptake through cut shoots leads to a genetic
transformation of Solanum plants. These experiments were related and to some extent based on the work of plant
geneticists like Arnd Michaelis and Rigomar Rieger who were closely tied to the Martin-Luther Universität Halle/
Saale.
Later work by these scientists demonstrated that the existence of extracellular nucleic acids is not limited to the
plant kingdom but seems to be a natural phenomenon in the animal world as well. We are beginning to understand that they have the ability to transfer physiological and pathological relevant information between cells and
thereby play a pathogenetic role. Furthermore, we conceive the clinical-practical possibilities in terms of diagnosis
and prognosis – and are often sobered by the challenges encountered on this translational avenue.
Today the analysis and application of CNAPS opens a variety of fascinating opportunities both in basic research
and in clinical applications alike. We are therefore proud to host this conference which builds a bridge from the
research lab to patient needs.
To that end I wish all participants of the CNAPS IX meeting exciting days in Berlin.
Prof. Dr. Michael Gekle
Dean of the Medical Faculty
5
Congress organization
Scientific Organization
Dr. rer. nat. Michael Fleischhacker
Department of pneumology, laboratory of molecular biology
University medical center and clinic for internal medicine I
University medical center Halle/Saale
Ernst-Grube-Str. 40
06120 Halle/Saale
Germany
Priv.-Doz. Dr. med. habil. Bernd Schmidt
Manager of Pneumology
University medical center and clinic for internal medicine I
University medical center Halle/Saale
Ernst-Grube-Str. 40
06120 Halle/Saale
Germany
Planning Committee
Philippe Anker, Beaumont (France)
Luis Diaz, Baltimore (USA)
Michael Fleischhacker, Halle/Saale (Germany)
Stefan Holdenrieder, Bonn (Germany)
Dennis Lo, Hong Kong (China)
Bernd Schmidt, Halle/Saale (Germany)
Heidi Schwarzenbach, Hamburg (Germany)
Alain R. Thierry, Montpellier (France)
Local Organizing Agency
Agency KONSENS GmbH
Germany
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PlasmaSelect -R
™
Highly sensitive and accurate detection of somatic
mutations and rearrangements with allele fractions
as low as 0.10%
PlasmaSelect™-R analyzes circulating tumor DNA in
plasma for genetic alterations in 63 well-characterized
cancer genes, eliminating the need for an invasive
biopsy. Our proprietary capture process and high
coverage next-generation sequencing identifies
tumor specific (somatic) mutations with biologic or
clinical implications as well as key amplifications and
translocations with a high sensitivity and specificity.
Analyses for sequence mutations or rearrangements
can be performed together or separately, depending
on the specific alterations of interest.
To learn more about PlasmaSelect™-R
visit us at personalgenome.com
or call 443-602-8833.
©2015 Personal Genome Diagnostics, Inc. All Rights Reserved. Key Benefits
•
Accommodates low abundance
cell-free DNA samples.
•
Identifies somatic mutations with
allele fractions as low as 0.10%.
•
High resolution analysis of copy
number alterations utilizing
proprietary capture design and
patented Digital Karyotyping.
•
Detects structural changes in
tumor-specific DNA, including
translocations using PARE
(Personalized Analysis of
Rearranged Ends) technology.
•
Design a custom, target-specific
assay to detect and quantify small
fractions of ctDNA in the plasma.
Sponsors/Exhibition
We express our gratitude to all who supported and helped the organization of the 9th edition of the CNAPS
congress:
Gold sponsor
Bio-Rad Laboratories
Booth number: 6
Silver sponsors
GATC Biotech AG
Booth number: 4
Personal Genome Diagnostics
Booth number: 8
Qiagen GmbH
Booth number: 7
Roche Pharma AG
Booth number: 9
Volition Rx
Booth number: 5
Other sponsors
Analytik Jena AG
Booth number: 1
Epigenomics AG
Booth number: 2
Exiqon
Booth number: 11
RainDance Technologies, Inc
Booth number: 3
Sysmex Inostics GmbH
Booth number: 10
FSA Code of conduct:
Roche Pharma AG:
8
15.500 €
2
1
Cate
ring
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6
ring
Cate
ring
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Cate
„Saphir“
Main conference room
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„Ame
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„Bern
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Congressoffice
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Exhibition map
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Scientific Program
Thursday, September 10, 2015
Morning
08:45-09:00
Welcome
09:00-10:30 CNAPS Basics / Technical Applications
Chairmen: Alain R. Thierry, Svetlana Tamkovich
09:00-09:30
KL 1: Biology and nature of circulating nucleic acids
Alain R. Thierry
09:30-09:45 OP 107: Methodological variables in the analysis of cell-free DNA
Abel Bronkhorst
09:45-10:00
OP 141: A historical and evolutionary perspective on circulating nucleic acids and
extracellular vesicles: Circulating nucleic acids as homeostatic genetic entities
Janine Aucamp
10:00-10:15
OP 39: Comparative analysis of ionizing radiation and low-frequency noise on genome of the cell
Irina Vasilyeva
10:15-10:30
OP 125: Use of high conductance dielectrophoresis to recover exosomes containing circulating
cell-free RNA from undiluted human plasma
Stuart Ibsen
10:30-11:00
Coffee break and visit of the exhibition
11:00-11:30
Chairmen: Alain R. Thierry, Maria Giraldez
11:00-11:15
OP 157: Utility of a synthetic RNA reference standard for optimizing extracellular RNA
sequencing methods
Maria Giraldez
11:15-11:30
OP 69: Tandem repeats of heterochromatin in the extracellular DNA
Irina Vasilyeva
11:30-13:15
Poster session
Cancer A/B, Fetal Medicine, Non-malignant Disease
Chairmen:
Ugur Gezer, Michael Heller (Cancer A/B)
Michael Parks (Fetal Medicine)
Susanne Helmig (Non-malignant Disease)
13:15-14:15
10
Lunch and visit of the exhibition
Afternoon
14:15-17:00 CNAPS in Maternal Fetal Medicine
Chairmen: Dennis Lo, Mareike Pendzialek
14:15-14:45
KL 2: High resolution size analysis of circulating DNA in pregnancy and cancer
Dennis Lo
14:45-15:00
OP 119: Implementing non-invasive prenatal diagnosis (NIPD) in a national health service
laboratory; from dominant to recessive disorders
Suzanne Drury
15:00-15:30
15:30-17:00
Chairmen: Dennis Lo, Ana Bustamente-Aragones
15:30-15:45
OP 177: Noninvasive prenatal diagnosis of feto-maternal platelet incompatibility by cold high
resolution melting analysis
Hada C. Macher
15:45-16:00
OP 192: Non-invasive prenatal diagnosis of duchenne and becker muscular dystrophies by
relative haplotype dosage
Michael Parks
16:00-16:15
OP 210: Towards the non-invasive prenatal diagnosis of maternal mutations in clinical practice:
validation of the digital PCR technique
Ana Bustamante-Aragones
16:15-16:30
OP 144: Construction of cfDNA-like reference materials for use in non-invasive prenatal screeing
(NIPS) tests
Seth Harkins
16:30-17:00 KL 3: Lost in translation? Ethical challenges of implementing a new diagnostic procedure
Dagmar Schmitz
17:00
Departure for Welcome reception (via public transport/no shuttle service provided)
19:00
Welcome reception at the Palais
11
Friday, September 11, 2015
Morning
12
09:00-10:30 CNAPS in Cancer I
Chairmen: Nitzan Rosenfeld, Heidi Schwarzenbach
09:00-09:30
KL 4: Multi-panel cfDNA approach demonstrating utility in monitoring cutaneous
melanoma progression
Dave Hoon
09:30-09:45 OP 5: Histone methylation marks in circulating nucleosomes as novel biomarker in colorectal
cancer
Ugur Gezer
09:45-10:00
OP 14: Analysis of circulating tumour DNA to monitor tumour burden and treatment resistance
in metastatic melanoma
Stephen Wong
10:00-10:15
OP 41: Evaluation of circulating cell-free DNA as a molecular monitoring tool during cancer
therapy
Clemens Hufnagl
10:15-10:30
OP 57: Rapid aneuploidy screening of plasma DNA samples from cancer patients using a
modified FAST-SeqS approach
Ellen Heitzer
10:30-11:00
11:00-12:45
Chairmen: Dave Hoon, Stefan Holdenrieder
11:00-11:30
IL 1: Clinical applications of ctDNA
Luis Diaz
11:30-11:45
OP 92: Circulating tumour DNA to monitor treatment response and resistance in chronic
lymphocytic leukaemia
Devbarna Sinha
11:45-12:00
OP 109: Low cost, broad panel (100kb) liquid biopsy with reduced DNA sequencing by depletion
of wild-type sequence
Andre Marziali
12:00-12:15
OP 211: Targeted sequencing reveals clonal genetic changes in the progression of early
glandular neoplasms of the lung and paired circulating DNA
David Sidransky
12:15-12:30
OP 140: Rapid isolation, detection and analysis of hematological cancer and solid tumor related
cell free nucleic acids in blood, plasma and serum
Michael Heller
12:30-12:45
OP 221: The clinical relevance of circulating, exosomal microRNAs as biomarkers for
gynecological tumors
Heidi Schwarzenbach
12:45-13:45
Poster Session
Cancer C/D, Basic Science, Technical Issues
Chairmen: Dimo Dietrich, Florent Mouliere (Cancer C/D),
Abel Bronkhorst (Basic Science, Technical Issues)
13:45-14:45
Lunch and visit of the exhibition
Afternoon
14:45-16:00
Industrial Session
Chairmen: N.N., N.N.
14:45-15:05
Droplet Digital PCR: Ally in Precision Cancer Diagnosis and Treatment
George Karlin-Neumann
15:05-15:25 Volition RX
“Nucleosomics”- translating epigenetic biomarkers into clinical diagnostics
Mark Eccleston
15:25-15:35
GATC Biotech AG
The molecular X-Ray of the 21st century
Tobias Paprotka
15:35-15:45
Qiagen GmbH
From Sample to Insight: QIAGEN´s Approach Pioneering the Liquid Biopsy Revolution
Michael Kazinski
15:45-15:55
Personal Genome Diagnostics (PGDx) –
turning The Promise of Personalized Medicine into Reality
Antony Newton
16:00-17:15
Non-malignant Disease
Chairmen: Luis Diaz, Elena Rykova
16:00-16:15
OP 31: Circulating microRNAs in the diagnosis of gestational diabetes mellitus
Eloise Lawrence
13
14
16:15-16:30
OP 162: Discovering the tissue origins of cell-free DNA
Roni Werman
16:30-16:45
OP 195: Exercise-induced cell free DNA in human plasma is predominantly released from cells
of the haematopoietic lineage
Susanne Helmig
16:45-17:00 OP 220: Comparison of different preanalytical workflows for isolation of intact exosomes and
other extracellular vesicles
Gabriele Christoffel
17:00-17:15 OP 121: An enquiry concerning the characteristics of cell-free DNA released by cultured
cancer cells
Abel Bronkhorst
17:15-17:30
17:30-19:00
Panel discussion / Academia meets industry
Future and unmet needs in liquid biopsy:
Mutual interests in the development of methods and applications, Future of CNAPS
Moderation: Christian Schäfer
Tobias Paprotka, GATC Biotech AG
Mark Eccleston, Volition RX
Johannes Noe, Roche Pharma AG
Frank Diehl, Sysmex Inostics GmbH
Ellen Heitzer, Medical University Graz
Alain R. Thierry, Institut de Recherche en Cancérologie de Montpellier
Stefan Holdenrieder, University Clinic Bonn
19:00
Departure for Theatre performance (via public transport/no shuttle service provided)
20:00
RambaZamba
Saturday, September 12, 2015
Morning
09:00-11:00 CNAPS in Cancer II
Chairmen: Dave Hoon, Ellen Heitzer
09:00-09:30
IL 2: Genomic analysis of circulating tumour DNA: pushing the limits for cancer
applications
Nitzan Rosenfeld
09:30-09:45
OP 151: Whole genome deep sequencing analysis of plasma DNA in breast cancer
Devika Ganesamoorthy
09:45-10:00
OP 197: Detection of tumor-specific mutations in circulating cell-free tumor DNA from patient
blood using a novel, barcoded and multiplexed NGS library construction approach
Jennifer Jackson
10:00-10:15
OP 202: Chromosomal rearrangement as cancer-specific biomarker for monitoring of lung
cancer after surgery
Hongdo Do
10:15-10:30
OP 204: Monitoring response to therapy in melanoma by quantifying circulating tumour DNA
with droplet digital PCR
Alexander Dobrovic
10:30-10:45
OP 208: Non-invasive genomic profiling of urothelial bladder cancer using urinary cfDNA
Fiona Togneri
10:45-11:00
OP 58: Clinical utility of circulating tumor DNA for molecular assessment and precision
medicine in pancreatic cancer
Erina Takai
11:00-11:30
11:30-13:00
Chairmen: Nitzan Rosenfeld, Adam Szpechcinski
11:30-11:45
OP 217: Liquid profiling of KRAS-status on circulating plasma-DNA in patients with pancreatic
cancer – a novel tool for therapy response prediction and prognosis
Stefan Holdenrieder
11:45-12:00
OP 116: Individual circulating DNA CpG-methylation profiling: unbiased approach for
identification of cancer related molecules
Anna Bondar
15
12:00-12:15
OP 182: Non-invasive analysis of glioblastoma with circulating nucleic acids
Florent Mouliere
12:15-12:30
OP 213: Free-circulating methylated DNA in blood for diagnosis, staging prognosis and
monitoring of head and neck squamous cell carcinoma patients : an observational prospective
cohort study
Luka de Vos
12:30-12:45
OP 193: Effects of collection and processing on plasma cell-free DNA in EDTA versus
cell-stabilizing tube
Dana Tsui
12:45-13:00
OP 219: Optimized plasma collection procedures for cell-free DNA analyses in advanced
malignancies
Sonya Parpart-Li
13:00-14:00
Pre-analytical Standards for CNAPS
Chairmen: N.N, N.N
The aim of this session is to discuss and propose the steps needed to achieve an agreement
on the best methods for pre-analytic handling of blood and other body fluids.
All participants of the CNAPS IX meeting are invited to contribute to this goal by suggesting
topics to discuss, setting up cooperations between laboratories or introducing SOPs with which
they had good experiences.
Feel free to contact us ahead of the conference or make your statement on the spot.
14:00-14:15
16
Closing remarks
Partner der Pathologie –
gemeinsam in die Zukunft
HER2
2000
EGFR
2011
BRAF
2012
In der Onkologie findet derzeit ein Paradigmenwechsel statt: Mithilfe von Biomarkern können einzelne Tumorentitäten
genauer charakterisiert und die Patienten einer stratifizierten Therapie zugeführt werden.
Roche ist in der einzigartigen Position, Spitzenexpertise aus den Bereichen Diagnostik und zielgerichtete Therapie unter
einem Dach zu vereinen. Mit HER2 wurde der Grundstein gelegt, EGFR und BRAF folgten als weitere Biomarker für
neue Behandlungsansätze.
Sie als Pathologe nehmen eine Schlüsselposition ein: Nur diejenigen Patienten, die zuverlässig getestet werden, können
auch von zielgerichteten Therapien profitieren.
Biomarkers of Excellence
Zuverlässig testen. Zielgerichtet therapieren.
Welcome reception at the Palais
The Kulturbrauerei:
The appealing structure of the well preserved KulturBrauerei shows it’s versatility with it’s 6 courtyards, over 20
buildings and it’s distinctive architecture.
Being one of the most popular event locations in Berlin, situated in the bustling district of Prenzlauer Berg, the
lavishly redeveloped area of the former Schultheiss Brewery is appreciated as a cultural hotspot and attracts over
1 million visitors per year with it’s wide variety of activities and beautiful facades.
Product presentations, banquets, weddings, conferences or corporate events – the KulturBrauerei offers the
special ambiance, a unique variety, a useful network and all-round combinations in the interior or outdoor areas –
and all this for 50 up to 5.000 persons. The complete building is situated on a total area of 25.000 sqm.
The Palais:
In the Palais events are conducted e.g. banquets, weddings, receptions, conventions, readings and opening
nights.
The Palais possesses excellent equipment including a variable stage, first-class light and sound engineering as
well as theatre style seating arrangement for up to 250 people.
A cloakroom for the artists and handicapped accessible lavatories are available.
In spring and summer the courtyard in front of the Palais can be fitted with deckchairs and potted plants and the
event area can be extended by roofing accommodating up to 1.000 persons.
Price winners:
Philippe Anker and Maurice Stroun will announce the prize winners for the best abstracts in the Palais.
The winners will be honored with a free congress ticket.
How to get there:
KulturBrauerei
Schönhauser Allee 36
10435 Berlin - Prenzlauer Berg
For directions to Kulturbrauerei
(Palais and RambaZamba) see separate
flyer and map with U-/S-Bahn available
at Registration desk.
18
Innovation is
in the blood
Let liquid biopsies tell you their secrets.
As a minimally invasive technology for detecting signs of cancer and other
diseases, the liquid biopsy can sidestep stressful, costly surgical procedures.
QIAGEN’s liquid biopsy solutions enable you to sensitively, specifically and
quickly analyze circulating free nucleic acids (cfDNA), exosomes and circulating
tumor cells (CTCs) from blood and other biofluids. This means you can find and
study elusive low-abundance DNA fragments, evaluate valuable biomarkers
from cellular vesicles, or even analyze a whole genome or transcriptome from
a single tumor cell.
Liquid biopsies promise to revolutionize the way serious diseases are detected
and monitored. Let QIAGEN help you unlock the molecular mysteries within
them.
Discover their secrets at www.qiagen.com/liquidbiopsy.
Sample to Insight
LIST OF ORAL PRESENTATIONS (OP)
CNAPS Basics / Technical Applications
OP 107
Methodological variables in the analysis of cell-free DNA
Abel Bronkhorst, Janine Aucamp, Piet Pretorius
OP 141
A historical and evolutionary perspective on circulating nucleic acids and extracellular vesicles:
Circulating nucleic acids as homeostatic genetic entities
Janine Aucamp, Abel Bronkhorst, Piet Pretorius
OP 39
Comparative Analysis of Ionizing Radiation and Low-Frequency Noise on Genome of the Cell
Irina Vasilyeva, Valery Zinkin, Vladimir Bespalov
OP 125
Use of High Conductance Dielectrophoresis to Recover Exosomes Containing Circulating Cell-Free RNA
from Undiluted Human Plasma
Stuart Ibsen, Jennifer Wright, Johnny Akers, Clark Chen, Michael Heller
OP 157
Utility of a synthetic RNA reference standard for optimizing extracellular RNA sequencing methods
Maria Giraldez, Alton Etheridge, Kai Wang, David Galas, Muneesh Tewari
OP 69
Tandem Repeats of Heterochromatin in the Extracellular DNA
Irina Vasilyeva, Olga Podgornaya, Vladimir Bespalov
CNAPS in Maternal Fetal Medicine
OP 119
Implementing non-invasive prenatal diagnosis (NIPD) in a National Health Service laboratory; from
dominant to recessive disorders
Suzanne Drury, Sarah Mason, Fiona McKay, Kitty Lo, Christopher Boustred, Melissa Hill, Samantha Edwards,
Naomi Gibson, Clinda Puvirajasinghe, Lucy Jenkins, Lyn Chitty
OP 177
Non invasive prenatal diagnosis of feto-maternal platelet incompatibility by cold high resolution melting
analysis
Marta Ferro, Hada C. Macher, Pilar Noguerol, Pilar Jiménez-Arriscado, Patrocinio Molinero, Juan Miguel
Guerrero, Amalia Rubio
20
OP 192
Non-invasive prenatal diagnosis of Duchenne and Becker muscular dystrophies by relative haplotype
dosage
Michael Parks, Samantha Court, Siobhan Cleary, Samuel Clokie, Julie Hewitt, Denise Williams, Trevor Cole,
Fiona MacDonald, Mike Griffiths, Stephanie Allen
OP 210
Towards the non-invasive prenatal diagnosis of maternal mutations in clinical practice: validation of the
digital PCR technique
Sara Perlado-Marina, Ana Bustamante-Aragones, Marta Donas, Maria José Trujillo Tiebas, Isabel Lorda-Sanchez,
Marta Rodriguez de Alba
OP 144
Construction of cfDNA-Like Reference Materials for Use in Non-Invasive Prenatal Screening (NIPS) Tests
Seth Harkins, Yves Konigshofer, Alice T. Ku, Farol L. Tomson, Katherine Bianco, Russell K. Garlick, Barathi
Anekella
CNAPS in Cancer I
OP 5
Histone methylation marks in circulating nucleosomes as novel biomarker in colorectal cancer
Ugur Gezer, Ebru E. Yörüker, Emre Özgür, Metin Keskin, Stefan Holdenrieder
OP 14
Analysis of circulating tumour DNA to monitor tumour burden and treatment resistance in metastatic
melanoma
Stephen Wong, Jeanette Raleigh, Jason Callahan, Athena Hatzimihalis, Ismael Vergara, Tony Papenfuss, Rodney
Hicks, Carleen Cullinane, Mark Shackleton, David Gyorki, Damien Kee, Ben Brady, Grant McArthur, Shahneen
Sandhu, Sarah-Jane Dawson
OP 41
Evaluation of circulating cell-free DNA as a molecular monitoring tool during cancer therapy
Clemens Hufnagl, Lukas Weiss, Thomas Melchardt, Martin Moik, Daniela Asslaber, Philipp Steininger, Thomas
Meißnitzer, Daniel Neureiter, Richard Greil, Alexander Egle
OP 57
Rapid aneuploidy screening of plasma DNA samples from cancer patients using a modified FAST-SeqS
approach
Jelena Belic, Marina Koch, Martina Auer, Peter Ulz, Jochen Geigl, Michael Speicher, Ellen Heitzer
OP 92
Circulating tumour DNA to monitor treatment response and resistance in chronic lymphocytic leukaemia
Devbarna Sinha, Sarah Ftouni, Tane A. Hunter, Paul Yeh, Elise Wallach, Stephen Wong, Enid Lam, Pasquale
Petrone, John F. Seymour, Mark A. Dawson, Constantine Tam, Sarah-Jane Dawson
21
OP 109
Low cost, broad panel (100kb) liquid biopsy with reduced DNA sequencing by depletion of wild-type
sequence.
Joel Pel, Milenko Despotovic, Patrick Davies, Laura Gelinas, Andre Marziali
OP 211
Targeted sequencing reveals clonal genetic changes in the progression of early glandular neoplasms of
the lung and paired circulating DNA.
Evgeny Izumchenko, Xiaofei Chang, Mariana Brait, Elana Fertig, Luciane T. Kagohara, Atul Bedi, Luigi
Marchionni, Sian Jones, Mohammad O. Hoque, William H. Westra, David Sidransky
OP 140
Rapid Isolation, Detection and Analysis of Hematological Cancer and Solid Tumor Related Cell Free
Nucleic Acids in Blood, Plasma and Serum
Michael Heller, Jennifer Marciniak Wright, Stuart Ibsen
OP 221
The clinical relevance of circulating, exosomal microRNAs as biomarkers for gynecological tumors
Heidi Schwarzenbach
Non-malignant Disease / pre-analytics
OP 31
Circulating microRNAs in the diagnosis of Gestational Diabetes Mellitus
Eloise Lawrence, Ramasamyiyer Swaminathan, Anthony Marinaki, Anna Brackenridge, Barbara McGowan
OP 162
Discovering the Tissue Origins of Cell-Free DNA
Roni Werman, Daniel Neiman, Hai Zemmour, Joshua Moss, Judith Magenheim, Adi Vaknin-Dembinsky, Kirsty
Spalding, Markus Grompe, Aviad Zick, Ayala Hubert, Volker Fendrich, Talia Golan, Iris Lavon, Benjamin Glaser,
Ruth Shemer, Yuval Dor
OP 195
Exercise-induced cell free DNA in human plasma is predominantly released from cells of the
haematopoietic lineage
Susanne Helmig, Klaus Bender, Suzan Tug, Eva Ricarda Deichmann, Anna Schmeier-Jürchott, Eva Wagner, Tim
Zimmermann, Markus Radsak, Mauro Giacca, Simon Perikles
OP 220
Comparison of Different Preanalytical Workflows for Isolation of Intact Exosomes and Other Extracellular
Vesicles
Gabriele Christoffel, Karolin Spitzer, Daniel Enderle, Johan Skog, Mikkel Noerholm, Markus Sprenger-Haussels,
Martin Schlumpberger
22
OP 121
An enquiry concerning the characteristics of cell-free DNA released by cultured cancer cells
Abel Bronkhorst, Jaco Wentzel, Etresia Van Dyk, Janine Aucamp, Lissinda du Plessis, Piet Pretorius
CNAPS in Cancer II
OP 151
Whole genome deep sequencing analysis of plasma DNA in breast cancer
Devika Ganesamoorthy, Alan Robertson, Wenhan Chen, Kaltin Ferguson, Peter Simpson, Lachlan Coin
OP 197
Detection of tumor-specific mutations in circulating cell-free tumor DNA from patient blood using a novel,
barcoded and multiplexed NGS library construction approach
Jennifer Jackson, Anders Ståhlberg, Paul Krzyzanowski, Lincoln Stein, Tony Godfrey
OP 202
Chromosomal rearrangement as cancer-specific biomarker for monitoring of lung cancer after surgery
Hongdo Do, Thomas John, Paul Mitchell, Carmel Murone, Bibhusal Thapa, Daniel Cameron, Ramyar Molania,
Tony Papenfuss, Alexander Dobrovic
OP 204
Monitoring response to therapy in melanoma by quantifying circulating tumour DNA with droplet digital
PCR
Simon Tsao, Jonathan Weiss, Tom Witkowski, Hongdo Do, Christopher Hudson, Christopher Christophi, Jonathan
Cebon, Andreas Behren, Alexander Dobrovic
OP 208
Non-invasive genomic profiling of urothelial bladder cancer using urinary cfDNA
Fiona Togneri, Douglas G. Ward, Joseph M. Foster, Adam J. Devall, Paula Wojtowicz, Sofia Alyas, Fabiana
Ramos Vasques, Assa Oumie, Nicholas D. James, K.K. Cheng, Maurice P. Zeegers, Nayneeta Deshmukh,
Brendan O’Sullivan, Phillipe Taniere, Karen G. Spink, Dominic J. McMullan, Mike Griffiths, Richard T. Bryan
OP 58
Clinical utility of circulating tumor DNA for molecular assessment and precision medicine in pancreatic
cancer
Erina Takai, Yasushi Totoki, Hiromi Nakamura, Mamoru Kato, Tatsuhiro Shibata, Shinichi Yachida
OP 217
Liquid profiling of KRAS-status on circulating plasma-DNA in patients with pancreatic cancer – a novel
tool for therapy response prediction and prognosis
Stefan Holdenrieder, Ina Prinz, Steffen Ormanns, Sibylle Bächmann, Michael Haas, Carina Ross, Philipp
Angenendt, Volker Heinemann, Frank Diehl, Stefan Boeck
23
OP 116
Individual circulating DNA CpG-methylation profiling: unbiased approach for identification of cancer
related molecules
Anna Bondar, Alexander Kurilshikov, Evgeny Morozkin, Marat Zaripov, Marsel Kabilov, Vladimir Voytsitskiy,
Valentin Vlassov, Pavel Laktionov
OP 182
Non-invasive analysis of glioblastoma with circulating nucleic acids
Richard Mair, Florent Mouliere, Davina Gale, Dana Tsui, Colin Watts, Kevin Brindle, Nitzan Rosenfeld
OP 213
Free-circulating Methylated DNA in Blood for Diagnosis, Staging, Prognosis and Monitoring of Head and
Neck Squamous Cell Carcinoma Patients: An Observational Prospective Cohort Study
Luka de Vos, Andreas Schröck, Annette Leisse, Friedrich Bootz, Glen Kristiansen, Dimo Dietrich
OP 193
Effects of collection and processing on plasma cell-free DNA in EDTA versus cell-stabilizing tube
Dana Tsui, Bente Risberg, Andrea Ruiz-ValdepenasMartindeAlmagro, Sarah-Jane Dawson, Heather Biggs,
Charlotte Hodgkin, Linda Jones, Christine Parkinson, Anna Piskorz, Francesco Marass, James Morris, Vincent
Plagnol, Nitzan Rosenfeld, Carlos Caldas, James D. Brenton, Davina Gale
OP 219
Optimized plasma collection procedures for cell-free DNA analyses in advanced malignancies
Sonya Parpart-Li, Bjarne Barlett, Julie Brahmer, Nilo Azad, Sarah Bonerigo, Ilene Browner, Amy Ryan, Victor E.
Velculescu, Mark Sausen, Luis Diaz
24
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List of Poster Presentations (P)
Cancer A
P 48
Capturing early changes in the cancer genome of patients under experimental therapeutic trials with
circulating tumour DNA and adaptive TAm-Seq
Florent Mouliere, Javier Garcia Corbacho, T. Hedley Carr, James Morris, Francesco Marass, Davina Gale, Brian
Dougherty, Elizabeth Harrington, J. Carl Barrett, Simon Pacey, Richard Baird, Nitzan Rosenfeld
P 145
Noninvasive cell-free DNA-based detection of copy-number variations and single-nucleotide variations in
cancer samples using single-nucleotide polymorphism-targeted massively multiplexed PCR
Bernhard Zimmermann, Eser Kirkizlar, Tudor Constantin, Ryan Swenerton, Bin Hoang, Nicholas Wayham,
Zachary Demko, Robert Pelham, Styrmir Sigurjonsson, Matthew Hill
P 181
Screening of kras mutation in pre- and post-surgery serum of patients sufferin colon cancer by cold-pcr
hrm
Elena Trujillo, Hada C. Macher, Pilar Jiménez-Arriscado, Noelia García-Fernández, Juan Miguel Guerrero, Amalia
Rubio
P 201
Detection and Quantification of KIT mutations in ctDNA by Safe-SeqS
Johannes Fredebohm, Daniel Mehnert, Ann-Kathrin Löber, Vanessa VanRahden, Frank Holtrup, Frank Diehl
P 30
Small non-coding RNAs of human blood plasma of healthy donors and patients with non-small cell lung
cancer
Dmitri Baryakin, Dmitry Semenov, Eugeniy Brenner, Alexander Kurilshikov, Vadim Kozlov, Elena Kuligina, Vladimir
Richter
P 42
A rapid and sensitive method for detection of T790M mutations of EGFR in plasma DNA
Hideharu Kimura, Shingo Nishikawa, Hayato Koba, Taro Yoneda, Takashi Sone, Kazuo Kasahara
P 82
Plasma microRNA profiles: identification of miR-744 as a novel prognostic and chemoresistant biomarker
in pancreatic cancer
Shuhei Komatsu, Daisuke Ichikawa, Mahito Miyamae, Tsutomu Kawaguchi, Wataru Okajima, Takuma Ohashi,
Taisuke Imamura, Jun Kiuchi, Tomohiro Arita, Hirotaka Konishi, Ryo Morimura, Atsushi Shiozaki, Hisashi Ikoma,
Toshiya Ochiai, Kazuma Okamoto, Hiroki Taniguchi, Eigo Otsuji
26
P 209
Evaluation of different blood collection tubes and blood storage conditions for the preservation and
stability of cell-free circulating DNA for the analysis of the methylated mSEPT9 colorectal cancer
screening marker
J. Distler, R. Tetzner, G. Weiss, T. König, A. Schlegel, Michal Bagrowski
Cancer B
P 90
RNA of blood circulating complexes of healthy donors and non-small cell lung cancer patients
Anna Savelyeva, Dmitry Semenov, Dmitri Baryakin, Vadim Kozlov, Elena Chikova, Elena Kuligina, Vladimir
Richter
P 91
Plasma microRNA profiles; down-regulation of plasma tumor suppressive miRNA level contributes poor
outcomes in pancreatic cancer
Taisuke Imamura, Shuhei Komatsu, Daisuke Ichikawa, Jun Kiuchi, Wataru Okajima, Takuma Ohashi, Mahito
Miyamae, Tomohiro Arita, Ryo Morimura, Hisashi Ikoma, Hirotaka Konishi, Yasutoshi Murayama, Atsushi
Shiozaki, Yoshiaki Kuriu, Masayoshi Nakanishi, Hitoshi Fujiwara, Kazuma Okamoto, Hiroki Taniguchi, Eigo Otsuji
P 97
Circulating miR-18a in plasma contributes to cancer detection and monitoring in patients with gastric
cancer.
Jun Kiuchi, Shuhei Komatsu, Daisuke Ichikawa, Masahiro Tsujiura, Mahito Miyamae, Wataru Okajima, Takuma
Ohashi, Taisuke Imamura, Tomohiro Arita, Toshiyuki Kosuga, Hirotaka Konishi, Atsushi Shiozaki, Kazuma
Okamoto, Eigo Otsuji
P 112
Plasma microRNAs as noninvasive biomarkers for lung cancer
Ivan Zaporozhchenko, Evgeny Morozkin, Tatyana Skvortsova, Rykova Elena, Anastasia Ponomaryova, Nadezhda
Cherdyntseva, Valentin Vlassov, Pavel Laktionov
P 118
The methylation profiling of multiple tumor suppressor genes in plasma cell-free DNA of patients with
chest radiological findings: resectable NSCLC versus benign lung nodules – pilot study
Adam Szpechcinski, Monika Gos, Renata Langfort, Wlodzimierz Kupis, Piotr Rudzinski, Jolanta Zaleska, Tadeusz
Orlowski, Kazimierz Roszkowski-Sliz, Joanna Chorostowska-Wynimko
P 126
Plasma microRNA profiles: Identification of novel microRNA-based biomarkers for chemoresistance in
esophageal squamous cell carcinoma
Takuma Ohashi, Shuhei Komatsu, Daisuke Ichikawa, Tsutomu Kawaguchi, Mahito Miyamae, Wataru Okajima,
Taisuke Imamura, Jun Kiuchi, Tomohiro Arita, Hirotaka Konishi, Atsushi Shiozaki, Hitoshi Fujiwara, Kazuma
Kazuma Okamoto, Eigo Otsuji
27
P 130
Detection of p53 mutations in circulating DNA of transplanted HCC patients as a biomarked of tumor
recurrence
Noelia García-Fernández, Hada C. Macher, Amalia Rubio, Pilar Jiménez-Arriscado, Carmen Bernal, Maria Luz
Bellido-Diaz, Gonzalo Suarez-Artacho, Juan Miguel Guerrero, Miguel Angel Gómez-Bravo, Patrocinio Molinero
P 133
Circulating DNA as a monitoring marker for patients with EBV-associated gastric cancer
Katsutoshi Shoda, Daisuke Ichikawa, Yuuji Fujita, Tomohiro Arita, Hirotaka Konishi, Shuhei Komatsu, Eigo Otsuji
P 222
The diagnostic potential of extracellular microRNAs in bronchial lavage samples
Grit Rehbein, Bernd Schmidt, Michael Fleischhacker
Fetal Medicine
P 117
Real-Time and Droplet PCR quantification for non-invasive determination of RHD incompatibility between
mother and fetus
Sarka Pospisilova, Iveta Svobodova, Eva Pazourkova, Ales Horinek, Marie Korabecna
P 128
Aberrant microRNA expression in maternal plasma and preimplantation embryos of diabetic rabbits- a
promising biomarker for trophoblast function in early pregnancy?
Mareike Pendzialek, Julia M. Knelangen, Katarzyna Grybel, Jacqueline Gürke, Maria Schindler, Bernd Fischer,
Anne Navarrete Santos
P 158
Characterization of human Pregnancy Specific Glycoprotein (PSG) gene copy number variations in preeclampsia patients
Chia Lin Chang, Chia Yu Chang, Da Xian Lee, Po Jen Cheng
P 56
The role of circulating mir-1 in Diabetic Retinopathy
Tanuka Palit, Ramasamyiyer Swaminathan, Anthony Marinaki, Janaka Karalliedde
P 120
Evaluation of the state of transplanted liver health by monitoring of organ-specific genomic marker in
circulating DNA from receptor
Hada C. Macher, Gonzalo Suarez-Artacho, Pilar Jiménez-Arriscado, Sara Alvarez-Gómez, Noelia GarcíaFernández, Juan Miguel Guerrero, Patrocinio Molinero, Elena Trujillo, Miguel Angel Gómez-Bravo, Amalia Rubio
28
P 146
Cell free DNA levels in healthy subjects
Miriam Albus, Alexandra Brahmer, Suzan Tug, Susanne Helmig, Daniela Zahn, Thomas Kubiak, Simon Perikles
P 174
Circulating DNA in rheumatoid arthritis development
Elena Rykova, Aleksey Sizikov, Oksana Antonenko, Leonid Bryzgalov, Evgeny Morozkin, Valentin Vlassov, Pavel
Laktionov, Vladimir Kozlov
P 180
Multiple ways of cfDNA reception and following ROS production in endothelial cells
Anna Alekseeva, Larisa Kameneva, Tatiana Smirnova, Svetlana Kostyuk, Natalia Veiko
Cancer C
P 134
Assessment of the clinical applications of circulating tumour DNA in non-small cell lung cancer using an
enhanced TAm-SeqTM assay
Davina Gale, Jordi Remon, Andrew Lawson, Sarah Smalley, Esperanza Perez, Karen Howarth, Michelle Pugh,
Abdelaziz Fahem, Tim Forshew, Ludovic Lacroix, Vincent Plagnol, Benjamin Besse, Nitzan Rosenfeld
P 138
Evaluation of Streck cfDNA blood collection tubes (BCTs) for liquid biopsy testing
Inga Medina Diaz, Erica Maldonado, Johannes Fredebohm, Daniel Mehnert, Florian Schiffel, Johanna Weiland,
Bianca Friese, Ann-Kathrin Löber, Frank Diehl, Frank Holtrup
P 143
Plasma miR-224 enable sensitive cancer screening and therapy monitoring with liver functionindependent manner in hepatocellular carcinoma
Wataru Okajima, Shuhei Komatsu, Daisuke Ichikawa, Mahito Miyamae, Tsutomu Kawaguchi, Taisuke Imamura,
Takuma Ohashi, Jun Kiuchi, Eigo Otsuji
P 147
Microvesicules and miRNA in urine of healthy and prostate cancer patients
Olga Bryzgunova, Evgeniy Lekchnov, Tatyana Skvortsova, Evgeny Morozkin, Ivan Zaporozhchenko, Alina
Grigorieva, Marat Zaripov, Elena Ryabchikova, Valentin Vlassov, Pavel Laktionov
P 155
The Potential of Circulating Tumour DNA as a Diagnostic Aid in Urological Cancers
Keval Patel, Dana Tsui, Charlie Massie, James Morris, Francesco Marass, Vincent Gnanapragasam, Nitzan
Rosenfeld
29
P 156
Unbiased detection of somatic copy number aberrations in cfDNA of lung cancer cases with low coverage
whole genome sequencing
Fiona Taylor, James Bradford, M. Dawn Teare, Penella Woll, Angela Cox
P 163
Using Cell Free DNA Reference Standards to evaluate the analytical performance of circulating tumor
DNA testing and solid organ transplant health surveillance
Hadas Amit, Shen Wei, Javier Armisen-Garrido, Bernice Edgeworth, Robert Woodward, John Sninsky, Colin
Barker, Karin Schmitt
P 224
Quantification of Cell-Free mSHOX2 Plasma DNA for Therapy Monitoring in Advanced Stage Non-Small
Cell (NSCLC) and Small-Cell Lung Cancer (SCLC) Patients
Bernd Schmidt, Julia Beyer, Dimo Dietrich, Ines Bork, Volker Liebenberg, Michael Fleischhacker
Cancer D
P 166
Malignant potential in pancreatic neoplasm; new insights provided by circulating miR-223 in plasma
Mahito Miyamae, Shuhei Komatsu, Daisuke Ichikawa, Tsutomu Kawaguchi, Ryo Morimura, Wataru Okajima,
Takuma Ohashi, Taisuke Imamura, Jun Kiuchi, Hisashi Ikoma, Hiroki Taniguchi, Eigo Otsuji
P 170
Plasma miR-141 and miR-205 as potential biomarkers of prostate cancer
Ivan Osipov, Evgeny Morozkin, Ivan Zaporozhchenko, Anna Bondar, Marat Zaripov, Vladimir Voytsitskiy, Valentin
Vlassov, Pavel Laktionov
P 172
Tracking KRAS mutation in circulating cell free DNA as biomarker for anti-VEGF and EGFR colorectal
cancer therapies
Shiro Kitano, Takeshi Yamada, Takuma Iwai, Masato Nakayama, Uchida Eiji
P 173
Circulating microRNA expression level during the lung cancer combined therapy: chemotherapy followed
by surgical resection
Anastasia Ponomaryova, Evgeny Morozkin, Elena Rykova, Ivan Zaporozhchenko, Tatyana Skvortsova, Aleksey
Dobrodeev, Alexander Zavyalov, Sergei Tuzikov, Nadezhda Cherdyntseva, Valentin Vlassov, Pavel Laktionov
P 194
Clinical validation of the analysis of circulating DNA for theragnostics and multiparametric strategy for
cancer patients’ prognosis
Safia El Messaoudi, Brice Pastor, Cynhia Sanchez, Stanislas Du Manoir, Florent Mouliere, Caroline Mollevi,
Charles Theillet, Brigitte Gillet, Michelle Nouaille, Denis Pezet, Muriel Mathonnet, Marc Ychou, Alain R. Thierry
30
P 203
Clinical implications of genomic alterations in the tumor and circulation of pancreatic cancer patients
Mark Sausen, Jillian Phallen, Vilmos Adleff, Sian Jones, Rebecca J. Leary, Michael T. Barrett, Valsamo
Anagnostou, Sonya Parpart-Li, Derek Murphy, Qing Kay Li, Carolyn A. Hruban, Rob Scharpf, James R. White,
Peter J. O’Dwyer, Peter J. Allen, James R. Eshleman, Craig B. Thompson, David S. Klimstra, David C. Linehan,
Anirban Maitra, Ralph H. Hruban, Luis Diaz, Daniel D. Von Hoff, Julia S. Johansen, Jeffrey A. Drebin, Victor E.
Velculescu
P 206
Multiplex KRAS G12/G13 mutation testing of 16ng of unamplified cell-free DNA from plasma of patients
with advanced cancers using Droplet Digital PCR
Helen J. Huang, Dawne Shelton, Siqing Fu, Sarina Anne Piha-Paul, Apostolia M. Tsimberidou, Jennifer J. Wheler,
Aung Naing, David Hong, Gerald S. Falchook, Scott Kopetz, Raja Luthra, Bryan K. Kee, George Karlin-Neumann,
Funda Meric-Bernstam, Filip Janku
Basic Science / Technical Issues
P 21
Purification of Circulating Cell-Free DNA from plasma using the automated Large-Volume Extraction on
the QIAsymphony SP Instrument
Alexander Wolf, Manuel Frietsch, Sebastian Groemminger, Wera Hofmann, Matthias Sachse, Jana Fassunke,
Katharina Beller
P 47
Artificial analogues of circulating box C/D RNAs induce microRNA activation in human adenocarcinoma
cells
Grigory Stepanov, Julia Filippova, Anna Nushtaeva, Elena Kuligina, Olga Koval, Vladimir Richter, Dmitry Semenov
P 61
cirDNA size and termini in blood of healthy females and breast cancer patients
Svetlana Tamkovich, Natalia Kirushina, Valentin Vlassov, Pavel Laktionov
P 115
A New Blood Collection Tube and Extraction Method for the Analysis of circulating cell-free DNA (ccfDNA)
Andrea Ullius, Thorsten Voss, Joachim Bonnet, Wera Hofmann, Markus Stumm, Nadine Dettmann, Katharina
Pfaff, Franziska Heese, Daniel Grölz
P 136
Comparison of microRNA content in plasma and urine suggests the existence of a transrenal passage of
selected microRNAs.
Eva Pazourkova, Sarka Pospisilova, Iveta Svobodova, Ales Horinek, Antonin Brisuda, Viktor Soukup, Jan
Hrbacek, Otakar Capoun, Jaroslav Mares, Tomas Hanus, Marek Babjuk, Marie Korabecna
31
P 139
A quantitative assessment of circulating nucleic acids utilizing several housekeeping genes:
Measurements from four different cell lines
Janine Aucamp, Abel Bronkhorst, Piet Pretorius
P 148
Circulating DNA-mediated immunoinhibiting involves Ku protein complexing
Anna Cherepanova, Ivan Zaporozhchenko, Valentin Vlassov, Pavel Laktionov
P 168
DNA/protein content of circulating nucleoprotein complexes
Svetlana Tamkovich, Oleg Tutanov, Danil Serdukov, Tatyana Duzhak, Natalia Kirushina, Valentin Vlassov, Pavel
Laktionov
P 175
Oxidized and GC-rich extracellular DNA have different effect on NF-kB and NRF2 signalling pathways in
hMSC, in HUVEC, in MCF7 and in HELF.
Vasilina Sergeeva, Natalia Veiko, Elizaveta Ershova, Larisa Kameneva, Tatiana Smirnova, Anna Alekseeva,
Svetlana Kostyuk
P 176
Non-invasive detection of tissue-specific cell death in ALS
Hai Zemmour, Roni Werman, Daniel Neiman, Joshua Moss, Judith Magenheim, Marc Gotkine, Benjamin Glaser,
Ruth Shemer, Yuval Dor
P 199
Quantitative detection method of DNA without PCR
Yoichi Makino, Masato Nakayama
P 215
Circulating, exosomal microRNAs as biomarkers for cancer and their clinical relevance
Heidi Schwarzenbach
P 216
Screening for epigenetic tumor markers circulating in blood: pitfalls and way out
Evgeny Morozkin, Elena Rykova, Anna Bondar, Tatyana Skvortsova, Ivan Zaporozhchenko, Alexander Kurilshikov,
N.P. Krasnova, E.S. Polovnikov, O.A. Pashkovskaya, E.A. Pokushalov, Anastasia Ponomaryova, Nadezhda
Cherdyntseva, Pavel Laktionov
P 218
A novel technology for enrichment of cell free DNA. Factors of influence for the successful extraction
Heidi Schwarzenbach, Bettina Steinbach, Vipul Patel, Magdalena Grunt, Timo Hillebrand
32
33
Abstracts of Keynote Lectures (KL)
KL 1 / Biology and Nature of Circulating Nucleic Acids
Alain R. Thierry
IRCM, Institut de Recherche en Cancérologie de Montpellier, Montpellier, France
Extracellular nucleic acids (NA) may be liberated outside of the cell in the man physiological extracellular milieu:
e.g. blood, lymph, bile, milk, urine, spinal fluid. These extracellular NA molecules are found also in the animal and
plant kingdoms. Extracellular circulating nucleic acids released in blood are designated by the term circulating NA
(cirNA). Because they were earlier discovered, cirDNA was principally investigated in comparison with the RNA
species but received nonetheless a very late development. In particular, two areas are the focus of most of the study
in the past decade: fetal and tumor cirDNA.
CirDNA present elevated concentrations in cancer patients as opposed to healthy individuals and bear molecular
alterations relating specifically to the tumour and so initiating the concept of a “liquid biopsy”. In parallel, circulating
DNA had become of interest in another clinical domain, since DNA of fetal origin was detected in the blood of
pregnant women so permitting the identification of fetal genetic anomalies through a simple maternal blood sample
and to avoid amniocentesis and other invasive techniques presenting risks and complications. CirDNA concerns
both nuclear and/or mitochondrial DNA and both species exhibit different structural characteristics potentially
revealing different biological stability or diagnostic significance. The small RNAs, mRNA or miRNA from both serum
and plasma has been exploited across a range of studies with some success as possible early markers of various
disorders, probably as a panel of early markers
There are many obstacles towards the transfer of the analysis to the patient’s bedside. It is mainly the lack of both a
complete understanding of the properties of circulating DNA and good practice that are responsible. The determination
of structural characteristics of circulating DNA is still being researched. Nevertheless, some structures have been
identified and circulating DNA has been shown to be composed of many “species”. This structural description
goes hand-in-hand with the mechanisms of its origins such as: apoptosis, necrosis, active release, phagocytosis,
exocytosis…There are multiple structural forms of cirDNA depending upon the mechanism of release: particle
structures (exosomes, microparticles, apoptotic bodies) or macromolecular structures (nucleosomes, virtosomes/
proteolipidonucleic complexes, DNA traps, link with serum proteins or to the cell surface,…).
Differentiation of the various structures and better knowledge of the fate of cirDNA would considerably expand
the diagnostic power of cirDNA analysis for diagnosis or for patient surveillance especially in regards of enlarging
the scope of the personalized medicine. The better understanding subsequent fate of cirNA would also help in
deciphering their functional aspects such as for their capacity of genometastasis or for their pro-inflammatory and
immunological effects.
34
KL 2 / High resolution size analysis of circulating DNA in pregnancy and
cancer
Y. M. Dennis Lo
Li Ka Shing Institute of Health Sciences, The Chinese University of Hong
Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR,
China
Through the use of paired-end sequencing on plasma DNA obtained from pregnant women, we have demonstrated
that circulating fetal DNA molecules are shorter than the maternally derived ones. Prompted by these results, we
have developed a novel approach for the noninvasive prenatal detection of fetal chromosomal aneuploidies, one that
is based on the measurement of the size of plasma DNA. Hence, in pregnant women carrying fetuses with trisomy
21, the size distribution of chromosome 21-derived DNA sequences in maternal plasma is shorter than those from
the other chromosomes, due to the release of an extra dose of short DNA molecules from the trisomic chromosome
of the fetus. This size-based diagnostics approach is synergistic with the current generation of sequencing-based
noninvasive prenatal tests which are based on the counting of DNA molecules in plasma.
We have also explored the use of paired-end sequencing in analyzing the size distribution of DNA molecules in
patients with hepatocellular carcinoma (HCC). We reasoned that genomic regions in which HCC tumor cells exhibit
amplifications would have an enrichment in tumor-derived DNA in plasma. Conversely, for genomic regions in which
the HCC tumor cells exhibit deletions would have a depletion in tumor-derived DNA in plasma. Hence, by comparing
the size distribution of plasma DNA molecules derived from the regions amplified versus those deleted in tumor
cells, we could obtain valuable information concerning the size distribution of cancer DNA in plasma. Our data
indicated that plasma DNA molecules derived from HCC tumor cells are shorter than those from non-tumor cells.
Apart from their intrinsic biologic interest, these data have implications for helping us to develop improved assays
for the detection of tumor-derived DNA in plasma.
35
KL 3 / Lost in translation? Ethical challenges of implementing a new diagnostic
procedure
Dagmar Schmitz
Institute for History, Theory and Ethics in Medicine, RWTH Aachen University,
Medical School, Aachen, Germany
Since cell-free fetal (cff) DNA fragments can be isolated and analyzed from the blood of pregnant women, applications
of this finding have been developed and implemented in clinical care pathways worldwide in an unprecedented pace
and manner. Implementation patterns, however, exhibit considerable insufficiencies. Empirical data demonstrates
that there is a significant loss of information in translating clinical research into the clinical reality of NIPT.
Recommendations of professional organizations are perceived incompletely especially by general practitioners.
Blindsided by the new diagnostic procedure in their clinical practice, professionals in prenatal care express their
insecurities with regard to its handling. While these deficits all are typical for implementation processes of many new
molecular diagnostic procedures, especially in NIPT they show a high variability among different nations.
Mainly, three different “motors” of implementation processes can be identified on a national level: (1) the market,
(2) internal regulatory institutions and (3) external regulatory institutions. Each “motor” entails characteristic
ethical challenges, which are exemplified impressively by a rising number of case reports. Whereas a marketdriven implementation typically lacks sufficient safeguards for pregnant women (or patients), its counterpart – an
implementation guided by external regulatory institutions – for example often comes along with a considerable loss
of time. A critical assessment of the preferred strategy of implementation against the background of already existing
national ethical frameworks is indispensable, if potential adverse effects are to be diminished.
36
KL 4 / Multi-panel cfDNA Approach Demonstrating Utility In Monitoring
Cutaneous Melanoma Progression
Dave SB Hoon
Director Dept of Molecular Oncology and Sequencing Center John Wayne
Cancer Institute, Providence Health Care, Santa Monica, CA USA
Over the last decade we have shown the clinical utility of cfDNA in different forms such as mutation, LOH, methylation,
and integrity. Recently new approaches involving modern techniques have improved in technical feasibility as well
as sensitivity and specificity for assessment of cfDNA. Melanoma in general has shown to release significant
amounts of different forms of cfDNA during tumor progression. With the onset of new therapeutics efficient blood
biomarkers are becoming more important as companion diagnostics. A new highly sensitive approach developed
with Guardant Health in massive parallel sequencing(MPS) digital sequencing and bioinformatics. The approach
utilizes a panel of >70 known cancer related gene mutations. As tumors progress particularly in onset of systemic
metastasis accumulation of multiple gene mutations occurs. Assessment of a multiple panel of cancer gene mutations
in assessing melanoma progression provides a more comprehensive analysis of multiple genomic event changes
that may be theranostic. Analysis of cfDNA panel in serial bleeds over several years of follow up in melanoma
patients can very informative on events ongoing during tumor progression. The highly sensitive and approach
of multiple gene mutation panel assessment by MPS of cfDNA provides a very comprehensive analysis to allow
correlation to realtime clinical status of patients. The retrospective studies demonstrate that cfDNA mutations can
arise at different time points during tumor progression. Retrospective analysis of patients’ serial bleeds for cfDNA
can allow rapid determination of the pattern and significance during melanoma progression. Long-term follow up
of cfDNA and clinical demographics are needed to better evaluate the utility of multi-panel cfDNA mutations and
determine potential role as a companion diagnostic for treatment decision.
37
Abstracts of Introduction Lectures (IL)
IL 1 / Clinical Applications of ctDNA
Luis Diaz
The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of
Medicine, Baltimore, MD 21287, USA
Several malignancies are known to shed DNA fragments into the blood stream. Cancer specific mutations can
distinguish these tumor-derived fragments, termed circulating tumor DNA (ctDNA), from non-tumor DNA. We
developed a dynamic biomarker based on this premise utilizing a highly sensitive digital PCR-based and nextgeneration sequencing-based assays. These assays allow for precise measurement of tumor cell dynamics by
quantifying circulating tumor DNA (ctDNA) levels in plasma from patients with cancer. This technology has been
studied extensively in a variety of tumor types and scenarios. Ongoing efforts are expanding the role of ctDNA
measurements in a variety of clinical scenarios and for the genotyping of patients enrolled in clinical trials. This
technology is also being incorporated into the human clinical trials as a companion diagnostic measuring key
predictive mutations in the blood.
38
IL 2 / Genomic analysis of circulating tumour DNA: pushing the limits for
cancer applications
Nitzan Rosenfeld
Cancer Research UK Cambridge Institute, University of Cambridge,
Cambridge, UK, and Inivata Ltd., Cambridge, UK
Cancer is driven by genomic alterations, and can evolve in response to selective pressures. Sampling of tumour
material however is a limiting factor for both diagnostics and research. Circulating tumour DNA can be found
in plasma and other body fluids, and with advanced genomic techniques it can be used as an effective source
of information for oncology. On one hand, sensitive detection of mutations, rearrangements and copy number
changes can be used for genomic characterisation of cancer using non-invasive sampling. Targeted molecular
profiling tests of “liquid biopsies” in blood plasma are now entering clinical use, and are emerging as an informative
clinical research tool to track response to treatment, cancer progression and emergence of resistance to therapy.
Wider-scale analysis can be used to study new drivers and mechanisms of resistance. In parallel, the specificity
of genomic alterations makes these excellent markers to quantify cancer dynamics and disease burden. Improved
methods and strategies can allow us to stretch the boundaries of analysis to detect smaller amounts of tumour
DNA and to obtain more information from limited samples. These can be used to support an expanding range of
applications for both earlier and later stage cancers.
39
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ABSTRACTS OF ORAL PRESENTATIONS (OP)
CNAPS Basics / Technical Applications
OP 107
Methodological variables in the analysis of cell-free DNA
Abel Bronkhorst1, Janine Aucamp1, Piet Pretorius1
1
North-West University
Since the discovery of cell-free DNA (cfDNA) in human blood, the focal point of most cfDNA studies was to scrutinize it
as a potential non-invasive diagnostic and prognostic marker for solid tumours. Although considerable progress has
been made in this regard, it is as yet a partial victory. Except for some prenatal tests and BEAMing, cfDNA analysis
has not yet been translated to clinical practice and routine application seems distant. This can be ascribed to three
factors that overlap: (i) a lack of knowledge regarding the origin and function of cfDNA, (ii) insufficient molecular
characterization, and (iii) the absence of an analytical consensus. In this work we address the latter determinant and
focus specifically on quantitative analysis of cfDNA. Although it is clear from the literature that a single quantitative
assessment is of limited value, it is discernible that an analysis of the kinetics of cfDNA concentration will be a
strong auxiliary component to qualitative characterization. In order to confidently use quantitative analysis for this
purpose a major proviso becomes crucial, i.e., the optimization and standardization of procedures. Thusly, we
aimed to elucidate the most confounding variables at each preanalytical step that need to be considered in this
endeavour. Using the growth medium of cultured cells as a source of cfDNA, we found variations to the centrifugation
regime, storage temperature, thawing temperature and storage tube type to affect the yield of cfDNA considerably.
Furthermore, regarding the isolation of cfDNA, we found variations to the type of denaturing agent, binding buffer,
elution buffer volume, elution regime, and the tube in which isolated cfDNA is stored to greatly affect the yield. We
contend that many of these variations have not yet been considered, and that it should be useful considerations
when optimizing protocols and setting up a standard operating procedure.
OP 141
A historical and evolutionary perspective on circulating nucleic acids and extracellular vesicles: Circulating
nucleic acids as homeostatic genetic entities
Janine Aucamp1, Abel Bronkhorst1, Piet Pretorius1
1
The quantitative and qualitative differences of circulating nucleic acids (cirNAs) between healthy and diseased
individuals have motivated researchers to utilize these differences in the diagnosis and prognosis of various
pathologies. Although great progress has been made, cirNAs research has not yet been translated to clinical
practice in great part due to a lack of knowledge regarding its function and biological and evolutionary origins. The
position maintained here is that reviewing the rather neglected early work associated with cirNAs and extracellular
vesicles (EVs) is required to fully describe the nature of cirNAs. This review consists of an empirically up-to-date
schematic summary of the major events that developed and integrated the concepts of heredity, genetic information
and cirNAs. This reveals a clear pattern implicating cirNAs as a homeostatic entity or messenger of genetic
information. Homeostasis is a critical prerequisite for biological function, as it provides balance, stability, synchrony
and order while adjusting to conditions that are optimal for survival. DNA is susceptible to change or damage during
transcription and replication and any form of cellular stress or change can promote epigenetic changes. These
stresses or changes, however, do not always affect whole tissues or organs, but can be transferred to unaffected
areas. In the bystander effect, for example, information is transferred from targeted cells exposed to damaging
41
agents of physical or chemical nature to adjacent, non-irradiated cells. It is therefore safe to assume that if the body
utilizes homeostasis to synchronize and regulate organ and tissue function there must also be a similar mechanism
to equilibrate or synchronize genetic information between the cells of organs and tissues as well as between
different organs and tissues. The schematic summary paints a picture of how cirNAs may serve as homeostatic
genetic entities that promote synchrony of both adaptation and damage in tissues and organs depending on the
source of the message.
OP 39
Comparative Analysis of Ionizing Radiation and Low-Frequency Noise on Genome of the Cell
Irina Vasilyeva1, Valery Zinkin2, Vladimir Bespalov1
1
N.N. Petrov Research Institute of Oncology
2
Research Institute of the Air Force of Defense of the Russian Federation
An action of ionizing radiation (IR) and low-frequency noise (LFN) on the integrity of cell genome was studied.
Genomic integrity was assessed by the frequency of chromosomal aberrations (ChA) and the content of extracellular
low-molecular-weight (exlm) DNA. Wistar male rats were irradiated on the unit IGUR137 Cs at a dose of 2 Gy (dose
rate of 1.9 Gy/min) or exposed to LFN on the installation JBL 2225, 2000 impulses for 17 min with sound pressure
levels of 120 and 150 dB. ChA at metaphase plates of bone marrow cells were counted by the standard method 24
h after exposures. ExlmDNA was isolated from blood plasma and measured by standard technique 5–24 h after the
exposures. The frequency of ChA after the exposures to the both IR and LNF were respectively 10.0% and 11.3%
(vs. 0.9% in intact control, p<0.05). The characteristics of ChA after the exposures were different. Dicentrics were
almost absent in control, but after the impacts their rate was 1.5‒2.2%. Proportions of single/pair fragments after IR
(2.8/7.3) and LFN (3.2/4.7) were similar. Thus, the LFN as well as IR is capable to create the double-strand DNA
breaks. The influences of IR and LFN on release of DNA into blood plasma were different. IR caused the maximum
increase of exlmDNA content 5 h after exposure and the content was decreased to initial level 24 h after exposure.
Elevated levels of exlmDNA were detected 24 h after LFN and remained stable during 7 days. The maximum
contents of exlmDNA after IR and LFN were 11.9 ng/ml and 84 ng/ml (vs. 5.5 ng/ml in control, p<0.05). Prolonged
circulation of high level exlmDNA in blood indicates the continuing death of many cells of a body, which can result
in significant disorders of homeostasis. Thus LFN, by analogy with the IR, is able to provide a direct DNA damage
that creates certain conditions for mutagenic effects. In addition, LFN at high levels due to direct and indirect actions
causes damage of internal organs, especially the lungs, and disturbance of microcirculation.
OP 125
Use of High Conductance Dielectrophoresis to Recover Exosomes Containing Circulating Cell-Free RNA
from Undiluted Human Plasma
Stuart Ibsen1, Jennifer Wright1, Johnny Akers2, Clark Chen2, Michael Heller1
1
Department of Nanoengineering, University of California San Diego
2
Moores Cancer Center, University of California San Diego
Circulating cell-free RNA (ccf-RNA) is a biomarker that can yield valuable information about various disease states
within the body. Long RNA strands that exist for a significant period of time in circulation must be encapsulated
or otherwise protected in a complex with proteins and lipids to avoid endogenous RNase activity in the blood.
Current recovery methods for this protected ccf-RNA require large volumes of plasma and time-intensive extraction
processes. Here we demonstrate for the first time the successful use of an electrokinetic technique to recover
exosomes containing RNA after having been spiked into undiluted human plasma samples from healthy donors.
42
These exosomes were produced by a glioblastoma cell line (U87) modified to express the epidermal growth factor
receptor variant III (EGFRvIII). We use a newly designed microfluidic chip with an electrode array at the bottom
that is capable of performing dielectrophoresis (DEP) in a 30 µl sample of a high conductance medium like human
plasma. We show that these spiked exosomes are preferentially pulled into the DEP high-field region around the
electrodes with enough force to be held in place as a buffer wash removes the bulk plasma sample. Some of these
exosomes then stained positively for RNA content. The exosomes were then recovered and RT-PCR analysis
revealed that the exosomes contained both general housekeeping RNA (beta-actin) and RNA that contained the
EGFRvIII mutation specific to the spiked exosomes produced by the cell line and not found in healthy human
plasma. Control plasma samples that were not spiked with the exosomes did not show the existence of either of
these two RNA types. These proof of concept results show the applicability of DEP to streamline recovery of ccfRNA from human plasma in a two-step collection and wash process, allowing RNA analysis for sequencing and for
future quantification. Future work will explore the use of this technique to recover naturally occurring ccf-RNA from
cancer patients as well as patients who have experienced a traumatic brain injury.
OP 157
Utility of a synthetic RNA reference standard for optimizing extracellular RNA sequencing methods
Maria Giraldez1, Alton Etheridge2, Kai Wang3, David Galas2, Muneesh Tewari1
1
Department of Internal Medicine, Division of Hematology/Oncology, University of Michigan
2
Pacific Northwest Diabetes Research Institute
3
Institute for Systems Biology
The discovery of extracellular RNAs (exRNAs) in biofluids has sparked considerable interest in their use as
disease-specific diagnostic and prognostic biomarkers. Their characterization using next generation sequencing
is challenging and prone to bias because the concentration of RNA is very low and long exRNAs are largely
degraded. However, the discovery of specific exRNAs as ‘disease-specific’ biomarkers assumes that differential
profiles of exRNAs across different samples are largely reflective of disease related changes, and not by differences
arising from sequencing bias. Therefore, the process of discovery of disease-specific exRNAs as clinically relevant
biomarkers would be greatly enhanced by standardized techniques for sequencing that minimizes variability. A
powerful approach for optimizing protocols and assessing sequence-specific bias, length bias, sensitivity and
reproducibility is to use a mixture of chemically synthesized RNA molecules present at defined concentrations
as a reference standard. We have developed a synthetic reference standard that comprises 476 RNA molecules
designed to simulate exRNA present in plasma or serum by containing a diverse range of RNA species of different
length (from 15 nt to 90 nt) and sequence. Synthetic RNA reference pools have been designed which comprise an
equimolar pool of all 476 synthetic RNAs, as well as additional pools in which the RNAs are present at a range of
concentrations. More specifically, the 476 RNAs comprise 286 human microRNAs, 164 fragments of mRNA and
lncRNAs ranging from 15 nt- 90 nt, 8 plant miRNA sequences and 18 artificial miRNA sequences as controls.
We will discuss the impact of our synthetic RNA pools in the optimization of small RNAseq protocols within the NIH
ExRNA Communication Consortium.
OP 69
Tandem Repeats of Heterochromatin in the Extracellular DNA
Irina Vasilyeva1, Olga Podgornaya2, Vladimir Bespalov1
1
Institute of Oncology, N.N. Petrov Research, St. Petersburg
2
Institution of Cytology, St. Petersburg
43
Extracellular DNA (ecDNA) composition altered in pathological conditions, probably as a result of the activation of
apoptosis. EcDNA composition determination becomes possible after genomes reading and assembly to such an
extent as the genomic DNA sequences are annotated. An asymmetric enrichment was shown in the distribution
of the repetitive sequences in ecDNAafter experimental apoptosis: (1) significant enrichment in pericentromeric
(periCEN) tandem repeats (TR), the main of which is human satellite 3 (HS3) but decrease of centromeric (CEN)
alpha satellite; (2) enrichment in Alu (SINE) but decrease of LINEs. PeriCEN TR are attached to the inner part
of the cell membrane as an ~ 6 6kb fragments and comes to the apoptotic ecDNA first of all. It is known that
TR begins to be transcribed and enters the bloodstream during pathological, stress conditions in the organism.
CREST (serum of patients with autoimmune diseases) contains antibodies against the kinetochore (CEN) and other
chromosomes’ scaffold proteins. Scaffold proteins provide structural integrity of the chromatin, and possibly they are
associated with heterochromatin sequences in ecDNA. Application of CREST serum make it possible to study the
protein part of kinetochore of many organisms - maize, Y chromosomes mouse, human neoCEN, but the motives
of the kinetochore proteins binding the DNA mainly unknown, with the exception of CENPB. CEN proteins are
conservative, but CEN DNA is the most variable part of the genome. EcDNA of apoptotic cells is highly enriched of
heterochromatin regions of normally transcriptional inactive regions of TR. In spite of the data, that ecDNA enriched
with mainly periCEN TR, including periCEN alpha-satellite type, ecDNA appears to be enriched with CEN regions
also, as evidenced by the existence and the successful application of serum CREST. The study of ecDNA-protein
complexes is necessary to prove this supposition.
CNAPS in Maternal Fetal Medicine
OP 119
Implementing non-invasive prenatal diagnosis (NIPD) in a National Health Service laboratory; from dominant
to recessive disorders
Suzanne Drury1, Sarah Mason1, Fiona McKay1, Kitty Lo2, Christopher Boustred1, Melissa Hill1, Samantha
Edwards1, Naomi Gibson1, Clinda Puvirajasinghe1, Lucy Jenkins1, Lyn Chitty1
1
Great Ormond Street Hospital
2
Ucl Genetics Institute
Our UK National Health Service regional genetics laboratory offers NIPD for autosomal dominant and de novo
conditions (achondroplasia, thanataphoric dysplasia, Apert syndrome) paternal mutation exclusion (cystic fibrosis)
and a range of bespoke tests. NIPD avoids the risks associated with invasive testing, making prenatal diagnosis
more accessible to families at high genetic risk. In 2014, 32% of definitive molecular prenatal diagnostic testing in
our laboratory was based on NIPD (67% if fetal sex determination is included). However, there is a need to offer
definitive diagnosis for autosomal recessive (AR) diseases. Such testing is complicated by the predominance of the
maternal allele in the cell-free DNA sample and requires a variety of different approaches.
We have overcome the challenges presented both for validation and diagnostic implementation for congenital
adrenal hyperplasia (CAH) due to the presence of a pseudogene by using a Sureselect Custom Enrichment assay
targeting approximately 6700 heterozygous SNPs around the CAH gene (CYP21A2) to construct the high-risk
parental haplotypes. In all five cases tested with follow-up we have shown that inheritance of the parental alleles
can be correctly identified non-invasively. This assay has been submitted to the regulatory body for approval for
clinical use.
We have developed a haemoglobinopathy sequencing panel and are employing a measure of fetal fraction to help
determine inheritance of parental mutations. We are currently exploring the utility of an NIPD multi-disorder panel
44
for AR disease, to streamline our workflow and make testing more cost-efficient and more widely applicable to
families with a variety of serious genetic conditions
OP 177
Non invasive prenatal diagnosis of feto-maternal platelet incompatibility by cold high resolution melting
analysis
Marta Ferro1, Hada C. Macher2, Pilar Noguerol3, Pilar Jiménez-Arriscado2, Patrocinio Molinero2, Juan Miguel
Guerrero2, Amalia Rubio2
1
Universitary Hospital Virgen del Rocío of Seville
2
Dpt. of Clinical Biochemistry, Instituto de Biomedicina de Sevilla (Ibis /Csic /Sas /University of Seville)
3
Immunohematology Unit of Universitary, Hospital of Seville
Fetal and Neonatal alloinmune thrombocytopenia (FNAIT) is a condition which approximately occurs in 1 of 1000
live births, and it is due to maternal alloinmunization against paternally inherited antigens of the fetal platelets.
HPA-1 is the most commonly antigen implicated in Caucasian population. The 80% of incompatibilities are caused
by pregnancies of heterozygous HPA-1a fetuses in HPA-1b homozygous women, even though the only difference
between the two alleles is a single nucleotide change (rs5918).
The development of a non-invasive prenatal test for the diagnosis of FNAIT attempts to prevent the intracranial
hemorrhage and therefore, to reduce the morbidity and mortality of the disease. In addition, current techniques as
the amniocentesis nowadays used for the diagnosis might be replaced.
Methods: HPA-1 was studied in serum of three homozygous HPA-1b gestant women and two homozygous for
HPA-1a, all carrying heterozygous fetuses. We performed a COLD High Resolution Melting PCR (COLD-HRMPCR) to distinguish fetal genotypes from the mothers.
Results: The differences in the melting curve shapes allow us to clearly distinguish correctly fetal genotypes from
each homozygous mother. These results were confirmed by sequencing. Once a homozygous pregnant women
was found, the risk of incompatibility because of an heterozygous fetus may be evaluated using a COLD-PCR
analysis to detect the free fetal DNA in her serum.
Conclusion: The results show the utility of HRM-COLD-PCR analysis to asses feto-maternal platelet HPA-1
incompatibility. Both techniques together represent a fast and cost-effective non-invasive method that may be
helpful to provide clinical advice about FNAIT possibility.
OP 192
Non-invasive prenatal diagnosis of Duchenne and Becker muscular dystrophies by relative haplotype
dosage
Michael Parks1, Samantha Court1, Siobhan Cleary1, Samuel Clokie1, Julie Hewitt1, Denise Williams1, Trevor
Cole1, Fiona MacDonald1, Mike Griffiths1, Stephanie Allen2
1
West Midlands Regional Genetics Service
2
West Midlands Regional Genetics Service; Birmingham Women’s NHS Foundation Trust
As part of the NIPSIGEN project (Non-Invasive Prenatal diagnosis for Single Gene disorders), we are aiming to
develop and validate a clinical test for the non-invasive prenatal diagnosis (NIPD) of Duchenne and Becker muscular
dystrophies (DMD/BMD) in at-risk pregnancies. Cell free DNA (cfDNA) was extracted from maternal blood; DNA
sample libraries were prepared for massively parallel sequencing on an Illumina MiSeq by enriching specific target
regions (200 Kb in total) on chromosome X with a custom built capture probe library; sequencing data was analysed
by relative haplotype dosage (RHDO) on 300-400 heterozygous SNPs across a 2.4 Mb long region containing the
45
dystrophin gene. Blood samples from seven healthy pregnant donors and three pregnant DMD carriers all bearing
a male fetus and undergoing invasive prenatal testing were tested. Fetal genomic DNA from the CVS obtained from
the healthy donors was used to identify the haplotype of interest on chromosome X. For the DMD pregnancies,
the affected haplotype was identified from genomic DNA obtained from a previously affected sibling. Using RHDO
analysis, the allelic imbalance observed in heterozygous SNPs was used to determine over or under-representation
of independent haplotype blocks (containing ≥25 SNPs) across the region of interest. The combined outcomes
of the haplotype blocks determined the final result. The results suggest a very high sensitivity and specificity for
this test based on the samples tested so far. The fetal portion of cfDNA inputted in the RHDO analysis was also
calculated using the same allelic imbalance. This new assay for NIPD of DMD has shown great promise in the
initial stages of validation and is highly affordable (2-3 patients can be multiplexed in a single sequencing run). It is
also capable of detecting recombination events within the DMD gene by splitting the RHDO analysis into multiple
statistically independent haplotype blocks across the targeted region.
OP 210
Towards the non-invasive prenatal diagnosis of maternal mutations in clinical practice: validation of the
digital PCR technique
Sara Perlado-Marina1, Ana Bustamante-Aragones1, Marta Donas1, Maria José Trujillo Tiebas1, Isabel LordaSanchez1, Marta Rodriguez de Alba1
1
Genetics Service. Fundacion Jiménez Diaz. Avda. Reyes Catolicos, 2. 28040. Madrid
Introduction: Non-invasive prenatal diagnosis (NIPD) based on the analysis of maternal blood is currently offered
worldwide in prenatal diagnosis units. However, NIPD of maternally inherited fetal diseases is not yet available in
clinical practice. This work shows a validation study of digital PCR (ddPCR) technology for the analysis of both
paternally and maternally inherited fetal alleles.
Mat&Methods: NIPD analysis of a single nucleotide polymorphism (SNP) was performed by ddPCR and Taqman
chemistry in 55 maternal plasma samples. In 36 out of 55, diagnosis of the maternally inherited fetal allele was
performed by relative mutation dosage (RMD) analysis. In 19 out of 55 cases, analysis of the paternal allele was
performed by presence/absence criteria.
Results: Detection of maternal alleles: Balanced and imbalanced allelic ratio was observed in 23 and 13 out of 36
cases, respectively. Presence of fetal DNA was confirmed by different strategies in those samples with a balanced
RMD. Thirty out of 36 fetuses were correctly diagnosed, showing an accuracy of 93% (1 false positive (FP) and 1
false negative (FN) case detected). Detection of paternal alleles: Accuracy of 100% with no FP or FN.
Discussion: While genotyping of fetal alleles not present in the maternal genome is affordable by different
techniques, fetal genotyping of maternal alleles is more challenging and requires highly precise allelic quantification.
ddPCR counting technology allows the study of both paternally and maternally inherited fetal conditions. Balanced/
Imbalanced allelic ratio calculated using RMD analysis can be associated to a fetal genotype. In this study, ddPCR
has been shown to be accurate for the detection of both paternal and maternal fetal alleles in maternal plasma.
OP 144
Construction of cfDNA-Like Reference Materials for Use in Non-Invasive Prenatal Screening (NIPS) Tests
Seth Harkins1, Yves Konigshofer1, Alice T. Ku1, Farol L. Tomson1, Katherine Bianco2, Russell K. Garlick1,
Barathi Anekella1
1
Seracare Life Sciences
2
Department of Obstetrics, Gynecology, and Reproductive Sciences, Ucsf School of Medicine, University
of California
46
Cell-free DNA (cfDNA) is scarce in human plasma and despite its characteristic elevation during pregnancy,
sequestering of the significant quantities needed for a “timeless” reference are not practically attainable. Clinical
utilization of cfDNA in NIPS tests has become commonplace for early indication of syndromes characterized by
chromosomal aneuploidies. Our goal in this study was bypass these inherent limitations and generate a fabricated,
patient like sample which is sufficiently equivalent to the cfDNA found during an aneuploid pregnancy. Hence, we
designed a pool of analyte DNA embodying the characteristic fragment size, fetal fraction percent, and chromosomal
imbalance (Trisomy 13, 18, or 21) and packaged it into a nucleosome mimetic to enhance stability and function.
Cultured human trophoblast progenitor cell (hTPC)lines from confirmed trisomic placentas were used as the source
fetal DNA and were place into a background of female euploid DNA at various concentrations. DNA isolates were
independently sheared to ~160 bp using a Covaris ultrasonicator followed by loaded into the nucleosome mimetic.
The nucleosome mimetic has a bilayer construction with a 100-200 nm in diameter. Unincorporated DNA was
removed from the systems via one-step anion exchange purification methodology. A particular set of reference
materials was prepared and samples at 100%, 16% and 7.7% fetal:maternal fraction were found have a normalized
chromosome count more than 1 SD above background when interrogated with by massively parallel sequencing;
samples at 3.7% & 1.8% could not be distinguished from background. We expect the underlying methods of this
study to be broadly applicable to the generation of patient-like samples containing minute quantities of nucleic
acids.
CNAPS in Cancer I
OP 5
Histone methylation marks in circulating nucleosomes as novel biomarker in colorectal cancer
Ugur Gezer1, Ebru E. Yörüker1, Emre Özgür1, Metin Keskin2, Stefan Holdenrieder3
1
Oncology Institute, Istanbul University
2
General Surgery, Istanbul University
3
Institute of Clinical Chemistry and Pharmacology, University Bonn
As stabile structure in circulation nucleosomes could be valuable sources for detection of cancer-associated
alterations in histone modifications. We are interested in the relevance of histone methylation marks in circulating
nucleosomes (cirNUCs) in cancer with a focus on colorectal cancer (CRC), one of the leading cancers respective
the incidence and mortality. Our previous work based on chromatin immmunoprecipiation-related PCR (ChIPPCR) revealed that trimethylations of H3lys9 (H3K9me3) and H4lys20 (H4K20me3) are found at reduced levels
in cirNUCs of CRC patients Here we asked whether global measurement of histone marks in circulatiın could
be a suitable approach to show their potential as biomarkers. In addition to H3K9me3 and H4K20me3 we also
measured H3K27me3 in plasma samples from CRC patients (n=63) and clonoscopy-based CRC free individuals
(n=40) by ELISA-based methylation assays. Our results show that of three marks, the amounts of H3K27me3
(p=0.04) and H4K20me3 (p=0.0002) were significantly lower in CRC patients than in cancer-free individuals. For
H3K9me3 similar amounts were measured. In conclusion, this explanatory study indicates the potential of ELISAbased measurement of histone marks in cirNUCs as potential cancer biomarker.
“This work was supported by the Scientific and Technological Research Council of Turkey (TUBITAK): Project no
113S881”
47
OP 14
Analysis of circulating tumour DNA to monitor tumour burden and treatment resistance in metastatic
melanoma
Stephen Wong1, Jeanette Raleigh1, Jason Callahan1, Athena Hatzimihalis1, Ismael Vergara1, Tony Papenfuss2,
Rodney Hicks1, Carleen Cullinane1, Mark Shackleton1, David Gyorki1, Damien Kee1, Ben Brady1, Grant
McArthur1, Shahneen Sandhu1, Sarah-Jane Dawson1
1
Peter Maccallum Cancer Centre
2
Walter and Eliza Hall Institute of Medical Research
Melanoma is an aggressive skin cancer, however, immunotherapy and MAPK targeted therapies have significantly
improved patient outcome in recent years. The analysis of circulating tumour DNA (ctDNA) in melanoma patients
to profile genomic alterations, monitor tumour burden and detect the early emergence of treatment resistance has
not been fully explored. In this study we performed serial ctDNA analysis on plasma samples from patients with
metastatic melanoma (n=34) commencing systemic therapy (BRAF inhibitor, BRAF/MEK inhibitor or immunotherapy).
In BRAF mutant patients (n= 19), BRAFV600 mutant copies were measured using microfluidic digital PCR and
were detected in 74% of patients at baseline (64-96 x 103 copies/ml plasma, median 1112) encompassing 1.8%86% (median 10.4%) of the total circulating DNA levels. Known measures of disease burden including LDH and FDGPET metabolic disease volume, correlated significantly with mutant BRAF copies in plasma (r=0.7818, p=0.0032)
and (r=0.63, p=0.0001), respectively. In BRAF wildtype patients (n=15) targeted sequencing of ctDNA, identified
mutations in genes such as NRAS, RAC1, MAP2K1, PTEN and TP53 allowing serial ctDNA levels to be monitored
during treatment. Targeted sequencing of serial plasma samples also identified genomic changes occurring following
the selective pressure of MAPK targeted therapy. In several cases where acquired resistance to BRAF inhibition
developed, ctDNA analysis revealed the emergence of mutations known to be associated with treatment resistance.
This included evidence for a subclonal population resistant to BRAF inhibitor treatment observed in one patient,
where a rapid decrease in mutant BRAF ctDNA levels occurred in parallel to the emergence of a PI3K pathway
mutation. These findings reveal the potential of ctDNA analysis to track clonal evolution during disease progression
and highlight the potential of ctDNA profiling as a non-invasive biomarker in the clinical management of melanoma
patients.
OP 41
Evaluation of circulating cell-free DNA as a molecular monitoring tool during cancer therapy
Clemens Hufnagl1, Lukas Weiss1, Thomas Melchardt1, Martin Moik1, Daniela Asslaber1, Philipp Steininger2,
Thomas Meißnitzer3, Daniel Neureiter4, Richard Greil1, Alexander Egle1
1
Department of Internal Medicine III
2
Institute for Research and Development on Advanced Radiationtechnologies
3
Institute of Radiology, Paracelsus Medical University Salzburg
4
Institute of Pathology, Paracelsus Medical University Salzburg
Introduction: The management of metastatic cancer requires new specific, sensitive and ideally low invasive
biomarkers to determine an early response to treatment. Circulating cell-free DNA (cfDNA) may represent such a
non-invasive biomaker, as it can be found in higher concentrations in the plasma of cancer patients sharing some
characteristics with DNA of tumor cells (cft-DNA) like specific oncogene or tumor-suppressor gene mutations.
The aim of this work was to compare different established serum-biomarkers with our analysis of cfDNA and cftDNA in correlation to disease monitoring by CT-imaging.
Materials and Methods: We analyzed the amount of cfDNA/cft-DNA in 15 patients with metastatic breast, colon
48
or pancreatic cancer during their different treatments. We analyzed the ratio of level changes of cfDNA/cft-DNA
and classical biomarkers, respectively, and compared these analysis to the disease phases as diagnosed by CT
imaging according to RECIST criteria.
Results: We observed a relevant correlation of cfDNA (p<0.0001) and cft-DNA (p=0.0072) with RECIST defined
response on longitudinal volumetric analysis of metastases in a subset of patients. In contrast to the excellent
correlation at defined staging time-points, we observed a large signal of unexplained noise when analyzing samples
obtained during treatment phases.
In comparison to cfDNA/cft-DNA all other tumor marker did not show a consistent change in the quantitative values,
corresponding the outcome of the CT-analysis.
Conclusion: Consecutive quantitative analyses of cfDNA and cft-DNA seem to be able to reflect the tumor
burden during chemotherapy in patients with metastatic disease and pose an interesting field of current and future
research.
OP 57
Rapid aneuploidy screening of plasma DNA samples from cancer patients using a modified FAST-SeqS
approach
Jelena Belic1, Marina Koch1, Martina Auer1, Peter Ulz1, Jochen Geigl1, Michael Speicher1, Ellen Heitzer1
1
Institute of Human Genetics, Medical University of Graz
Introduction: Recent progress in the analysis of cell-free DNA fragments now allows monitoring of tumor genomes
by non-invasive means. However, previous studies with plasma DNA from patients with cancer demonstrated highly
variable allele frequencies of ctDNA. The comprehensive analysis of tumor genomes is greatly facilitated when
plasma DNA has increased amounts of ctDNA. Therefore, a fast and cost-effective pre-screening method to identify
such plasma samples without previous knowledge about alterations in the respective tumor genome could assist in
the selection of samples suitable for further extensive qualitative analysis.
Methods: To address this, we adapted the recently described FAST-SeqS method, which was originally established
as a simple and effective, non-invasive screening method for fetal aneuploidy from maternal blood.
Results: We show that our modified FAST-SeqS method (mFAST-SeqS) can be used as a pre-screening tool for
an estimation of the ctDNA percentage. Plasma samples with an mFAST-SeqS z-score above 5 showed highly
concordant results compared to copy number profiles obtained from our previously described plasma-Seq approach.
Therefore, mFAST-SeqS revealed a general overview about the aneuploidy status of the tumor genome on the
resolution of chromosome arms.
Conclusion: Advantages of this approach include that no prior knowledge about the genetic composition of tumor
samples is necessary in order to identify between plasma DNA samples with more than 10% of ctDNA content, and
the speed and cost-effectiveness of the assay.
OP 92
Circulating tumour DNA to monitor treatment response and resistance in chronic lymphocytic leukaemia
Devbarna Sinha1, Sarah Ftouni1, Tane A. Hunter1, Paul Yeh1, Elise Wallach1, Stephen Wong1, Enid Lam1,
Pasquale Petrone1, John F. Seymour1, Mark A. Dawson1, Constantine Tam1, Sarah-Jane Dawson1
1
The Peter Maccallum Cancer Centre
49
Several highly active targeted therapeutics are poised to change the treatment landscape in chronic lymphocytic
leukaemia (CLL). The advent of new therapeutic options has highlighted the deficiencies of current strategies
for disease monitoring and brought into focus the need for improved methods to accurately assess treatment
responses. We sought to prospectively assess the role of circulating tumour DNA (ctDNA) as a molecular tool for
disease monitoring in CLL. ctDNA analysis was undertaken in 8 patients with relapsed / refractory CLL receiving
one of several novel therapies; Ibrutinib, Venetoclax or Obinutuzumab. Targeted sequencing (TS) of plasma and
matched leukaemic cellular DNA collected pre-treatment was undertaken to identify somatic mutations across a
panel of genes known to be recurrently mutated in CLL and key driver mutations in TP53, SF3B1 and Notch1 were
identified in all individuals. ctDNA was quantified using TS and digital PCR in 37 serial plasma samples across the
cohort and comprised between 0.2-45% of total circulating DNA. Dynamic changes in ctDNA correlated closely with
conventional treatment responses across the series. In one case, rising ctDNA levels coincided with progressive
lymphadenopathy despite negative minimal residual disease immunophenotyping on peripheral blood and bone
marrow, highlighting the potential of this technique to detect compartmentalized disease progression following
treatment. In 3 patients receiving ibrutinib, the initial peripheral lymphocytosis, which often confounds conventional
disease monitoring, was not associated with a parallel rise in ctDNA, which reflected the effects of therapy on
disease burden. In the context of 2 individuals who developed Richter’s transformation (RT) following treatment,
paired whole exome sequencing was performed on plasma and bone marrow/lymph node tissue DNA at baseline
and RT. In each case, ctDNA analysis identified the emergence of mutations associated with disease transformation,
revealing the potential of this approach for non-invasive molecular disease monitoring in CLL.
OP 109
Low cost, broad panel (100kb) liquid biopsy with reduced DNA sequencing by depletion of wild-type
sequence.
Joel Pel1, Milenko Despotovic1, Patrick Davies1, Laura Gelinas1, Andre Marziali2
1
Boreal Genomics Inc.
2
University of British Columbia, Engineering Physics; Boreal Genomics Inc.
The most significant barrier to widespread clinical deployment of a highly sensitive circulating tumor DNA (ctDNA)
assay (liquid biopsy) is the high cost of the assay compared to potential reimbursement. Assay cost is currently
dominated by the large amount of DNA sequencing required to achieve coverage of a broad gene panel, and by the
high read depth required for high clinical sensitivity. The common practice of attaching unique molecular barcodes
to ctDNA fragments for the purpose of error reduction further increases the sequencing requirements, making liquid
biopsy commercialization in many clinical applications impractical.
Since the majority of reads are wasted re-sequencing wild-type cell free DNA (cfDNA) fragments, a powerful
solution to this challenge is to somehow separate mutant cfDNA from wild-type cfDNA so that only relevant tumor
molecules are sequenced. Given that in many clinical cases ctDNA represents only 0.1% – 0.01% of the total
cfDNA, this approach has the potential to reduce required sequencing by 1,000 – 10,000 fold. While we have
previously demonstrated such separation for specific mutations, the larger challenge is to achieve this separation
and enrichment of ctDNA without any prior knowledge of the mutation site or sequence, and over a broad set of
relevant genes.
We present a new version of our electrophoretic enrichment technology that can deplete a sample of wild-type
genomic DNA prior to sequencing without a priori knowledge of the mutation sequence or location, and over large
regions of the genome, in the range of 100kb or more. By narrowing the sample to contain primarily mutant ctDNA,
both the sequencing cost and false positive sequencing errors are greatly reduced.
We present a methodology used to deplete wild-type sequence over large genomic regions, as well as data from
clinical samples. The addition of wild-type DNA depletion to NGS assays enables lower cost, high sensitivity,
50
and high specificity liquid biopsy tests to be developed, enabling commercialization in applications with limited
reimbursement, potentially including early cancer detection.
OP 211
Targeted sequencing reveals clonal genetic changes in the progression of early glandular neoplasms of the
lung and paired circulating DNA.
Evgeny Izumchenko1, Xiaofei Chang1, Mariana Brait1, Elana Fertig2, Luciane T. Kagohara1, Atul Bedi1, Luigi
Marchionni3, Sian Jones4, Mohammad O. Hoque1, William H. Westra1, David Sidransky1
1
Department of Otolaryngology-Head and Neck Surgery and Oncology, Johns Hopkins University School of
Medicine, Baltimore, MD.
2
Division of Biostatistics and Bioinformatics, Johns Hopkins University School of Medicine, Baltimore,
MD.
3
Center for Computational Genomics, Johns Hopkins University School of Medicine, Baltimore, MD.
4
Personal Genome Diagnostics, Inc. 2809 Boston St, Suite 503, Baltimore, MD.
Lungs resected for primary adenocarcinomas often harbor minute discrete foci of cytologically atypical pneumocyte
proliferations designated as atypical adenomatous hyperplasia (AAH). Evidence suggests that AAH represents
an initial step in the progression to adenocarcinoma in situ (AIS), minimally invasive adenocarcinoma (MIA) and
ultimately fully invasive adenocarcinoma. Despite efforts to identify predictive markers of malignant transformation,
genetic changes driving this progression are not yet understood. We performed targeted next generation sequencing
(NGS) on DNA isolated from multifocal AAHs and different zones of histologic progression within AISs and MIAs.
Multiregion sequencing approach discovered different genetic mutation drivers within the same tumor and revealed
that clonal expansion is an early event of lung tumorigenesis. We also found that KRAS mutations or loss of P53
activity and EGFR activation were a harbinger of malignant transition. Utilizing an ultra-sensitive digital droplet PCR
(ddPCR), genetic alterations in early glandular neoplasms could be detected in paired circulating DNA (ctDNA). This
study provides unique insight into the heterogeneity of clonal events in the progression of early lung neoplasia and
demonstrates proof of concept for the detection of these events even before neoplasms have invaded and acquired
malignant potential.
51
OP 140
Rapid Isolation, Detection and Analysis of Hematological Cancer and Solid Tumor Related Cell Free Nucleic
Acids in Blood, Plasma and Serum
Michael Heller1, Jennifer Marciniak Wright2, Stuart Ibsen2
1
Department of Nanoengineering, University of California San Diego
2
University of California San Diego
Cell free (cf) DNA, RNA and exosomes offer great
promise for liquid biopsy based cancer diagnostics,
therapy monitoring and patient management.
Unfortunately, the isolation of cf-nucleic acids
from plasma and serum remain a relative long and
involved process, and isolation directly from whole
blood has not been feasible until recently. We report
on using a dielectrophoretic (DEP) device that allows
rapid isolation of cf-DNA/RNA and exosomes from
small volumes (20ul-50ul) of whole blood, plasma
and serum. These DEP devices have now been
used for the isolation of cf-nucleic acids from chronic
lymphocytic leukemia (CLL) patient blood, plasma
and serum samples; as well as lung, brain, breast,
colorectal, pancreatic and ovarian cancer patient
samples. Figure 1 shows fluorescent image analysis results for cf-DNA isolated by the DEP device in less than ten
minutes from a normal blood sample and five CLL patient blood samples (left side), and a normal plasma sample
and five CLL patient plasma samples (right side). Subsequent PCR and DNA sequencing results for cf-DNA isolated
from the CLL patient blood and plasma samples by the DEP process were exactly comparable to results from “gold
standard” conventional methods, which take hours to complete and require much larger sample volumes.
Figure 2 shows an example of fluorescent stained cfDNA which was isolated from a 50ul plasma sample
from a NSC lung cancer patient. In addition to PCR
and DNA sequencing mutation analysis, extensive
work is also being carried out on determining the size
distribution of cf-DNA (apoptotic and necrotic) in the
various cancer patient samples. Figure 3 shows the
isolation of cf-RNA containing glioblastoma exosomes
from human plasma samples. DEP devices are also being used to determine presence of injury related cf-RNA in
traumatic brain injury patient samples. Overall the stage is being set for “seamless sample to answer” and liquid
biopsy cancer diagnostics. Sonnenberg A, et al., “Rapid Electrokinetic Isolation of Cancer-Related Circulating Cell
Free DNA Directly from Blood”, Clinical Chemistry, 60:3, pp.500-509, 2014.
52
OP 221
The clinical relevance of circulating, exosomal microRNAs as biomarkers for gynecological tumors
Heidi Schwarzenbach1
1
Department of Tumor Biology, University Medical Center Hamburg-Eppendorf
MicroRNAs are a family of evolutionary conserved, small non-coding RNA molecules that post-transcriptionally
inhibit protein expression of their target mRNAs. As microRNAs loci frequently map to fragile chromosomal regions
harboring DNA amplifications, deletions or translocations, their expression is often deregulated during tumorigenesis,
contributing to tumor progression, metastasis and drug resistance. Apart from their release by apoptotic and necrotic
cells, microRNAs can also be actively secreted into the blood circulation by exosomes. Exosomal microRNAs are
thought to play an important role in cell-to-cell communication. Based on their biological functions and the possibility
of quantifying microRNAs in patient blood in real-time, these small RNA molecules, in size of approx. 22 nucleotides,
may be a new promising class of potential blood-based biomarkers.
Our investigations have shown a prevalence of microRNAs in exosomes. In breast cancer patients, the serum levels
of circulating exosomal miR-373 were found to be higher in triple- and hormone receptor-negative than luminal and
receptor-positive tumors, respectively. Overexpression of miR-373 by transfection of MCF-7 cells downregulated
protein expression of the estrogen receptor, and inhibited apoptosis induced by camptothecin. In ovarian cancer,
increased serum levels of miR-200b and miR-200c were detected to be associated with FIGO IV stage, lymph
node-positive status, the tumor marker CA125 and a shorter overall survival.
Our findings show the relevance of exosomal microRNAs as diagnostic and prognostic tumor markers and their
association with a particular biology of gynecological carcinomas favoring tumor progression and metastatic
spread.
Non-malignant Disease / pre-analytics
OP 31
Circulating microRNAs in the diagnosis of Gestational Diabetes Mellitus
Eloise Lawrence1, Ramasamyiyer Swaminathan2, Anthony Marinaki3, Anna Brackenridge4, Barbara
McGowan4
1
King’s College London
2
Department of Chemical Pathology, Guy’s and St. Thomas’ NHS Foundation Trust
3
Purine Research Laboratory, Guy’s and St. Thomas’ NHS Foundation Trust
4
Guy’s and St. Thomas’ NHS Foundation Trust
Background: Aberrant microRNA expression is speculated to be of use as a diagnostic biomarker for Gestational
Diabetes Mellitus (GDM). GDM affects 3-9% of women during pregnancy and increases the risk of maternal and
neonatal adverse outcomes. Studies have reported the difficulty in diagnosing GDM at an earlier gestational age,
as pregnancy is a period of progressive insulin resistance which is not detectable until the late second to early third
trimester. Early detection of GDM is imperative to prevent maternal and perinatal morbidity and mortality.
microRNAs are small ~22nt non-coding RNAs which post-transcriptionally regulate gene expression. There is
growing interest in the possibility that circulating microRNAs may be useful as minimally-invasive diagnostic and
prognostic biomarkers in molecular diagnostics as expression levels are dysregulated in numerous pathological
states, including cancer.
53
Methodology/principle findings: The aim of this study was to identify circulating plasma miR-29a, 132 and 222 in
GDM positive, GDM negative and healthy subjects to determine whether these microRNAs may serve as candidate
biomarkers for the diagnosis of GDM. 35 participants were recruited into this study (14 GDM, 12 non-GDM, 9
controls). Plasma preparation, RNA extraction, reverse transcription, pre-amplification, Real Time-quantitative
Polymerase Chain Reaction and data analysis were performed. Lower expression of miRNAs were found in cases
than controls. The fold difference of miR-132 was significantly decreased in GDM compared to non-GDM patients
(P<0.001). miR-222 was significantly down-regulated in pregnant participants compared to non-pregnant controls
(P<0.001) but not between cohorts (P<0.431). miRNA expression was also significantly correlated with clinical
parameters associated with GDM.
Conclusion/significance: These findings suggest that circulating plasma miR-132 is a biomarker for GDM detection;
however larger studies are required to confirm and evaluate the potential clinical use of miRNA expression in the
detection and diagnosis of GDM.
OP 162
Discovering the Tissue Origins of Cell-Free DNA
Roni Werman1, Daniel Neiman1, Hai Zemmour1, Joshua Moss1, Judith Magenheim1, Adi Vaknin-Dembinsky2,
Kirsty Spalding3, Markus Grompe4, Aviad Zick2, Ayala Hubert2, Volker Fendrich5, Talia Golan6, Iris Lavon2,
Benjamin Glaser2, Ruth Shemer1, Yuval Dor1
1
Hebrew University
2
The Hebrew University-Hadassah Medical School
3
Karolinska Institute
4
Oregon Health & Science University
5
Marburg University
6
Sheba Medical Center
Cell-free circulating DNA (cfDNA) released from dying cells is emerging as a powerful minimally-invasive biomarker
in prenatal diagnosis and in cancer. However its utility is limited to cases where the tissue of interest (e.g. fetus or
tumor) differs genetically from the host. Here we present a novel approach to identify the tissue origins of cfDNA,
based on tissue-specific methylation patterns that are retained in circulating DNA fragments. We identified multiple
CpG clusters whose methylation pattern is unique to specific organs, and utilized this information to non-invasively
detect cell death deriving from these organs. Here we demonstrate elevations in white matter derived cell free DNA
in the circulation of multiple sclerosis and glioma patients, and pancreatic derived DNA in chronic pancreatitis and
pancreatic cancer patients.
OP 195
Exercise-induced cell free DNA in human plasma is predominantly released from cells of the haematopoietic
lineage
Susanne Helmig1, Klaus Bender2, Suzan Tug1, Eva Ricarda Deichmann1, Anna Schmeier-Jürchott3, Eva
Wagner3, Tim Zimmermann4, Markus Radsak3, Mauro Giacca5, Simon Perikles1
1
Department of Sports Medicine, Disease Prevention and Rehabilitation, Johannes Gutenberg-University of
Mainz, Germany
2
Department of Legal Medicine, University Medical Center, Johannes Gutenberg-University of Mainz,
Germany
3
Department of Internal Medicine III, Hematology, Oncology and Pneumology, University Medical Center,
54
Johannes Gutenberg-University of Mainz, Germany
4
Department of Medicine, Johannes Gutenberg-University of Mainz, Germany
5
Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology, Trieste
and Department of Medical, Surgical, and Health Sciences, University of Trieste, Italy
Strenuous exercise induces a rapid and transient elevation of cell free DNA (cfDNA) concentration in blood plasma.
We investigated the origin of cfDNA in sex-mismatched haematopoietic stem cell transplantation (HSCT) and
liver transplantation (LT) patients by determining the relative proportion of Y-chromosomal to total cfDNA and by
chimerism analysis.
7 HSCT and 5 LT patients were analysed. Concentrations of total nuclear and Y-chromosomal cfDNA were measured
in blood plasma before and after an incremental exercise test by quantitative real-time PCR (qPCR). Chimerism
of cfDNA in HSCT patients was determined by STR analysis. Spearman correlations were calculated with JMP 11
(SAS Institute Inc., Cary, NC, USA).
HSCT patients showed proportions of donor-derived cfDNA of mean (±SD) 59.9 (24.3) before, 64.1 (27.8) immediately
after and 57.8 (26.6) % 90 min after exercise as determined by qPCR. Chimerism analysis revealed 71.8 (16.5),
85.7 (17.4) and 73.3 (24.8) % of donor-derived DNA in the circulation. In female HSCT patients total nuclear and
Y-chromosomal cfDNA increased in a highly correlated fashion due to exercise (r= 0.98, p < 0.0001). In male HSCT
patients < 10% of cfDNA was of Y-chromosomal origin and even though total cfDNA concentrations increased
during exercise, no elevations in Y-chromosomal DNA could be detected. The percentage of donor-derived cfDNA
in LT patients was low (2.1 (1.4) %) and levels remained unchanged during exercise.
Despite quantitative differences between both methods the results suggest that cells from the haematopoietic
lineage are the main source of cfDNA released at rest and during acute bouts of exercise. Differences in cellular
origin of the cfDNA pool in resting state and during physical exercise have to be elucidated in further studies.
OP 220
Comparison of Different Preanalytical Workflows for Isolation of Intact Exosomes and Other Extracellular
Vesicles
Gabriele Christoffel1, Karolin Spitzer1, Daniel Enderle2, Johan Skog3, Mikkel Noerholm2, Markus SprengerHaussels1, Martin Schlumpberger1
1
Qiagen GmbH
2
Exosome Diagnostics GmbH
3
Exosome Diagnostics Inc.
Introduction: The scientific literature on exosomes and other extracellular vesicles (EVs) continues to be
characterized by a wide variety of vesicle isolation and characterization procedures, as well as nomenclature,
resulting in considerable difficulty comparing results between independent studies. An additional layer of variability
is added by sample handling and pretreatment. This may include the type of blood collection tube, time between
blood draw and generation of plasma or serum, as well as measures taken to remove residual cells and cell
fragments, such as pre-centrifugation or filtration
steps.
In this study, we compare different handling and pretreatments, and how they affect physical characteristics, as well
as RNA content of vesicle preparations resulting from a spin column-based purification approach.
Methods: Blood from healthy donors was collected in different collection tubes. After generation of plasma and
removal of residual cells and fragments, vesicular RNA was generated and relative abundance of selected RNAs
compared by qPCR. In addition, effects of the time between blood collection and generation of plasma was tested on
the RNA level. Finally, we compared different approaches to remove residual cells and cell fragments by additional
55
centrifugation and filtration steps prior to vesicle isolation, and analyzed the resulting vesicle size distribution and
RNA content.
Results: In all tested preprocessing workflows, intact vesicles and vesicular RNA could be isolated. However,
the choice of collection tube, anticoagulant, etc. does have an influence on RNA representation, so it is strongly
recommended to not switch between different collection tubes within the same study.
Summary/Conclusion: Storage of blood prior to plasma generation can result in release of additional vesicular
RNA from blood cells, which in most cases represents unwanted background. Centrifugation and filtration steps
affect representation of different classes of vesicles.
OP 121
An enquiry concerning the characteristics of cell-free DNA released by cultured cancer cells
Abel Bronkhorst1, Jaco Wentzel1, Etresia Van Dyk1, Janine Aucamp1, Lissinda du Plessis1, Piet Pretorius1
1
The most prominent determinant that delays the translation of cell-free DNA (cfDNA) analyses to clinical practice
is the lack of knowledge regarding its origin and composition. The elucidation of the former is complicated by
the seemingly random fluctuation of quantitative and qualitative characteristics of cfDNA in healthy and diseased
individuals. Besides methodological discrepancies, this could be ascribed to a web of cellular responses to various
environmental cues and stressors. Since all cells release cfDNA, it follows that the cfDNA in the blood of cancer
patients is not only representative of tumour derived DNA, but also of DNA released by “healthy” cells under
otherwise conditions. Additionally, cfDNA released by malignant cells is not necessarily just aberrant. This may
cause false positive/negative results. Although many have acknowledged that this is a major problem, few have
addressed it. We believe that many of the current stumbling blocks encountered in in vivo cfDNA studies can be
partially circumvented by in vitro models. Accordingly, the purpose of this work was to evaluate the release of
cfDNA from cultured cells and to gauge its potential use for elucidating the nature of cfDNA. Firstly, release of
cfDNA was characterized over time. After growth medium renewal the amount of cfDNA increased incrementally,
peaked considerably after 24 hours, and plateaued thereafter. Electrophoresis after 4 hours of incubation showed
a prominent peak at 166 bp, which is consistent with the literature. This peak then diminished incrementally and
disappeared after 24 hours when a new peak of approximately 2000 bp formed. A flow cytometric assay indicated
that a significant fraction of cells are apoptotic after 4 hours, whereas 24 hours showed almost no apoptosis. These
results were corroborated by another flow cytometric method based on the detection of nicked DNA. Additionally,
the cell cycle phase of the cells at each of the times was also analyzed by flow cytometry using a cell proliferation
assay. These observations suggest that the bulk of cfDNA released is not derived from apoptosis.
CNAPS in Cancer II
OP 151
Whole genome deep sequencing analysis of plasma DNA in breast cancer
Devika Ganesamoorthy1, Alan Robertson1, Wenhan Chen1, Kaltin Ferguson2, Peter Simpson2, Lachlan
Coin1
1
Institute for Molecular Bioscience, University of Queensland, St Lucia, QLD, Australia 2University of
Queensland Centre for Clinical Research, Royal Brisbane & Women’s Hospital, Herston, QLD, Australia
56
Analysis of circulating tumour DNA (ctDNA) in the plasma holds the potential for the development of non-invasive
diagnostic tools for cancer. The recent improvements in sequencing technologies have enabled the detection of
somatic genomic alterations in the plasma of cancer patients. To date there are several reports on the use of
targeted genomic approaches to noninvasively detect somatic alterations by ctDNA analysis. However, there are
limited reports on the use of whole genome sequencing analysis of ctDNA. Here we present whole genome deep
sequencing analysis of ctDNA in the plasma of breast cancer patients to detect somatic genomic alterations. Plasma
DNA samples from 4 breast cancer patients were sequenced to at least 120x coverage on Illumina HiSeq X Ten.
The matched germline and tumour samples were sequenced to at least 30x and 60x coverage respectively. The
deep sequencing analysis of the plasma DNA assisted with the discovery of various somatic genomic alterations,
including single nucleotide variants, copy number changes, and chromosomal aneuploidies. Furthermore, tumour
sequence data validated majority of the somatic genomic alterations identified by deep sequencing analysis of
plasma DNA and also hinted the presence of tumour heterogeneity. The results indicated that the complete genetic
architecture of the tumour genome in the plasma can be determined with increased sequence depth across the
whole genome. Non-invasive determination of the tumour genome in plasma could ultimately guide the diagnosis,
monitoring and treatment of cancer.
OP 197
Detection of tumor-specific mutations in circulating cell-free tumor DNA from patient blood using a novel,
barcoded and multiplexed NGS library construction approach
Jennifer Jackson1, Anders Ståhlberg2, Paul Krzyzanowski3, Lincoln Stein4, Tony Godfrey1
1
Department of Surgery, Medical Center, Boston University
2
Sahlgrenska Cancer Center, Department of Pathology, Sahlgrenska; Academy at University of
Gothenburg
3
Ontario Institute for Cancer Research, Mars Centre
4
Department of Molecular Genetics-University of Toronto; Informatics and Biocomputing-Ontario Institute
for Cancer Research
Mounting evidence has shown that tumor cells release cell-free DNA containing tumor-specific alterations into
the blood of cancer patients and that detection of these mutations in plasma may serve as a clinically relevant
biomarker. Detection of these mutations, which are typically at <1% in plasma DNA, is challenging however as
standard next generation sequencing (NGS) lacks the required sensitivity and digital PCR requires prior knowledge
of the mutations in the tumor. Limited (typically <100ng) and fragmented DNA from plasma is also an issue as it
restricts the number of mutations that can be tested with digital PCR and its use is challenging for NGS library
construction. To enable detection of low frequency mutations in multiple genes simultaneously, we have developed
a novel approach to NGS library construction that simplifies barcoding of high-order multiplex NGS libraries. Our
approach uses a molecular hairpin to hide the barcodes and adapter sequences in target primers during the
first PCR cycles when DNA fragments are being barcoded. Barcoding enables background noise reduction and
sensitive mutation detection while our molecular hairpin approach enables high-order multiplexing as it minimizes
non-specific PCR product formation from promiscuous binding of the random barcode sequences. Furthermore,
our workflow is simple with two rounds of PCR and a single bead-based purification step. With this approach we
are able to detect variant allele frequencies of 0.1% or lower in several kilobases of DNA targeting multiple cancerassociated genes when starting with ≤50ng of plasma DNA. This approach may provide a tool for cancer screening,
monitoring tumor heterogeneity and evolution, directing targeted therapies, monitoring response to therapy and
monitoring for disease recurrence.
57
OP 202
Chromosomal rearrangement as cancer-specific biomarker for monitoring of lung cancer after surgery
Hongdo Do1, Thomas John2, Paul Mitchell3, Carmel Murone4, Bibhusal Thapa5, Daniel Cameron6, Ramyar
Molania5, Tony Papenfuss7, Alexander Dobrovic4
1
Olivia Newton-John Cancer Research Institute, University of Melbourne, La Trobe University
2
Austin Hospital, La Trobe University
3
Austin Hospital
4
Olivia Newton-John Cancer Research Institute
5
Olivia Newton-John Cancer Research Institute, University of Melbourne
6
The Walter and Eliza Hall Institute of Medical Research, University of Melbourne
7
The Walter and Eliza Hall Institute of Medical Research, Peter Maccallum Cancer Centre
Clinical outcomes for patients undergoing lung cancer surgery are suboptimal even when the disease is caught
early. Circulating tumour DNA (ctDNA) in patient blood has shown to be promising for detection of early relapse,
enabling timely therapies. Somatic mutations have been used as markers for ctDNA, but this approach is only
applicable to a subset of patients. Chromosome rearrangements are ubiquitous in lung cancer, and can thus be
used as tumour specific markers. This pilot study tested the feasibility of using chromosome rearrangements
for monitoring minimal residual disease in blood from lung cancer patients. Chromosomal rearrangements were
searched in two lung tumours by whole genome sequencing and were detected at a high frequency (109 and
45 events). Four chromosomal rearrangements from each tumour were chosen for an initial validation. Primers
were designed for specific amplification of the rearrangements. Seven out of the 8 rearrangements tested were
successfully validated by qPCR in their tumour DNA. The validated primer sets were used to quantify the level of
ctDNA using droplet digital PCR in plasma collected before and after surgery. Chromosomal rearrangements were
detectable in the baseline plasma of both patients. However, the level of ctDNA was decreased after surgery. This
pilot study proves that identification of chromosomal rearrangements is the effective approach to generate robust,
minimal residual disease markers for lung cancer. The proposed workflow can be used as a model for other types
of cancer with rearranged genomes.
OP 204
Monitoring response to therapy in melanoma by quantifying circulating tumour DNA with droplet digital
PCR
Simon Tsao1, Jonathan Weiss2, Tom Witkowski2, Hongdo Do3, Christopher Hudson4, Christopher Christophi5,
Jonathan Cebon3, Andreas Behren3, Alexander Dobrovic3
1
Olivia Newton-John Cancer Research Institute; Department of Surgery, University of Melbourne, Austin
Health. Melbourne, Australia
2
Olivia Newton-John Cancer Research Institute
3
Olivia Newton-John Cancer Research Institute; Department of Pathology, University of Melbourne; School
of Cancer Medicine, La Trobe University, Melbourne, Australia
4
Olivia Newton-John Cancer Research Institute; School of Cancer Medicine, La Trobe University, Melbourne,,
Australia
5Department of Surgery, University of Melbourne, Austin Health. Melbourne, Australia
We assessed the utility of droplet digital PCR (ddPCR) for mutant BRAF and NRAS alleles to evaluate the potential
of using circulating tumour DNA (ctDNA) as a post therapeutic monitoring tool in melanoma by comparing it to LDH
serum levels and RECIST scores. ddPCR was reliable in distinguishing mutant from wild type alleles . We quantified
58
ctDNA in stage IV melanoma patients during their treatment course. All tested patients had detectable ctDNA,
which exhibited dynamic changes corresponding to the changes in their disease status. The ctDNA levels fell
upon treatment response and rose with detectable disease progression. In our group of patients, ctDNA was more
consistent and informative than LDH as a blood-based biomarker. In addition, BRAF mutant ctDNA as detected by
ddPCR could be used diagnostically where the tumour block was unavailable. In conclusion, this study demonstrates
the applicability of using ddPCR to detect and quantify ctDNA in the plasma of melanoma patients.
OP 208
Non-invasive genomic profiling of urothelial bladder cancer using urinary cfDNA
Fiona Togneri1, Douglas G. Ward2, Joseph M. Foster3, Adam J. Devall2, Paula Wojtowicz1, Sofia Alyas1,
Fabiana Ramos Vasques1, Assa Oumie3, Nicholas D. James4, K.K. Cheng5, Maurice P. Zeegers6, Nayneeta
Deshmukh2, Brendan O’Sullivan7, Phillipe Taniere7, Karen G. Spink3, Dominic J. McMullan1, Mike Griffiths1,
Richard T. Bryan2
1
West Midland Regional Genetics Laboratory; Birmingham Women’s NHS Foundation Trust
2
Bladder Cancer Prognosis Programme, School of Cancer Sciences, University of Birmingham, B15 2tt
3
Affymetrix UK Ltd, High Wycombe
4
Cancer Research Unit, University of Warwick, Coventry, Cv4 7al
5
School of Health and Population Sciences, University of Birmingham, Birmingham, B15 2tt
6
Department of Complex Genomics, Nutrim School for Nutrition, Toxicology and Metabolism, Maastricht
University Medical Centre, Maastricht
7
Department of Histopathology, University Hospitals Birmingham NHS Foundation Trust, B15 2wg
Urothelial bladder cancers (UBCs) have heterogeneous clinical characteristics which are mirrored in their diverse
genomic profiles. Genomic profiling of UBCs has the potential to benefit routine clinical practice by providing prognostic
utility above and beyond conventional clinic-pathological factors, and allowing for prediction and surveillance of
treatment responses. Urinary DNAs representative of the tumour genome provide a promising resource as a liquid
biopsy for non-invasive genomic profiling of UBCs. We compared the genomic profiles of urinary cellular DNA and
cell-free DNA (cfDNA) from the urine to matched diagnostic formalin-fixed paraffin embedded (FFPE) tumour DNAs
for well-characterised UBC patients. Our data show urinary DNAs to be highly representative of patient tumours,
allowing for detection of recurrent clinically actionable genomic aberrations. Furthermore, a greater aberrant load
(indicative of tumour genome) was observed in cfDNA over cellular DNA (p<0.001), resulting in a higher analytical
sensitivity for detection of clinically actionable genomic aberrations (p<0.04) when using cfDNA. Our data show
cfDNA in the urine to be malignancy but not UBC-specific and to allow for characterisation of even single low grade
and stage UBC tumours less than 0.5cm in size when using the Affymetrix OncoScan assay. Thus, cfDNA extracted
from the urine of UBC patients has a higher tumour genome burden and allows greater detection of key genomic
biomarkers (90%) than cellular DNA from urine (61%) and provides a promising resource for robust whole-genome
tumour profiling of UBC with potential to influence clinical decisions without invasive patient interventions.
OP 58
Clinical utility of circulating tumor DNA for molecular assessment and precision medicine in pancreatic
cancer
Erina Takai1, Yasushi Totoki1, Hiromi Nakamura1, Mamoru Kato1, Tatsuhiro Shibata2, Shinichi Yachida1
1
Division of Cancer Genomics, National Cancer Center Research Institute
2
Laboratory of Molecular Medicine, Human Genome Center, The Institute of Medical Science, The University
of Tokyo
59
Background: Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies. The genomic
landscape of the PDAC genome features 4 frequently mutated genes (KRAS, CDKN2A, TP53, and SMAD4) and
dozens of candidate driver genes altered at low frequency, including potential clinical targets. Circulating cell-free
DNA (cfDNA) is a promising resource to detect and monitor molecular characteristics of tumours, supporting the
concept of “liquid biopsy”.
Methods: We determined the mutational status of KRAS in plasma cfDNA using multiplex droplet digital PCR
(RainDrop) in 259 patients with PDAC retrospectively. We next analysed the relationship between the detection of
KRAS mutations and clinicopathological features. Furthermore, we constructed a novel modified SureSelect-KAPAIllumina platform and an original panel of 60 genes in 48 patients who had ≥1% mutant allele frequencies of KRAS
in plasma cfDNA. We then performed targeted deep sequencing of cfDNA and matched germline DNA samples.
Results: Droplet digital PCR detected KRAS mutations in plasma cfDNA in 76 of 164 (46.3%) patients with UICCstage IV cancer, but in only 6 of 95 (6.3%) UICC-stage I-III cases. Multivariate analysis revealed that detection of
KRAS mutations before treatment was an independent poor prognostic factor (p<0.0001). Importantly, potentially
targetable somatic mutations were identified in 14 of 48 patients (29.2%) examined by cfDNA sequencing.
Conclusion: Our two-step approach with plasma cfDNA, combining droplet digital PCR and targeted deep
sequencing, is a feasible clinical approach. Assessment of mutations in plasma cfDNA may provide a new prognostic
and diagnostic tool, assisting decisions for optimal therapeutic strategies for PDAC patients.
OP 217
Liquid profiling of KRAS-status on circulating plasma-DNA in patients with pancreatic cancer – a novel
tool for therapy response prediction and prognosis
Stefan Holdenrieder1, Ina Prinz1, Steffen Ormanns2, Sibylle Bächmann3, Michael Haas4, Carina Ross5,
Philipp Angenendt5, Volker Heinemann4, Frank Diehl5, Stefan Boeck4
1
Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn
2
Department of Pathology, Ludwig-Maximilians-University of Munich
3
Department of Pathology, Department of Internal Medicine III and Comprehensive Cancer Center, LudwigMaximilians-University of Munich
4
Department of Internal Medicine III and Comprehensive Cancer Center, Ludwig-Maximilians-University of
Munich
5
Sysmex-Inostics
Liquid profiling of tumor biology in blood of cancer patients is a novel minimal invasive approach for therapy
stratification and monitoring, early recurrence detection and resistance monitoring.
In 32 patients with advanced pancreatic cancer receiving gemcitabine (±erlotinib)-based first-line chemotherapy, we
investigated the pretherapeutic KRAS mutation status on plasma DNA using using beads, emulsions, amplification
and magnetics (BEAMing) technology and correlated it with tissue KRAS status, therapy response, progressionfree (PFS) and overall survival (OS).
Staging using CT or MRT exams after 2 months revealed 16 patients with progression and 16 with stable disease
or partial remissions according RECIST criteria. KRAS mutations were found in tissue of 20 and in plasma of 16
patients.
While there was no correlation between KRAS status in plasma and tissue, nor an association between tissue
KRAS status with therapy response, presence of mutated KRAS in plasma significantly correlated with poor therapy
response (PD vs SD/PR; P=0.022) and was associated with a 3.3 fold risk of progression – independent from
tissue status. Similarly mutated KRAS in plasma clearly correlated with short PFS (P<0.001) and OS (P=0.002)
while tissue KRAS status did not. In addition, the courses of plasma levels of mutated KRAS during therapy were
60
indicative for response and tumor recurrence.
Our findings underline the high clinical relevance of liquid profiling approach for the individualized guidance of
cancer patients.
OP 116
Individual circulating DNA CpG-methylation profiling: unbiased approach for identification of cancer related
molecules
Anna Bondar1, Alexander Kurilshikov1, Evgeny Morozkin2, Marat Zaripov3, Marsel Kabilov1, Vladimir
Voytsitskiy3, Valentin Vlassov1, Pavel Laktionov2
1
Institute of Chemical Biology and Fundamental Medicine Sb Ras
2
Insitute of Chemical Biology and Fundamental Medicine of Sb Ras; Novosibirsk Research Institute of
Circulation Pathology of Academician E.N. Meshalkin
3
Novosibirsk Regional Cancer Center
Among numerous variants of circulating in blood cell-free DNA (cfDNA) methylation patterns only few reflect cancer
related changes. Single nucleotide resolution of methylated cytosine locations in the individual cfDNA molecules
would provide required information to identify those tumor-specific profiles.
We performed target bisulfite sequencing of potential prostate cancer (PC) cfDNA markers (GSTP1; RNF219)
isolated from blood plasma of 18 healthy donors (HD), 17 benign hyperplasia (BPH) and 20 PC patients using
MiSeq platform (Illumina). Selected loci were amplified after bisulfite conversion (Zymo Research) of cfDNA with
methyl-independent barcoded primers and sequenced with coverage ranging from 23509 to 143953.
To reveal diagnostically significant differences data was analyzed using several different approaches. Conventional
approach based on differences in methylation levels of CpG-sites and novel approach based on comparison of
correlation matrices (CCM) for HD, BHP and PC groups. Study population was randomly subsampled into training
and test cohorts. The logit regression model based on methylation levels of CpG-sites achieved an area under the
ROC curve (AUC) exceeding 0.94 in both cohorts for GSTP1 gene and 0.81 - for RNF219 gene. ROC curves for
binomial regression models based on correlation matrices (pairwise phi coefficient between methylation statuses of
CpG sites) with LASSO penalization yielded AUC, specificity and sensitivity as 0.99, 100 % and 92 % for GSTP1
gene and 0.99, 100 % and 90 % for RNF219 (test cohort).
CCM approach estimates diagnostic value of target genes and reveals cancer-related cytosine methylation. That
could potentially reflect the features of tumor cell physiology and on a par with conventional approaches provide
helpful information for rational design of noninvasive, methylation-specific cfDNA-based diagnostics for PC.
OP 182
Non-invasive analysis of glioblastoma with circulating nucleic acids
Richard Mair1, Florent Mouliere1, Davina Gale1, Dana Tsui1, Colin Watts1, Kevin Brindle1, Nitzan Rosenfeld1
1
Cancer Research UK Cambridge Institute, University of Cambridge
Non-invasive assessment of the cancer genome with circulating tumour DNA (ctDNA) is particularly attractive for
brain tumours as repeated biopsy risks neurological mortality. Previously, plasma ctDNA was detected in only 10%
of glioblastoma (GBM) patients. We hypothesized that this could be improved thus providing an effective noninvasive biomarker for monitoring tumour burden in the brain.
We developed an orthotopic xenograft rat model using cells derived from biopsy specimens obtained at surgery in
order to study nucleic acid release from GBM. MRI was used to obtain tumour size and degree of enhancement.
Plasma was collected in more than 40 rats and ctDNA concentration was assayed using digital PCR. We targeted
61
both single copy and multi copy human genome regions to identify ctDNA as well as sequences specific for
mitochondrial circulating tumour DNA (mctDNA).
ctDNA was detected in 25% of the rat plasma samples. Depending upon the type of cells engrafted, this varied
between from 0 % to 52%. For 17 rats engrafted with a mutant PTEN driven tumour-derived cell line (GBM1)
identified with a tagged-amplicon sequencing (TAm-Seq), ctDNA concentrations were positively correlated with
tumour burden (p=0.004). The detected ctDNA concentrations with the single copy assay however, remained
relatively low (mean 88 copies/mL).
However tumour mctDNA was detected more often than ctDNA (55% detection rate), especially for rats engrafted
with GBM1 cells (75 % detection rate) with a higher plasma concentration (mean 9500 copies / mL). mctDNA
concentration was also positively correlated with tumour burden (p=0.014).
Thus, ctDNA and mctDNA can be detected in the plasma of rat xenografted with human-derived GBM cells, and
their concentrations correlate positively with tumour burden.
OP 213
Free-circulating Methylated DNA in Blood for Diagnosis, Staging, Prognosis and Monitoring of Head and
Neck Squamous Cell Carcinoma Patients: An Observational Prospective Cohort Study
Luka de Vos1, Andreas Schröck2, Annette Leisse1, Friedrich Bootz2, Glen Kristiansen1, Dimo Dietrich1
1
Department of Otolaryngology, Head and Neck Surgery, University Hospital Bonn
2
Institute of Pathology, University Hospital Bonn
Biomarkers in minimal invasively obtained liquid biopsies facilitating outcome prediction and monitoring of disease
progression in cancer patients are clinically valuable in optimizing an individualized treatment. For this purpose,
methylated cell-free circulating tumor DNA in blood was evaluated in an observational prospective cohort study.
284 head and neck squamous cell carcinoma (HNSCC) patients and 122 patients with benign diseases were
recruited. DNA methylation of SHOX2 and SEPT9 in plasma was quantified prior to and after first-line treatment
(surgery with optionally adjuvant radio-chemotherapy or definitive radio-chemotherapy) and longitudinally during
surveillance.
59% (sensitivity) of the patients with HNSCC were positive for methylated cell-free DNA in blood at 96% specificity.
Plasma methylation was correlated with T stage and nodal status (p<0.001), and therefore representing a powerful
staging parameter. Patients with initially elevated SEPT9 methylation level had a higher hazard of death (HR=5.27,
p=0.001). Furthermore, SEPT9 methylation was predictive for loco-regional recurrence-free (p<0.001), distant
metastases-free (p=0.002), and progression-free (p<0.001) survival. Fifty-eight percent of deaths showed a positive
test result during surveillance 213±117 days before death. Disease progression was diagnosed up to 377 days
earlier compared to current clinical practice.
SHOX2 and SEPT9 methylation in plasma are powerful biomarkers for risk stratification and disease monitoring.
Patients at high risk to suffer from recurrence might benefit from an intensified surveillance. The early detection of a
recurrent tumor will allow for a timely initiation of a consecutive treatment thereby improving patients’ outcome.
OP 193
Effects of collection and processing on plasma cell-free DNA in EDTA versus cell-stabilizing tube
Dana Tsui1, Bente Risberg2, Andrea Ruiz-ValdepenasMartindeAlmagro1, Sarah-Jane Dawson3, Heather
Biggs4, Charlotte Hodgkin5, Linda Jones4, Christine Parkinson5, Anna Piskorz1, Francesco Marass1, James
Morris1, Vincent Plagnol6, Nitzan Rosenfeld1, Carlos Caldas1, James D. Brenton1, Davina Gale1
1
Cancer Research UK Cambridge Institute, LI Ka Shing Centre, University of Cambridge, UK
2
Institute for Cancer Research, Oslo University Hospital, Norway
62
Division of Research and Cancer Medicine, Peter Maccallum Cancer Centre, Melbourne, Australia
The Cambridge Breast Unit, Addenbrooke’s Hospital, Cambridge University Hospitals NHS Foundation
Trust, Addenbrooke’s Hospital, Cambridge, UK
5
Department of Oncology, Cambridge University Hospitals NHS Foundation Trust, Addenbrooke’s
Hospital, UK
6
Ucl Genetics Institute, University College London, UK
3
4
Circulating cell-free tumour DNA (ctDNA) in plasma offers potential for non-invasive cancer management. ctDNA
contributes to a minor fraction of the total cell-free DNA in plasma, therefore it is important to avoid release of total
DNA during cell lysis that would further reduce the tumour DNA fraction. Pre-analytical factors including processing
procedures and collection condition will affect the chance of cell lysis. It has been proposed that blood collection
tubes containing cell-stabilizing reagents may be used to prevent cell lysis for a longer period. We studied the
effects of (i) different blood collection tubes, (ii) delays in processing, (iii) storage temperature, and (iv) centrifugation
protocols on the yield of circulating total DNA levels and tumour-specific DNA from plasma samples of breast cancer
and high-grade serous ovarian cancer patients.
Blood samples were collected into either EDTA tubes or BCT tubes (STRECK, Omaha) and processed to separate
plasma either immediately (<1 hour) or after a delay of 6h, 24h, 48, 96h or 1 week, at room temperature and/or
4C. Additional samples were processed by different centrifugation protocols. When the processing was delayed,
the samples collected in EDTA tubes showed a gradual increase of total DNA levels and variations in mutant
allele fractions, more so than if the samples were collected in BCT tubes. Analysis by targeted sequencing did
not show differences in the background noise profiles between the two tube types. Storing of unprocessed blood
samples collected in EDTA tubes at 4C resulted in less variation compared to storage of those samples at room
temperature, but higher variation compared to storage at room temperature of samples collected in BCT. Our
results suggest that cell-stabilizing BCT tubes may overcome some of the pre-analytical variations associated with
a delay in processing. Comparison of different centrifugation protocols provides support for the use of less stringent
centrifugation protocols that would be compatible with most hospital settings.
OP 219
Optimized plasma collection procedures for cell-free DNA analyses in advanced malignancies
Sonya Parpart-Li1, Bjarne Barlett2, Julie Brahmer2, Nilo Azad2, Sarah Bonerigo2, Ilene Browner2, Amy
Ryan2, Victor E. Velculescu2, Mark Sausen1, Luis Diaz2
1
Personal Genome Diagnostics, Inc., Baltimore, MD 21224, USA
2
The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine,
Baltimore, MD 21287, USA
Analyses of genomic alterations in cell-free DNA (cfDNA) is rapidly advancing as a method to detect, monitor and
genotype solid tumor malignancies. Liquid biopsy analyses, using cfDNA, are a favorable alternative to invasive
tissue approaches, particularly for cancers in organs where tissue is difficult to obtain. As these approaches become
a clinical standard, the method for blood collection will be an important consideration to ensure optimal assay
performance especially since degradation of circulating tumor DNA (ctDNA) in the circulation is rapid (half-life ~
1-2 hours). To evaluate the blood collection process and its effects on ctDNA recovery, we compared K2EDTA with
cell-free DNA Streck™ BCT tubes to determine the stability over time of total cfDNA and ctDNA levels in blood.
Seventy-two plasma samples were collected from nine cancer patients in both K2EDTA and Streck™ BCT tubes.
Once collected, blood was processed into plasma immediately or kept at room temperature and processed to
generate plasma at day 1, 3, 5 or 7. In K2EDTA tubes, germline DNA was released into the blood (1,300% increase
63
over seven days), which significantly increased the number of total genomic equivalents. Additionally, we found that
the volume of plasma collected from both tube types during blood fractionation decreased over time when tubes
were stored at room temperature (an average of 3.5 mL vs. 1.0 mL, respectively). To evaluate the effect of storage
conditions on performance of each tube type, blood was also collected from six cancer patients with KRAS or EGFR
mutations and either maintained at room temperature or stored at 4°C for up to three days. Taken together, K2EDTA
tubes stored at 4°C prevented cell lysis and preserved the ctDNA in a manner equivalent to Streck™ BCT tubes.
These data demonstrate that liquid biopsies can be collected using either tube type with similar performance and
appropriate consideration of storage conditions.
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Abstracts of Poster Presentations (P)
Cancer A
P 48
Capturing early changes in the cancer genome of patients under experimental therapeutic trials with
circulating tumour DNA and adaptive TAm-Seq
Florent Mouliere1, Javier Garcia Corbacho2, T. Hedley Carr3, James Morris1, Francesco Marass1, Davina
Gale1, Brian Dougherty4, Elizabeth Harrington3, J. Carl Barrett4, Simon Pacey2, Richard Baird2, Nitzan
Rosenfeld1
1
2
Department of Oncology, University of Cambridge
3
Translational Science, Oncology Imed, AstraZeneca
4
AstraZeneca
The ability to track circulating tumour DNA (ctDNA) in plasma provides a new reactive capacity to understand in
real time what is happening in a patient’s body. Serial analysis of ctDNA with next generation sequencing offers
an opportunity to monitor on a large genomic scale changes in the relative abundance of mutations and to detect
emergence of resistance to treatment.
We set up an intensive longitudinal assessment of plasma collection for patients under experimental therapeutic
trials. At this date more than 200 samples from more than 20 patients from multiple early-phase trials have been
incorporated in this work. Notably, ctDNA sample collection has been incorporated to RADICAL [NCT01791985], a
phase 2 multisite clinical trial that explores the activity of an aromatase inhibitor in combination with an anti-FGFR
inhibitor in ER positive breast cancer.
We analysed ctDNA point mutations with a modified tagged-amplicon deep-sequencing (TAm-Seq) method
introducing DNA size selection. We developed highly multiplexed panels spanning a total of 40kbp of the genome,
covering regions in 28 genes of interest. We used a flexible design combining coverage of genes commonly mutated
in most cancer types, and a highly focused panel covering genes and pathways related to one specific study.
Preliminary results indicated a relationship between progressive disease and increase in ctDNA levels. Gene
mutations have been identified by TAm-Seq in all tested patients. Notably, ESR1 mutations, associated with
endocrine resistance in breast cancer were consistently detected. Moreover, we observed modifications in ctDNA
levels only 6 hours after the beginning of treatments in some patients.
Relationship between ctDNA mutational profiles and clinical outcomes will be explored. Gene-fusions and new
mutations associated to an emergence of resistance to treatment will be assessed by wider-scale sequencing.
P 145
Noninvasive cell-free DNA-based detection of copy-number variations and single-nucleotide variations in
cancer samples using single-nucleotide polymorphism-targeted massively multiplexed PCR
Bernhard Zimmermann1, Eser Kirkizlar1, Tudor Constantin1, Ryan Swenerton1, Bin Hoang1, Nicholas
Wayham1, Zachary Demko1, Robert Pelham1, Styrmir Sigurjonsson1, Matthew Hill1
1
Natera Inc.
C-class cancers, such as breast and ovarian, typically have a mutation profile predominated by copy-number
variations (CNVs). Methods incorporating CNV analysis may have significantly greater diagnostic coverage than
those using single-nucleotide variations (SNVs) alone. We developed a single-nucleotide polymorphism (SNP)based massively multiplexed PCR (mmPCR) assay to detect CNVs in cell-free DNA (cfDNA). This feasibility assay
targeted 3,168 SNPs on chromosomes 1 and 2 and the focal region 22q11.2 (average allelic imbalance (AAI)
65
<1%).
We analyzed nine patients with breast and ovarian cancer—four with breast cancer (stage II) and five with ovarian
cancer (stage III and IV). All patients had CNVs detected in tumor-tissue samples. Using a conservative threshold
of ≥0.45% AAI and ≥99.9% confidence, at least one CNV was detected in 75% (3/4) of breast-cancer plasmas and
80% (4/5) of ovarian-cancer plasmas. Furthermore, we assessed tumor heterogeneity by analyzing 4–6 subsections
of tumor tissue in the four patients with breast cancer. We were able to detect some subclonal CNVs in plasma that
were not present in all tumor sections, whereas CNVs present in some tissue sections were not detected in the
matched plasma.
CNV detection was then combined with a PCR panel targeting the 584 most frequent SNVs in breast cancer. Among
41 patients, variants were detected in 83% (60% stage I, 88% stage II, and 100% stage III), highlighting the fact
that disease load coverage can be increased using an analysis that incorporates both CNVs and SNVs. SNVs
alone were detected in 60% of stage I, 72% of stage II, and 83% of stage III (71% overall) patients. CNVs alone,
using ≥0.45% AAI threshold, were detected in 20% of stage I, 64% of stage II, and 50% of stage III (51% overall)
patients.
In summary, we used SNP-based mmPCR to detect both chromosome-arm level and focal CNVs in cfDNA at very
low AAI, and detected CNVs in plasma that may have gone undetected via biopsy. Futher optimization of both SNV
and CNV probe pools should allow for increased detection of patients with cancer using this methodology.
P 181
Screening of kras mutation in pre- and post-surgery serum of patients sufferin colon cancer by cold-pcr
hrm
Elena Trujillo1, Hada C. Macher1, Pilar Jiménez-Arriscado1, Noelia García-Fernández1, Juan Miguel
Guerrero1, Amalia Rubio1
1
Background: Tumor heterogeneity have a challenge for personalized cancer medicine since a single needle biopsy
or surgical excision is unlikely to accurately capture the complete genomic landscape of a patient’s cancer. Genomic
characterization of cell-free circulating tumour DNA (ctDNA) may offer an opportunity to assess clonal dynamics
throughout the course of a patient’s illness. The existence of KRAS drivers mutations in colon cancer patients is
determinant to decide their treatment and to predict their outcome. The aim of this study was to discriminate, in a
non-expensive way, between patients with mutations in the KRAS gene in serum cf-DNA by Cold High resolution
melting PCR (COLD-PCR HRM) Methods: 3 patients were included in the study diagnosed of colorectal cancer.
Positive control was DNA from a cellular line with the most common KRAS gene mutations in colorectal cancer
(c.35G>T) and negative controls were wild type patients for KRAS gene (diagnosed by the Department of Pathology).
One sample of 10 mL serum was obtained from each patient before surgical resection and another one 24 h after
surgery. DNA was automatically extracted from serum, COLD-PCR HRM was performed in the Light Cycler 480.
Results: After analysis 3 different situations were found A) patient had pre- and post-surgical samples grouped with
negative controls B) the pre-surgical sample of a patient appears grouped with positive control and the post-surgical appears
with the negative control and C) both samples, pre- and post-surgical ones, appear grouped with the positive control.
In the first situation it should not be necessary to analysed KRAS gene in the biopsy. In the second case (B) sequencing
KRAS of the biopsy should be necessary to confirm the mutation. In the third case (C) metastasis should be considered.
Conclusions: The results show that the slope of the melting curve of patients with one of the KRAS mutations is
homogeneous and different from healthy controls. COLD-PCR HRM is a cost-effective way for screening one of the
most common driver mutations to predict the worst prognosis in colorectal cancer.
66
P 201
Detection and Quantification of KIT mutations in ctDNA by Safe-SeqS
Johannes Fredebohm1, Daniel Mehnert1, Ann-Kathrin Löber1, Vanessa VanRahden1, Frank Holtrup1, Frank
Diehl1
1
Objective: Gain-of-functions mutations in the KIT gene are frequently detected in GIST tumors. These primary KIT
mutations cluster in specific domains and play an important role in tumor development. Furthermore, secondary
mutations have been identified that only occur during treatment with targeted therapies and vary between patient
and metastatic site. Determining these secondary resistance mutations is limited by the need to have serial tissue
samples available. In addition, the genomic heterogeneity of secondary mutations is poorly represented in single
tissue specimens. For these reasons, we sought to develop a ctDNA based sequencing assay that covers the
relevant regions of the KIT gene.
Methods: We have developed a KIT sequencing assay based on Safe-SeqS. Specifically, two multiplex primer
panels were established to cover >89% of the reported primary and secondary KIT mutations. Plasma from healthy
donors as well as lymphocyte DNA spiked with synthetic DNA was utilized to establish analytical performance.
Furthermore, plasma from advanced GIST patients enrolled in a clinical trial was tested with Safe-SeqS.
Results: The performance of a KIT ctDNA sequencing assay was accessed. For example, the limit of detection was
determined to be 0.02% or lower depending on the variant position. Spike-in controls were used to demonstrate
the quantitative nature of the KIT Safe-SeqS assay. Safe-SeqS was able to identify novel secondary resistance
mutations. Lastly, the clonal evolution of KIT mutations in response to treatment was measured.
Conclusion: We have developed a highly sensitive Safe-SeqS assay to identify KIT mutations in ctDNA. This
sequencing assay can be applied to the genotyping of advanced GIST tumors prior to treatment as well as
subsequent monitoring of resistance mutations in patients receiving targeted therapy.
P 30
Small non-coding RNAs of human blood plasma of healthy donors and patients with non-small cell lung
cancer
Dmitri Baryakin1, Dmitry Semenov1, Eugeniy Brenner1, Alexander Kurilshikov1, Vadim Kozlov2, Elena
Kuligina1, Vladimir Richter1
1
2
Novosibirsk Regional Cancer Centre
Understanding structure of circulating RNA expands fundamental knowledge of cell communications, establishes
novel mechanism of physiological processes distant regulation, and allows developing new molecular diagnostic
and prognostic approaches.
The purpose of this study is profiling of circulating plasma RNA of healthy subjects and non-small cell lung cancer
patients using high-throughput sequencing technology for assembling comprehensive and unbiased transcriptome
of human blood plasma, aimed at identifying new species of regulatory RNA and the development of unique and
complex diagnostic markers of human diseases.
In this study we analyzed the diversity of short blood plasma RNA of 8 healthy donors, 4 patients with lung
adenocarcinoma and 4 patients with squamous cell lung carcinoma. In order to obtain cDNA libraries that encode
the most full-scale set of circulating RNAs, short (n > 19) plasma RNAs were dephosphorylated, 5’-phosphorylated,
ligated with adapters, reverse transcribed and amplificated. Individual cDNA libraries were sequenced on SOLiD
platform. The resulting data sets were analyzed using the Bowtie/Cufflinks software, human RefSeq RNA and
67
human genome hg19 sequence databases.
The abundance of the main classes of cellular RNA such as rRNA, tRNA, mRNA, mitochondrial transcripts, scRNA,
snRNA, snoRNA and mature miRNA were determined. Our data indicated that exosomes and microvesicles are
predominant types of circulating miRNA transport. Particular cell and tissue types that are potential sources of the
largest fraction of human blood plasma mRNA set were defined. Novel antisense transcripts and microRNA-like
species were found in the set of circulating RNA fragments.
Comparative analysis of plasma RNA of healthy donors and patients with non-small cell lung cancer allowed
characterizing a set of changes in extracellular human mRNAs and microRNAs expression profile in malignant
tumor growth.
The work was supported by the RFBR (grants № 13-04-01058 and 14-04-31468) and the Interdisciplinary Integration
Project of SB RAS № 84 (2012-2014).
P 42
A rapid and sensitive method for detection of T790M mutations of EGFR in plasma DNA
Hideharu Kimura1, Shingo Nishikawa1, Hayato Koba1, Taro Yoneda1, Takashi Sone1, Kazuo Kasahara1
1
Kanazawa University Hospital
Epidermal growth factor receptor (EGFR) T790M mutation is associated with EGFR tyrosine kinase inhibitors
(EGFR-TKIs) resistance in non-small cell lung cancer (NSCLC). However, tissue availability limits the genotyping
of EGFR T790M mutation in a clinical setting. The aims of this study are to develop a blood-based, non-invasive
approach to detecting the EGFRT790M mutation in advanced NSCLC patients, using PointMan™ EGFR DNA
Enrichment Kit, which is a novel method for selective amplification of genotype specific sequences.
Methods: Pairs of blood samples and tumor tissues were collected from NSCLC patients with activating EGFR
mutations, who were resistant to EGFR-TKIs treatment. EGFR T790M mutations in plasma DNA were detected
using the kit. The concentrations of plasma DNA were determined using quantitative real-time PCR.
Results: A total of 22patient were enrolled in this study. 9 (41%) of the patients had EGFR T790M mutations in
their plasma DNA as detected using the kit after progression desease to EFGR-TKI. In 10 cases detected T790M
mutations from tumor tissues, 8 (80%) ware detected also in plasma DNA. The concentrations of plasma DNA were
similar between in patients with T790M mutations and without the mutations.
Conclusions: The PointMan™ is a useful method for determining plasma EGFR T790M mutation status.
P 82
Plasma microRNA profiles: identification of miR-744 as a novel prognostic and chemoresistant biomarker
in pancreatic cancer
Shuhei Komatsu1, Daisuke Ichikawa1, Mahito Miyamae1, Tsutomu Kawaguchi1, Wataru Okajima1, Takuma
Ohashi1, Taisuke Imamura1, Jun Kiuchi1, Tomohiro Arita1, Hirotaka Konishi1, Ryo Morimura1, Atsushi
Shiozaki1, Hisashi Ikoma1, Toshiya Ochiai1, Kazuma Okamoto1, Hiroki Taniguchi1, Eigo Otsuji1
1
Division of Digestive Surgery, Department of Surgery, Kyoto Prefectural University of Medicine
Objective: This study aims to explore novel microRNAs in plasma for screening cancer and predicting clinical
outcomes in pancreatic cancer (PCa) patients using a microRNA array-based approach.
Methods: We used the Toray® 3D-Gene microRNA array-based approach to compare plasma levels between PCa
patients and healthy volunteers.
Results: (1) Six oncogenic microRNAs (miR-615-5p, 744, 575, 557, 675, and 550a) with high expression in plasma
were selected. (2) By quantitative RT–PCR using plasma samples from 94 PCa patients and 68 healthy volunteers,
a significantly higher level of plasma miR-744 in PCa patients than in healthy volunteers was validated in small68
scale analysis (P = 0.0038), two independent cohort analyses, and large-scale analysis (P <0.0001, AUC 0.8307).
(3) miR-744 expression was significantly higher in PCa tissues (P = 0.0069) and PCa cell lines (P = 0.0074) than
in normal tissues and fibroblasts, respectively. Preoperative plasma level of miR-744 was significantly reduced in
postoperative samples (P = 0.0063). (4) A high level of plasma miR-744, which was correlated with lymph node
metastasis (P = 0.0407) and recurrences (P = 0.0376), was an independent poor prognostic factor of PCa patients
after pancreatectomy (P = 0.0007, HR 21.2 (3.17-436)). Furthermore, a high level of plasma miR-744 contributed to
poorer progression-free survival of non-operable PCa patients who underwent gemcitabine-based chemotherapy
(P = 0.0533). Overexpression of miR-744 in PCa cells induced significant chemoresistance to gemcitabine in vitro.
Conclusions: Plasma miR-744 might be useful biomarker for screening PCa, monitoring and predicting poor
prognosis and chemoresistance in PCa patients.
P 209
Evaluation of different blood collection tubes and blood storage conditions for the preservation and
stability of cell-free circulating DNA for the analysis of the methylated mSEPT9 colorectal cancer
screening marker
J. Distler1, R. Tetzner1, G. Weiss1, T. König1, A. Schlegel1, Michal Bagrowski1
1
Epigenomics AG, Berlin
Background: mSEPT9 analysis for the early detection of colorectal cancer requires plasma generated from whole
blood samples. Due to different sample logistics and shipment procedures in the laboratory routine a blood collection
tube for long term storage and preservation of blood samples is required. We investigated long term storage of
blood samples at different temperature conditions in S-Monovette CPDA (Sarstedt), Cell-Free DNA BCT (Streck),
S-Monovette EDTA K2-Gel (Sarstedt), BD Vacutainer PPT (Becton, Dickinson and Company) blood collection tubes
for subsequent analysis of mSEPT9 with the Epi proColon 2.0 CE Early Detection Assay.
Methodology: Blood draws from healthy donors were collected with different type of blood collection tubes. The
storage and transport simulation was performed at conditions at room temperature (18 to 25 °C) for up to 73h.
For DNA stability analysis part of the samples were spiked with methylated DNA. The False positivity rate was
determined in unspiked samples.
The samples were analyzed according to the instructions for use of the Epi proColon 2.0 CE assay.
Results: It could be verified, that the Epi proColon test was designed for use of plasma prepared from blood
collected with S-Monovette,CPDA, 8.5 ml tubes and stored at 15 – 25 °C for up to 48 h. Blood draws collected in
Cell-Free DNA BCT , BD Vacutainer PPT 8.5 mL tubes and S-Monovette EDTA K2-Gel tubes in combination with
the evaluated storage conditions failed verification.
Conclusions: The study clearly demonstrates that mSEPT9 can be consistently detected in plasma samples derived
from whole blood samples from S-Monovette CPDA tubes stored at 18 to 25 ºC for 48 hours. Study results did not
support the usage of the other tested tubes for this application.
69
Cancer B
P 90
RNA of blood circulating complexes of healthy donors and non-small cell lung cancer patients
Anna Savelyeva1, Dmitry Semenov1, Dmitri Baryakin1, Vadim Kozlov2, Elena Chikova3, Elena Kuligina1,
Vladimir Richter1
1
Institute of Chemical Biology and Fundamental Medicine
2
3
Center of New Medical Technologies
The understanding of RNA content of exosomes, microvesicles and extracellular ribonucleoproteins, that are known
to be involved in the cell-to-cell signaling and communication pathways, is one of the crucial goals of the last decade.
In this study we focused on RNome of human blood exosomes, microvesicles and ribonucleoproteins of non-small
cell lung cancer patients and healthy donors. Blood samples of healthy donors, patients with squamous cell lung
cancer or lung adenocarcinoma were subjected to sequential centrifugation steps at 1 200, 16 000 and 160 000 g.
It was shown that 16 000 and 160 000 g pellets were enriched with membrane-covered structures of ~40–100 nm.
RNAs of blood plasma subfractions were extracted and sequenced with high-throughput SOLiD 5500xl platform.
It was determined that 16 000 and 160 000 g pellets as well as particle-depleted supernatant contain fragments of
all known types of cellular RNAs: rRNA, tRNA, mRNA, the variety of small and long non-coding RNAs, including
miRNAs. The fragments of mtRNA were found to have the maximum contribution to the 16 000 g pellet. The 16 000
g pellet subfractions were also enriched with cytoplasmic Y-RNAs (up to 5%). The exosome-containing 160 000
g pellets of squamous cell lung cancer patients were characterized with elevated level of miR-524, miR-1246 and
fragments of TIMELESS, ADAM29, IMPG1 and IK mRNAs. We also detected that the level of miR-150, miR-484,
miR-486 were rised in 16 000 g pellet and 160 000 g supernatant subfractions of patients with lung adenocarcinoma.
The independent validation of RNA distribution in blood subfractions using real-time RT-PCR is ongoing. This study
will help in better understanding of blood circulating complexes function in lung cancer progression.
The work was supported by the RFBR (13-04-01058 and 14-04-31468) and the Interdisciplinary Integration Project
of SB RAS № 84 (2012-2015).
P 91
Plasma microRNA profiles; down-regulation of plasma tumor suppressive miRNA level contributes poor
outcomes in pancreatic cancer
Taisuke Imamura1, Shuhei Komatsu1, Daisuke Ichikawa1, Jun Kiuchi1, Wataru Okajima1, Takuma Ohashi1,
Mahito Miyamae1, Tomohiro Arita1, Ryo Morimura1, Hisashi Ikoma1, Hirotaka Konishi1, Yasutoshi
Murayama1, Atsushi Shiozaki1, Yoshiaki Kuriu1, Masayoshi Nakanishi1, Hitoshi Fujiwara1, Kazuma
Okamoto1, Hiroki Taniguchi2, Eigo Otsuji1
1
2
Department of Surgery, Kyoto Second Red Cross Hospital
Background: This study aimed to explore depleted tumor suppressive microRNAs (miRNAs) in plasma of pancreatic
cancer (PCa) patients and detect their possible roles as a treatment target in PCa.
Methods: This study involved three steps: (1) microarray analysis comparing PCa patients and healthy volunteers
in plasma miRNAs; (2) validation of candidate miRNAs by quantitative RT-PCR; (3) evaluation of monitoring tumor
dynamics by the plasma selected miRNA assay.
Results: Five down-regulated miRNAs were selected using a miRNAs array approach. Test- and large scale
analyses revealed that plasma miRNA-X level in PCa patients was most down-regulated among candidates
70
compared to healthy volunteers (p < 0.001). Prognostic analysis demonstrated that low miRNA-X level in plasma
was an independent poor prognostic factor in PCa patients [p = 0.012, Hazard ratio 4.56 (95% CI: 1.38-17.4)].
Enforced expression of miRNA-X in PCa cell lines significantly inhibited cell proliferation in vitro. Fluorescenceactivated cell sorting analysis demonstrated that transfection of PCa cell lines with mimic-miRNA-X resulted in an
accumulation of cells in the G0–G1 phase compared with transfection with control miRNA. In addition, p21 mRNA
abundance was increased level in miRNA-X-transfected cells, suggesting that the miRNA-X directly or indirectly
induced the production of p21, which results in G0–G1 arrest.
Conclusion: Down regulation of tumor suppressive miRNA-X in plasma contribute to poor outcomes and miR-X
might be a treatment candidate.
P 97
Circulating miR-18a in plasma contributes to cancer detection and monitoring in patients with gastric
cancer.
Jun Kiuchi1, Shuhei Komatsu1, Daisuke Ichikawa1, Masahiro Tsujiura2, Mahito Miyamae1, Wataru Okajima1,
Takuma Ohashi1, Taisuke Imamura1, Tomohiro Arita1, Toshiyuki Kosuga1, Hirotaka Konishi1, Atsushi
Shiozaki1, Kazuma Okamoto1, Eigo Otsuji1
1
2
Gastroenterology Center, The Cancer Institute Hospital Of Jfcr
Background: Recently, circulating microRNAs have been reported to be stably detectable in plasma/serum and to
function as potent non-invasive biomarkers in various cancers. We hypothesized that miR-18a could contribute to
a novel plasma biomarker in patients with gastric cancer (GC).
Methods: We focused on miR-18a, which is a component of miR-17-92 cluster and has been reported as highly
expressed in GC tissues. The study involved three steps: (1) confirmation of the higher miR-18a expression in
primary GC tissues and GC cell lines than in normal gastric tissues and a fibroblast cell line; (2) evaluation of the
plasma miR-18a assay using quantitative RT-PCR by comparing 104 GC patients and 65 healthy volunteers; (3)
evaluation of monitoring tumor dynamics by the plasma miR-18a assay.
Results: (1) The miR-18a expressions were significantly higher in GC tissues than in normal gastric tissues
(P = 0.0286) and higher in all examined GC cell lines than in the fibroblast cell line. (2) The plasma miR-18a
concentrations were significantly higher in GC patients than in healthy controls (P < 0.0001). The value of the area
under the receiver-operating characteristic curve was 0.8059. (3) The plasma miR-18a levels were significantly
reduced in postoperative samples compared to in preoperative samples (P = 0.0002). In an miR-18a overexpressing
cell line, the miR-18a concentration of cultured medium increased in both cell number and time-course dependent
manners, suggesting microRNA might be released from cancer cells into the surrounding environment.
Conclusions: Circulating miR-18a could be a useful biomarker for screening GC and monitoring tumor dynamics.
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P 112
Plasma microRNAs as noninvasive biomarkers for lung cancer
Ivan Zaporozhchenko1, Evgeny Morozkin1, Tatyana Skvortsova2, Rykova Elena2, Anastasia Ponomaryova3,
Nadezhda Cherdyntseva4, Valentin Vlassov2, Pavel Laktionov1
1
Institute of Chemical Biology and Fundamental Medicine of Sb Ras; Novosibirsk Research Institute of
2
Institute of Chemical Biology and Fundamental Medicine of Sb Ras
3
Tomsk Cancer Research Institute of Sb Rams; National Research Tomsk Polytechnic University
4
Tomsk Cancer Research Institute of Sb Rams; National Research Tomsk State University
Lung cancer (LC) is currently the world’s leading cause of cancer-related mortality with overall mortality to incidence
ratio of 0,87. Most LC patients are diagnosed with advanced disease due to low efficiency of cancer screening,
therefore new strategies for preclinical lung cancer screening as well as monitoring of post therapy relapses are
required. Recently, specific subsets of miRNAs that reflect tumor properties and disease progression have been
found circulating in blood of cancer patients and suggested as potential biomarkers for lung cancer.
We investigated a signature of seven miRNAs (miR-21, miR-19b, miR-126, miR-25, miR-205, miR-183, miR-125b)
previously shown to be involved in tumor development, invasion and vascularization in blood plasma of LC patients
and healthy individuals using qRT-PCR.
Two miRNAs were significantly upregulated (miR-19b, miR-21) and two were downregulated (miR-25, miR183) in lung cancer patients (p<0.05). Four miRNAs (miR-19b, miR-126, miR-25, miR-205) were found to be
differentially regulated in squamous cell carcinoma (SCC) patients when compared to healthy controls (p<0.05).
In adenocarcinoma (AD) patients only two miRNAs (miR-19b, miR-183) were differently expressed (p<0.05). ROC
curve analysis demonstrated high predictive value of miR-19b for total population and SCC patients, while miR183 was more effective in discriminating patients with AD. Stepwise binary logistic regression has identified the
combination of miR-19b and miR-183 to be a strong predictor of LC in total population AUC: 0,990. This combination
can be used to detect LC with 94,7% sensitivity and 95,2% specificity thus demonstrating the efficacy of cell-free
miRNA-based non-invasive diagnostics of LC.
P 118
The methylation profiling of multiple tumor suppressor genes in plasma cell-free DNA of patients with
chest radiological findings: resectable NSCLC versus benign lung nodules – pilot study
Adam Szpechcinski1, Monika Gos2, Renata Langfort3, Wlodzimierz Kupis4, Piotr Rudzinski4, Jolanta
Zaleska5, Tadeusz Orlowski4, Kazimierz Roszkowski-Sliz5, Joanna Chorostowska-Wynimko 1
1
Department of Genetics and Clinical Immunology, National Institute of Tuberculosis and Lung Diseases
2
Department of Medical Genetics, National Research Institute of Mother and Child
3
Department of Pathomorphology, National Institute of Tuberculosis and Lung Diseases
4
Department of Thoracic Surgery, National Institute of Tuberculosis and Lung Diseases
5
III Department of Lung Diseases, National Institute of Tuberculosis and Lung Diseases
Background: While most solitary pulmonary nodules are benign, around 20% of cases represent an early stage
lung cancer. Upon their detection by imaging techniques the diagnostic dilemma araises: is the nodule benign or
malignant?, should it be investigated or followed?, should it be surgically resected? The presence of cell-free DNA
(cfDNA) in plasma of lung cancer patients demonstrates promising diagnostic implications and could be considered
as an auxiliary tool in the differential diagnosis of solid pulmonary nodules.
72
Methods: Plasma cfDNA of 66 resectable NSCLC (I-IIIa) patients, 15 subjects with benign lung nodules (hamartoma,
fibrosis, granuloma) and 40 healthy volunteers was quantified by qPCR. The simultaneous methylation profiling of
21 tumor suppressor genes (TSGs) in plasma cfDNA was performed using optimized MS-MLPA assay.
Results: Mean cfDNA level in 66 NSCLC patients (21.5 ng/ml) was significantly higher than in 40 healthy controls
(4.5 ng/ml; p<0.0001), but did not differ from values observed in 15 patients with non-malignant tumors (23.4 ng/ml).
Similarly, mean cfDNA integrity value significantly differed NSCLC (4.0) from healthy controls (1.0; p<0.0001), but
not from non-malignant group (4.0). 25/32 (78%) NSCLC and 8/8 (100%) benign-nodule cfDNA samples presented
at least one TSG methylation. APC (frequency 18% samples), MLH1 (18%), ATM (13.6%), DAPK1 (13.6%), HIC 1
(13.6%), and RARβ (9%) were the most frequently methylated genes in NSCLC, while TIMP3 (75%), MLH1 (25%)
and TP73 (37.5%) – in benign nodule patients.
Conclusions: plasma cfDNA quantification showed promising but clinically not satisfactory sensitivity/specificity
for NSCLC detection. The optimized MS-MLPA assay allowed detection of multiple methylated TSGs in plasma
cfDNA. The MS-MLPA showed good performance in samples with diverse cfDNA concentrations suggesting that
methylation detection rate depends on the methylated DNA content in a sample. The study is on-going.
P 126
Plasma microRNA profiles: Identification of novel microRNA-based biomarkers for chemoresistance in
esophageal squamous cell carcinoma
Takuma Ohashi1, Shuhei Komatsu1, Daisuke Ichikawa1, Tsutomu Kawaguchi1, Mahito Miyamae1, Wataru
Okajima1, Taisuke Imamura1, Jun Kiuchi1, Tomohiro Arita1, Hirotaka Konishi1, Atsushi Shiozaki1, Hitoshi
Fujiwara1, Kazuma Kazuma Okamoto1, Eigo Otsuji1
1
Background: Recent studies have demonstrated that microRNAs are stably detectable in plasma/serum because
of their binding to specific proteins or being packaged in secretory particles. However, little is known about clinical
significance of circulating microRNAs related to chemoresistance in plasma of patients with esophageal squamous
cell carcinoma (ESCC).
Methods: Through Toray® 3D-Gene microRNA array-based approaches to compare plasma microRNA levels
between patients with histopathologic high response (Grade 2 or 3) and histopathologic low response (Grade 0),
we identified novel plasma microRNAs for chemoresistance in preoperative chemotherapy of patients with ESCC.
Results: (1) Five candidate microRNAs (miR-223, 103a, 23a, 23b, 21), which were upregulated in pre-treatment
plasma of patients with histopathologic low response, were selected. (2) In a large-scale validation analysis, all
candidates were significantly higher in ESCC patients with histopathologic low response (Grade 0 or 1a) than that
with histopathologic high response (Grade 1b, 2 or 3). (3) miR-223, miR-23a and miR-21 were significantly higher
in pre-treatment ESCC tumor specimens than normal esophageal specimens. (4) Furthermore, the growth of ESCC
cells transfected with the control mimics was manifestly inhibited by 5-fluorouracil and cisplatin treatment, whereas,
the inhibitory effects of 5-FU and cisplatin in miR-23a and miR-21, and cisplatin in miR-223 were signiﬁcantly
reduced in each overexpressed ESCC cells.
Conclusion: Circulating miR-223, miR-23a and miR-21 in plasma could be a useful biomarker for predicting
chemoresistance in patients with ESCC.
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P 130
Detection of p53 mutations in circulating DNA of transplanted HCC patients as a biomarked of tumor
recurrence
Noelia García-Fernández1, Hada C. Macher1, Amalia Rubio1, Pilar Jiménez-Arriscado1, Carmen Bernal2,
Maria Luz Bellido-Diaz1, Gonzalo Suarez-Artacho2, Juan Miguel Guerrero1, Miguel Angel Gómez-Bravo2,
Patrocinio Molinero1
1
2
Hepatobiliary and Liver Transplantation Unit, Virgen del Rocío University Hospital
P53 is the most commonly mutated gene in human cancers including hepatocellular carcinoma (HCC). The mutated
p53 protein and anti-p53 antibodies are related to the clinical characteristics, diagnosis and therapeutic response
of HCC. Effective methods for treatment of liver cancer include liver transplantation. Because tumor recurrence
is the major obstacle to improve prognosis after this therapeutic option, novel tumor biomarkers are considered.
In this way, the use of circulating DNA as a tool for evaluation of p53 mutation in the patients selected for liver
transplantation will be an interesting approach. The objective of this study was to determine the diagnostic and
prognostic utility of mutated p53 in the tumor recurrence of transplanted HCC patients.
Methods: 40 liver transplanted HCC patients were included in the study. A group of healthy controls were also
included. Detection of the specific p53 mutation in cDNA was performed by high resolution meelting PCR (HRMPCR) and COLD-PCR immediately before the transplantation. Also was determined serum anti-p53 using p53autoantibody ELISA kit in TRITURUS.
Results: The results of the HRM and COLD-PCR showed two well-different groups of transplanted patients after
normalization by healthy controls. These data allow us to distinguish between patients with p53 mutated cDNA
and wild type ones. Moreover, we have found that most of p53 mutated patients also presented elevated anti-p53
antibodies.
Conclusion: The present results suggest that it is possible to monitor p53 gene in the circulating DNA that could be
used as a biomarker of tumor recurrence during the clinical evolution of the transplanted patients. Beside, this is a
non-expensive approach that may be performed in an easy and quick way.
P 133
Circulating DNA as a monitoring marker for patients with EBV-associated gastric cancer
Katsutoshi Shoda1, Daisuke Ichikawa1, Yuuji Fujita1, Tomohiro Arita1, Hirotaka Konishi1, Shuhei Komatsu1,
Eigo Otsuji1
1
Background: Recent comprehensive analysis has proposed new molecular classification of gastric cancer (GC)
dividing into four subtypes. In this study, we firstly determined the frequency of EBV-related GC, and investigated
the usefulness of circulating EBV DNA as individual tailor-made follow-up marker in clinical courses.
Methods: We enrolled 153 patients with GC who underwent surgery at our hospital. After RPPH1 had been selected
as a reference gene for quantitative assessment by real-time PCR in GC tumors and plasma, EBNA1-to-RPPH1
ratio (EBV ratio) was determined for each patients, and compared with clinicopathological factors.
Result: Twenty-one patients (14%) were found to have EBV-associated GC. Circulating EBV DNA could be detected
in 14 patients, and the sensitivity and specificity were 47.7% and 97.0%, respectively. In quantitative assessment,
plasma EBV ratio was correlated with tissue EBV ratio (p < 0.05, spearman’s correlation). The plasma EBV ratio
was found to be useful biomarker showing tumor dynamics in some patients with EBV-related GC.
74
Conclusion: This study demonstrated the potential clinical use of circulating cfDNA to detect EBV genome as a
monitoring marker in patients with EBV-associated GC.
P 222
The diagnostic potential of extracellular microRNAs in bronchial lavage samples
Grit Rehbein1, Bernd Schmidt1, Michael Fleischhacker1
1
KIM I, Department of pneumology, University medical center, Halle/Saale
Introduction: MicroRNAs (miRs) are small non-coding RNAs regulating expression of oncogenes as well as of
tumorsuppressorgenes. Their expression is tissue specific and it is changed by several diseases. MiRs are released
from the cell by different mechanisms and they can be detected in different body fluids. Therefore miRs are potential
non-invasive biomarkers for cancer detection. The detection of tumor associated molecular changes in bronchial
lavage (BL), could improve sensitivity and specificity of lung cancer diagnostics. Aim: The aim of this study is to
detect changes in the concentration of extracellular miRs in bronchial lavage (BL) samples of lung cancer patients
and patients with benign lung diseases, to analyze the use of miRs as potential non-invasive diagnostic biomarkers
for lung cancer.
Methods: Total-RNA was isolated from cell free supernatant of BL of patients with lung cancer and patients with
benign lung diseases. Using Taqman-qRT-PCR the presence of specific microRNAs was evaluated. For normalization
of the data the exogenous added synthetic cel-miR 39 was used.
Conclusion: Extracellular microRNAs can be detected in different concentrations in BL samples of lung cancer
patients and patients with benign lung diseases. Using a panel of four microRNAs (miR-21, miR-193b, miR-574 and
miR-100) in our patient cohort a differentiation between these two groups is possible with 89 % sensitivity and 82
% specificity. For evaluation further experiments with a larger patient cohort are necessary. Furthermore protocols
have to be standardized, universal normalization markers have to be found and a combination of tumor markers
should be considered for developing noninvasive screening tools for lung cancer diagnostics.
Fetal Medicine
P 117
Real-Time and Droplet PCR quantification for non-invasive determination of RHD incompatibility between
mother and fetus
Sarka Pospisilova1, Iveta Svobodova1, Eva Pazourkova1, Ales Horinek1, Marie Korabecna1
1
Institute of Biology and Medical Genetics of the First Faculty of Medicine, Charles University in Prague
and General University Hospital in Prague
The aim of this study was to compare two strategies of DNA quantification: Real-Time PCR (qPCR) and Droplet
PCR (dPCR). The benefit of dPCR against qPCR is represented by absolute quantification of target nucleic acid
molecules without the requirement of calibration curves.
The methods were compared by quantification of standard nuclear DNA of known concentration. DNA was eightfold
diluted (2-0.015ng/µl) and twelve replicates were realized for each dilution. Concentrations were then measured as
levels of amplicons of two genes, housekeeping gene GAPDH and human RHD gene (exon 10). The same genes
were analyzed in plasma cell-free DNA and also in cell-free fetal DNA isolated from maternal plasma.
Evaluation of these two methods performance was based on several parameters, such as degree of linearity (R²),
75
accuracy, detection limit, etc.
In case of standard nuclear DNA, both methods showed high accuracy and low detection limit, both genes were
detected even in most diluted samples. High linearity degree (R² > 0.99) has been reached by both methods. The
correct RHD status of tested women was immunologically verified. Regarding analysis of cell-free fetal DNA in
RHD negative maternal plasma, dPCR failed against the qPCR to convincingly distinguish RHD positive samples
from the negative ones. For the purpose of prenatal screening, the qPCR method would be probably the preferable
alternative due to very low fetal DNA concentrations (about 0.002ng/µl) in maternal plasma, which is under the
detection limit of dPCR. Supported by the Ministry of Health of the Czech Republic RVO VFN64165
P 128
Aberrant microRNA expression in maternal plasma and preimplantation embryos of diabetic rabbits- a
promising biomarker for trophoblast function in early pregnancy?
Mareike Pendzialek1, Julia M. Knelangen1, Katarzyna Grybel1, Jacqueline Gürke1, Maria Schindler1, Bernd
Fischer1, Anne Navarrete Santos1
1
Faculty of Medicine, Department of Anatomy and Cell Biology, Martin Luther University Halle-Wittenberg
Proper placenta development is vital for embryo survival and health. Impairment of placenta function and
development is associated with many pregnancy complications such as miscarriage, preterm birth and intrauterine
growth restriction. Metabolic disorders like diabetes mellitus adversely affect placentation and placenta function
already at the time of trophoblast differentiation with potentially long-lasting effects on offsprings’ health. Recent
studies suggest that microRNAs (miRs) are regulators and markers of placental development. We have analysed
the influence of a maternal type 1 diabetes mellitus (T1DM) on miR expression in vivo in maternal (plasma,
endometrium) and embryonic samples (blastocyst, blastocyst cavity fluid), using the rabbit as experimental model.
Diabetes was induced in female non-pregnant rabbits by alloxan treatment two weeks prior to mating. Furthermore,
in in vitro experiments we examined the effects of insulin, glucose and LIF on embryonic miR expression.
MiR-27b, -141, -191 and -222 were detected in maternal plasma and endometrium and in embryoblast and trophoblast
cells and blastocyst cavity fluid. Maternal diabetes mellitus was associated with a decrease of these miRs in both
maternal plasma and embryonic samples. Furthermore, in blastocysts from diabetic rabbits a decreased expression
of miR-27b, -141, -191 and -222 was related to an increased mRNA expression of target genes. In vitro experiments
revealed that external stimuli like LIF (10ng/ml) and to a lower extend glucose (10mM and 25mM) and insulin
(17nM) influenced expression of embryonic miR-27b, -141 and -191.
Our results show for the first time the incidence of miRs in blastocyst cavity fluid, indicating that they might serve as
communicator between embryoblast and trophoblast cells. Furthermore, metabolic diseases like diabetes mellitus
lead to distinct changes of miR expression in maternal blood and in the embryo, demonstrating that miRs could
serve as possible biological markers as early as during the preimplantation period.
P 158
Characterization of human Pregnancy Specific Glycoprotein (PSG) gene copy number variations in preeclampsia patients
Chia Lin Chang1, Chia Yu Chang1, Da Xian Lee1, Po Jen Cheng1
1
Department of Obstetrics and Gynecology, Chang Gung Memorial Hospital Linkou Medical Center
Pre-eclampsia is a pregnancy-specific hypertensive disorder that affects 2-8% of pregnancies. This disorder can
76
lead to seizure, multi-organ failure, and maternal death. The best approach to prevent pre-eclampsia-associated
adverse outcomes is to be able to prevent pre-eclampsia as early as possible. Unfortunately, current diagnosis
methods are ineffective at predicting the risk of pre-eclampsia during early pregnancy.
Recent discoveries have shown that a group of placenta-derived Pregnancy Specific Glycoproteins (PSGs) are
autocrine/paracrine hormones that regulate trophoblast cell invasion and vascular remodeling at the feto-maternal
interface. In humans, low levels of PSGs have been associated with intrauterine growth retardation and preeclampsia, and there is a significant enrichment of cases with deletions in the PSG gene locus in pre-eclampsia
patients. In addition, our recent study has shown that human PSG gene region exhibits a significantly higher density
of single nucleotide polymorphisms (SNPs) and copy number variations (CNVs) as compared to the genome
average. Accordingly, we hypothesize that genomic variations at the PSG gene locus of maternal and/or fetal
genomes may confer increased risks of pre-eclampsia.
Taking advantage of recent progresses in massively parallel sequencing of circulating cell-free fetus DNA (cffDNA)
for the diagnosis of diseases in the fetus, we are investigating the relationship between genomic variations at the
PSG gene locus and preeclampsia using circulating cell-free DNA. To sequence the highly polymorphic 550-kb PSG
gene locus, we adopted a targeted sequencing strategy in which dozens of overlapping 10-kb genomic fragments
were first amplified by long-range PCR, followed by the next generation sequencing with an Ilumina Miseq personal
sequencer. The identification of novel PSG CNV–disease relationships would provide a better understanding of
the pathology of pre-eclampsia and a novel opportunity to identify patients with a high risk of developing preeclampsia.
P 56
The role of circulating mir-1 in Diabetic Retinopathy
Tanuka Palit1, Ramasamyiyer Swaminathan2, Anthony Marinaki3, Janaka Karalliedde2
1
King’s College London
2
Guy’s Hospital
3
St Thomas’ Hospital
Diabetic Retinopathy (DR) is a major diabetic complication and is the main cause of blindness under the age of 74,
posing a major health risk in our society. Since 2003 the NHS has used fundus photography as a routine screening
procedure for DR detection. However there are downfalls to this screening process as it is highly subjective and
expensive. Using circulating biomarkers as a diagnostic tool has been suggested as an alternative method.
This study focuses on microRNAs as biomarkers for DR detection. miRNAs are non-coding molecules that negatively
regulate gene transcription. They are good candidates as biomarkers because they remain stable in bodily fluids
and are highly tissue and disease specific. Also miRNAs target multiple genes at once. This is important in DR as
the pathophysiology is multifactorial.
Previous in vitro studies have shown that mir-1 is involved in regulation of vasoconstrictor endothelin-1 (ET-1).
ET-1 dysfunction is a candidate for involvement in abnormal haemodynamics seen in DR. Therefore, we believe
dysregulation of mir-1 could be an important contributing factor in DR progression. We aimed to see if mir-1
concentration correlated with disease severity, to use as a potential biomarker.
Patients were recruited from the Diabetics Eye Complication Service and ophthalmology clinics at St. Thomas’ hospital
in South London. Serum samples of 61 diabetic patients were collected. Retinopathy status was characterised by
the NSC grading criteria and included the full spectrum of DR severity. qRT-PCR was then conducted on each
77
sample. Relative expression was calculated for mir-1 compared to the control gene mir-26b, and compared across
DR groups.
Findings showed a lower expression of mir-1 in cases compared to controls. We also identified a significant
downregulation of mir-1 (p<0.025) across disease severity, particularly between patients with severe retinopathy
and patients with maculopathy (p<0.029). Therefore, our results suggest that mir-1 is a potential biomarker for
determining DR severity. mir-1 could also be a potential target for treatment of DR.
P 120
Evaluation of the state of transplanted liver health by monitoring of organ-specific genomic marker in
circulating DNA from receptor
Hada C. Macher1, Gonzalo Suarez-Artacho2, Pilar Jiménez-Arriscado1, Sara Alvarez-Gómez3, Noelia GarcíaFernández1, Juan Miguel Guerrero1, Patrocinio Molinero1, Elena Trujillo1, Miguel Angel Gómez-Bravo2,
Amalia Rubio1
1
2
Hepatobiliary and Liver Transplantation Unit, Virgen del Rocío University Hospital
3
University of Seville
Health assessment of the transplanted organ to detect the onset of transplant rejection is critical for the long-term
survival of the organ. The evaluation of the transplanted liver health by non-invasive approaches may offer a
personalized follow up of the patient and an improvement in the early clinical intervention. As transplanted organs
have genomes that are distinct from the host’s genome, we may find gene polymorphisms (a “genomic signature”)
to distinguish donate liver DNA in the plasma recipient. Thus, the quantification of the specific DNA of the donated
organ in the patient serum will allow us obtaining information about its damage. The aim of this work was to evaluate
the state of transplanted liver by monitoring the donated organ DNA as a biomarker for early diagnosis of rejection.
With this purpose we monitoring serum cDNA from RH gene in 20 recipient and donor mismatched for this gene
Methods: 20 RH negative patients transplanted with a liver from RH positive donors were included in the study.
cDNA quantization of the RH gene was performed by RT- PCR before, at the moment of transplantation (day 0) and
during the stay at the intensive care unit. Beta-globin cDNA levels, a general cellular damage marker, were also
quantified. Patients were grouped based on clinical outcome. Group A: patients that accepted liver transplantation
without any complication; group B patients that accepted the male organ but suffer other complications; and group
C patients that suffered severe or mild organ rejection
Results: All patients showed an increase of cDNA levels at day 0 that decreased until patient stabilization. Patients
from group A and B showed low levels of RH gene cDNA during the follow up, but an increase of beta-globin
gene levels was observed at the moment of any clinical complication. Patients from group C showed an increase
in the RH gene at the moment of the rejection diagnosis or even 24h before the biopsy results were available
Conclusion: Donor-derived cDNA may be quantified in the serum of organ transplant recipients, and high levels of
donor DNA might be used as an indication of graft injury.
P 146
Cell free DNA levels in healthy subjects
Miriam Albus1, Alexandra Brahmer1, Suzan Tug1, Susanne Helmig1, Daniela Zahn2, Thomas Kubiak2, Simon
Perikles1
1
Department of Sports Medicine, Rehabilitation and Prevention, Johannes Gutenberg-University of Mainz
2
Department of Health Psychology, Johannes Gutenberg-University of Mainz
78
Background: Cell free DNA (cfDNA) is often used as a biomarker in context of several diseases, but the relevance
of cfDNA in healthy persons remain underinvestigated. We wanted to investigate the relation between metabolic
and immunological parameters with cfDNA levels in healthy subjects. We also evaluated the association of these
parameters with cfDNA concentrations in venous blood plasma and serum and capillary blood plasma.
Methods: 84 healthy subjects were examined. Subjects were characterized by clinical parameters (e.g. BMI, high
sensitivity CRP (hsCRP)). We measured levels of cfDNA in plasma, serum and capillary blood plasma by quantitative
real-time PCR (qPCR). We performed statistical analyses with venous blood plasma since cfDNA concentrations
showed the lowest interindividual differences and best correlation with other blood parameters.
Correlation analysis was performed according to spearman with JMP 8 (SAS Institute Inc., Cary, NC, USA).
Results: We measured mean cfDNA levels of 12,91 ng/ml in venous blood plasma, 47,6 ng/ml in serum and 10,39
ng/ml in capillary blood plasma.
We found a positive correlation between levels of cfDNA with BMI (p=0,0012), levels of uric acid (p=0,0032) , insulin
(p=0,0078), hs CRP (p=0,0045) and the complement factors C3 (p<0,001) and C4 (p=0,0216).
Serum cfDNA concentrations correlated significantly with levels of C3 (p=0,0038) and C4 (p=0,0096), but with none
of the other parameters mentioned above (insulin, BMI, uric acid, hsCRP : p>0,05).
Conclusion: We conclude that in healthy subjects the baseline levels of cfDNA are influenced by metabolical and
immunological parameters. In this context further investigation is required. Furthermore we recommend to use
blood plasma and not serum for the analysis of resting cfDNA levels.
P 174
Circulating DNA in rheumatoid arthritis development
Elena Rykova1, Aleksey Sizikov2, Oksana Antonenko3, Leonid Bryzgalov4, Evgeny Morozkin5, Valentin
Vlassov1, Pavel Laktionov5, Vladimir Kozlov2
1
2
Research Institute of Fundamental and Clinical Immunology
3
Institute of Molecular and Cellular Biology of Sb Ras
4
Institute of Cytology and Genetics of Sb Ras
5
Early diagnostics of rheumatoid arthritis (RA) hampered due to the unspecific clinical manifestations is necessary for
efficient treatment and recovery. This study was targeted to investigation of cell-free circulating DNA (cirDNA) level
as a factor of RA development and progression. Blood samples were taken from 57 healthy subjects (HS) and 74 RA
patients who fulfilled the ACR criteria, 1987. CirDNA was isolated from blood plasma and fraction of cirDNA bound
with cell surface (csb-DNA). Concentrations of nuclear and mitochondrial DNA (nDNA, mtDNA) were measured
by quantitative PCR specific for LINE-1 repetitive elements and a mitochondrial DNA segment. Rheumatoid factor
(RF), C-reactive protein (CRP), anti-ssDNA, anti-dsDNA antibodies were examined and involved in the assay.
Reliable increase of the cir-nDNA from plasma was found for RA patients compared with HS (12.0 versus 8.4 ng/ml,
Mann-Whitney test, p<0,01). Cell-surface-bound nDNA (csb-nDNA) concentration in RA patients was significantly
decreased (24 versus 50.8 ng/ml, p<0.01). Cell-surface-bound mtDNA (csb-mtDNA) concentration was increased
(1.44 x 106 copies/ml versus 0.58 x 106 copies/ml р<0.05). Significant elevation of RF (median 16.1 versus 6.8 U/
ml, p<0.01) and CRP (median 21.4 versus 2.6 U/ml, p<0.01) was detected in the patient group. According to ANOVA
test the most valid for the discrimination of RA patients from HS were CRP and csb-mtDNA levels, while RF and csbnDNA possessed lower power. Machine learning Random Forests test demonstrate that combination of 4 variables
79
[csb-nDNA, csb-mtDNA, RF, and CRP] allowed the high accuracy of RA patients discrimination from HS (97%
sensitivity and 91% specificity). Csb-nDNA level was elevated in RA patients who developed anti-ssDNA and/or
anti-dsDNA antibodies compared with the RA patients without anti-DNA antibodies (Mann-Whitney test, р<0,01). To
conclude RA development is accompanied by significant changes of the circulating nuclear DNA and mitochondrial
DNA levels found both in blood plasma and at the surface of blood cells.
P 180
Multiple ways of cfDNA reception and following ROS production in endothelial cells
Anna Alekseeva1, Larisa Kameneva1, Tatiana Smirnova1, Svetlana Kostyuk1, Natalia Veiko1
1
Federal State Budgetary Institution “Research Centre for Medical Genetics”
Heart attack and cardiovascular diseases are common reasons for high cell free DNA (cfDNA) burst in blood plasma.
There are few papers showing the effects of such circulating DNA on blood vessels. In our study we strived to
examine the mechanisms of possible interaction of cfDNA with endothelial cells and the following ROS production.
We performed experiments using different model cfDNA fragments as well as cfDNA extracted from blood plasma of
healthy individuals (cfDNAnorm) and people with heart attack (cfDNAinf). We incubated these fragments in optimal
concentration with HUVEC endothelial cells for 3-24 hours and then detected the expression of TLR9 receptors
on mRNA and protein levels. We also detected the ROS level and NOX expression to follow the oxidative stress
formation which is known to occur in endothelial cells after cfDNA treatment. For our experiments we used RT-PCR,
flow cytometry and fluorescent microscopy. In order to block the TLR-pathway we used Chloroquine which is an
endosomal acidification inhibitor. During our research a 2-5 fold increase of TLR9 expression in endothelial cells
treated by CG-rich and cfDNAinf was detected. Chloroquine significantly reduced this effect. Nevertheless, the
expression of NOX4 gene and NOX protein production either without or with chloroquine remained 2-6 times higher
after incubation with model and cfDNAinf fragments. Even the usage of SB203580, the TLR9 selective inhibitor, did
not block the effects of highly oxidized cfDNA on the oxidative stress and ROS production. These results made us
look for other potential cfDNA receptors and we detected a significant rise in AIM2 and RIG1 receptor expression.
This fact points to multiple ways of cfDNA reception by endothelial cells and suggests new possible ways of therapy
after heart attack.
Cancer C
P 134
Assessment of the clinical applications of circulating tumour DNA in non-small cell lung cancer using an
enhanced TAm-SeqTM assay
Davina Gale1, Jordi Remon2, Andrew Lawson1, Sarah Smalley1, Esperanza Perez2, Karen Howarth1,
Michelle Pugh1, Abdelaziz Fahem1, Tim Forshew1, Ludovic Lacroix2, Vincent Plagnol1, Benjamin Besse2,
Nitzan Rosenfeld1
1
Inivata Ltd
2
Institut Gustave-Roussy
Background: Conventional biopsies are costly, invasive and only provide a snapshot of the tumour’s mutational
profile. A promising alternative for assessing tumour burden and monitoring drug response in real time is the detection
of circulating tumour DNA (ctDNA), genomic material released from tumours into the blood plasma. ctDNA has been
80
detected for a wide range of solid tumours and can be distinguished from germline cell-free DNA by tumour-specific
DNA alterations.
Methods: We developed an enhanced tagged-amplicon deep sequencing (TAm-Seq™) assay that enables
detection of point mutations and indels in ctDNA with high levels of sensitivity and specificity. A standardised TAmSeq assay with rapid turnaround time was used to analyse regions of interest (hotspots or entire coding regions)
in 35 genes for plasma samples from 20 patients (18 treatment-naive; 1 Stage II, 5 Stage III and 14 Stage IV) with
non-small cell lung cancer. For 10 patients, a 2nd sample was collected 21 days after starting chemotherapy. When
possible, matched tumour biopsies were obtained prior to treatment and analysed by complementary techniques.
Results: 37 mutations in cancer-associated genes were identified in the initial timepoint samples. 0-4 mutations
were identified per sample, with only one sample with no detectable mutations. Allele fractions ranged from 0.1026.57%, with 17/37 at <1%. The only recurrent mutation was KRAS G12 in 7/20 samples. For the 2nd timepoint,
2/10 samples exhibited the same mutational profile at comparable allele fractions, 2/10 had lower allele fractions
and 6/10 had no detectable mutations. Concordance with matched tumour samples will be presented.
Conclusions: This work demonstrates the potential utility of using ctDNA to monitor treatment response in NSCLC
patients, and for determining the mutational profile of the tumour when a conventional biopsy cannot be obtained. It
highlights the importance of high sensitivity mutation detection and analysing multiple genomic regions, rather than
relying on assays targeting single hotspot mutations.
P 138
Evaluation of Streck cfDNA blood collection tubes (BCTs) for liquid biopsy testing
Inga Medina Diaz1, Erica Maldonado2, Johannes Fredebohm1, Daniel Mehnert1, Florian Schiffel1, Johanna
Weiland1, Bianca Friese1, Ann-Kathrin Löber1, Frank Diehl1, Frank Holtrup1
1
2
Sysmex Inostics Inc
Objectives: A major bottleneck for molecular oncology testing and monitoring based on circulating free DNA
(cfDNA) is the availability of a shipping concept for whole blood over periods of days to the testing laboratory. Most
physician offices and small hospitals are not equipped to prepare plasma with high speed centrifugation and send
it to the testing laboratory deep-frozen. Broad use of liquid biopsy testing thus requires a strategy to ship whole
blood at ambient temperatures while retaining the stability of cfDNA and blood cell integrity to prevent dilution of
circulating tumor DNA (ctDNA) with wild-type (wt) genomic DNA. Streck cfDNA BCTs claim to deliver these features.
The objective of this study was to examine whether the stabilizing reagent in the tube affects DNA amplifiability and
sequence integrity using the highly sensitive BEAMing and Plasma-SafeSequencing (PSS) technologies.
Methods: Venous blood was collected from healthy donors in Streck cfDNA as well as reference K2EDTA tubes
and stored for up to 3 days at various temperatures. Isolated cfDNA was analyzed for overall DNA yield (short and
long DNA fragments), PCR amplifiability and mutational background using qPCR, BEAMing and PSS technologies.
Additionally, detectability of low-frequency mutations spiked into blood samples was evaluated at different storage
times and temperatures.
Results: No significant difference in DNA yield, the ratio between long and short DNA fragments, and DNA
amplifiability was identified for blood stored between 6°C and 37°C in Streck cfDNA tubes. Lower and higher
storage temperatures led to increased genomic DNA release. BEAMing and PSS technologies confirmed that the
Streck preservative does not lead to increased background rates in wt DNA. Low-frequency mutations were reliably
detected after three days of storage.
Conclusion: Whole blood shipped in Streck cfDNA BCTs over several days is qualified for downstream liquid
81
biopsy testing using BEAMing. Since temperature is a critical factor, an insulated shipping box should be used to
protect blood samples under extreme weather conditions.
P 143
Plasma miR-224 enable sensitive cancer screening and therapy monitoring with liver functionindependent manner in hepatocellular carcinoma
Wataru Okajima1, Shuhei Komatsu1, Daisuke Ichikawa1, Mahito Miyamae1, Tsutomu Kawaguchi1, Taisuke
Imamura1, Takuma Ohashi1, Jun Kiuchi1, Eigo Otsuji1
1
Background: Recent increase of non-alcoholic fatty liver disease (NAFLD) and viral hepatitis from Asian to Western
countries lead to the increase of hepatocellular carcinoma (HCC) in the world. However, its prognosis remains still
poor. And no ideal and generally accepted blood-based molecular biomarkers have existed to enable a sensitive
cancer screening and monitoring in HCC.
Methods: We comprehensively selected 4 candidates (miR-151, 155, 191 and 214) from 121 microRNAs in NCBI
database up to August 2013, which were reported highly expressed in HCC tissue.
Results: Test- and large scale analyses by quantitative RT-PCR validated a significant higher levels of plasma miR224 in HCC patients than controls (P < 0.0001, AUC 0.914). Plasma miR-224 reflected tumor dynamics; plasma
miR-224 levels were significantly correlated with their paired miR-224 levels of HCC tissues (P = 0.0005), and
reduced in normal levels following curative hepatectopmy (P = 0.0058). Specifically, plasma miR-224 levels were
not correlated with conventional tumor markers such as AFP and PIVKA-II, and significantly discriminate HCC
patients in a liver function-independent manner. Plasma miR-224 level was significantly correlated with tumor size
(P =0.0150), and could accurately detect small tumor less than 18mm (AUC 0.832) and residual HCC tumor after
local or chemoembolization therapy (P = 0.0318).
Conclusion: These findings suggest that plasma miR-224 could be a molecular biomarker as a liquid-biopsy and
highlight its usefulness in HCC.
P 147
Microvesicules and miRNA in urine of healthy and prostate cancer patients
Olga Bryzgunova1, Evgeniy Lekchnov1, Tatyana Skvortsova1, Evgeny Morozkin2, Ivan Zaporozhchenko2,
Alina Grigorieva1, Marat Zaripov3, Elena Ryabchikova 1, Valentin Vlassov1, Pavel Laktionov2
1
Institute of Chemical Biology and Fundamental Medicine Sd Ras
2
3
The study is aimed to investigation of the microvesicules, their content, total and microvesicules packed miRNA in
urine of healthy donors (HD) and prostate cancer patients (PCP). The study population included 14 PCP (63-82
years, T2-3NxMx1) and 20 HD with no previous history of prostate disease (48-73 years). Urine was clarified by two
serial centrifugations at 400 and 1700g, both 20°C, 20 min and microparticles were precipitated by high-speed
centrifugation at 100000g, 18°C, 90 min. Microparticles were washed once or 0.1 μm filtered to obtain exosomeenriched fraction. Both fractions were investigated by transmission electron microscopy (TEM). MiRNAs were
isolated by one-step single-phase protocol (Zaporozhchenko et al., Anal. Biochem, 2015) or excess biopolymers
82
precipitation method (Rus. patent application No 2015117350). The size and quantity assessment of extracellular
RNA were performed using Agilent 2100 Bioanalyzer capillary electrophoresis. Concentrations of miRNAs (miR19b,
miR25, miR205, miR125b, miR126) were measured by qRT-PCR and normalized to miR-16 using dCq method.
TEM demonstrated the presence of 20-300 nm microparticles in urine of HD and PCP patients. The pool of urine
microvesicles consists of 50-70% exosomes and 30-50% particles larger than 100 nm.
The major part of extracellular RNA found both in exosomes and microparticles of HD and PCP, is 25-200 nt long.
Concentration of extracellular urine miRNA in microparticles varied from 100 pg/ml to 1740 ng/ml in depend from
donor and isolation protocol.
Receiver Operating Characteristic (ROC) curve analysis of all miRNAs in samples isolated from total urine did not
demonstrate any diagnostic significance. In contrast, in training cohort relative concentration of miR-19b in exosomes
and total microparticles fraction provide 95%/75% and 93%/100% sensitivity and specificity, respectively.
Thus microparticles, isolated from urine of PCa patients represent a valuable source of diagnostically significant
miRNAs, although the data obtained need to be confirmed using large testing cohort.
P 155
The Potential of Circulating Tumour DNA as a Diagnostic Aid in Urological Cancers
Keval Patel1, Dana Tsui2, Charlie Massie1, James Morris1, Francesco Marass1, Vincent Gnanapragasam3,
Nitzan Rosenfeld1
1
2
Cancer Research UK Cambridge Institute, LI Ka Shing Centre, University of Cambridge 3University
Lecturer in Uro-Oncology, Department of Surgery & Oncology, University of Cambridge
Prostate cancer (PCa) is the most common cancer in UK men and its incidence is rising. The current diagnostic gold
standard is needle biopsy. However, PCa is a notoriously heterogeneous cancer. This leads to clinical uncertainty,
especially when stratifying patients to conservative treatments. Recent evidence suggests that mutant circulating
tumour DNA (ctDNA) can represent tumour heterogeneity and that ctDNA can be detected when prostate cancer is
localised. Previously, we investigated archival plasma samples from men who developed TP53 driven metastatic
PCa and were able to demonstrate the presence of the same TP53 mutants in plasma samples taken at the time
of initial surgery.
In addition, the presence of urinary cell-free DNA (cfDNA) has long been established and mutant cfDNA has been
detected in urine of bladder cancer patients. This has often been achieved by employing single locus assays. For
urological cancers, direct shedding may increase the urinary mutant:wild type ratio detected and thus could be
more informative than plasma. In a pilot study of patients with muscle invasive bladder cancer using a targeted
sequencing panel to assess the status of multiple genes, we detected mutant cfDNA in the urine supernatant of
75% patients.
We are now investigating whether tumour heterogeneity can be represented at low volume disease and how urinary
mutant cfDNA levels compare to ctDNA levels in urological cancers. We hope that by tracking multiple clones at
an early stage, ctDNA analysis may be able to track tumour progression in PCa. In the future, such a test would
improve the confidence of clinicians to stratify patients to conservative therapy whilst reducing the need for invasive
biopsies.
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P 156
Unbiased detection of somatic copy number aberrations in cfDNA of lung cancer cases with low coverage
whole genome sequencing
Fiona Taylor1, James Bradford1, M. Dawn Teare1, Penella Woll1, Angela Cox1
1
Sheffield Cancer Research Centre, University of Sheffield
Background: Molecular profiling using low coverage whole genome sequencing of cfDNA represents a non-targeted
approach to identify somatic copy number alterations (SCNA). We aim to establish that SCNA are detected in cfDNA
of lung cancer cases, and to determine the limit of detection. We hypothesise that the number and magnitude of
SCNA might also serve as a discriminative test to aid early lung cancer detection.
Methods: Standard protocols were followed to process, extract, quantify and sequence matched cfDNA, FFPE
tumour and lymphocyte DNA. Read copy number profiles for cfDNA or FFPE DNA were normalised to profiles from
matched lymphocyte DNA with the software CNAnorm. Technical sensitivity was determined by spiking different
proportions of FFPE tumour DNA into cfDNA from healthy volunteers.
Results: The mean total number of reads for each sample was 14,605,710. Of these, 89% mapped to the human
reference genome hg19 after filtering. The median genome coverage was 0.26X (range 0.05X-0.97X). For 2
advanced stage cases there was a significant positive correlation between copy number ratio profiles of matched
cfDNA and FFPE DNA (Spearman Rank r=0.62, 0.75 respectively, p<0.0001). There was no correlation for 4
advanced and 2 early stage cases. There were variable frequency but low magnitude copy number aberrations
detected in high risk controls (N=5). The estimated tumour content identified in cfDNA from cases varied from 2.5%
to 8.0% (N=6). In comparison, we detected spiked FFPE DNA derived SCNAs with a tumour fraction as low as 10%
of cfDNA from healthy volunteers.
Conclusion: Our preliminary results demonstrate non-invasive detection of tumour derived copy number alterations
in advanced lung cancer cases with low coverage whole genome sequencing. Clinical characteristics may influence
whether SCNA are detected in cfDNA. Further optimisation of this approach will be required to identify SCNA in
cfDNA of early stage cases.
P 163
Using Cell Free DNA Reference Standards to evaluate the analytical performance of circulating tumor
DNA testing and solid organ transplant health surveillance
Hadas Amit1, Shen Wei1, Javier Armisen-Garrido1, Bernice Edgeworth1, Robert Woodward2, John Sninsky2,
Colin Barker1, Karin Schmitt1
1
Horizon Discovery
2
Caredx Inc
The development of novel platforms to assess the presence of circulating nucleic acids is challenging due to their
low abundance in human fluids and the difficulty in obtaining a sustainable source of patient-relevant material.
Translating the potential of circulating nucleic acids detection into the clinic requires highly precise and accurate
technologies to enable sensitive and specific measurements.
Utilising proprietary genome editing technology, Horizon’s diagnostics division has established a range of bestin-class, genetically defined genomic reference standards, including FFPE sections and purified DNA. These
standards offer a sustainable and highly defined source of reference material to laboratories, proficiency schemes
and technology developers. Here we present the design and validation of fit-for-purpose Cell Free DNA Reference
Standards to evaluate the analytical performance of cell free DNA assays for circulating tumor DNA and solid organ
transplantation.
84
Using our highly validated isogenic cell lines, a panel of Cell Free DNA Reference Standards were developed. These
genetically defined standards are fragmented to the approximate size of circulating DNA extracted from plasma.
Using Digital PCR for absolute quantification of mutant and wild type variants, we were able to show the creation of
extremely accurate reference material even at allelic frequencies down to 0.1% across a range of markers.
We demonstrate that the Cell Free DNA Reference Standards enabled assessment of assay specificity and sensitivity
across multiple PCR and NGS platforms. In addition, the standards were used to validate the limit of detection and
quantification of a recently described donor-derived cell free DNA NGS assay for solid organ transplant health
surveillance developed by CareDx Inc.
This study presents a proof-of-concept for the application of Reference Standards in defining analytical performance
of circulating DNA testing, enabling the development of new molecular profiling platforms, routine circulating DNA
testing and cross-platform comparison.
P 224
Quantification of Cell-Free mSHOX2 Plasma DNA for Therapy Monitoring in Advanced Stage Non-Small
Cell (NSCLC) and Small-Cell Lung Cancer (SCLC) Patients
Bernd Schmidt1, Julia Beyer1, Dimo Dietrich2, Ines Bork1, Volker Liebenberg3, Michael Fleischhacker1
1
KIM I, Department of pneumology, University medical center Halle/Saale
2
University Hospital Bonn, Department of Otolaryngology, Head and Neck Surgery
3
Thermo Fisher Scientific
Most patients suffering from advanced lung cancer die within a few months. To exploit new
therapy regimens we need better methods for the assessment of a therapy response.
In a pilot study we prospectively enrolled 36 patients with advanced NSCLC and SCLC
(34 stage IV, 2 stage IIIB) of whom 34 received standard platinum-based chemo/radiotherapy and two were treated
with a tyrosine kinase inhibitor. We measured the levels of extracellular methylated SHOX2 DNA (mSHOX2) in
plasma before and during therapy until restaging. The mSHOX2 analysis was blinded with respect to the clinical
data making it an observational study.
According to the re-staging of 31 first-line patients, 19 patients were classified as nonresponders while 12 patients
were in the responder group. We observed a tight correlation between radiological data and the change of plasma
mSHOX2 level as the equivalent for a therapy response. A ROC analysis showed a high discriminatory power
for both patient groups already one week after therapy start (AUC 0.844). Additionally, a Kaplan-Meier and Cox
Proportional Hazards analyses revealed a strong relationship between survival and plasma mSHOX2 value p#0.001
(hazard ratio 11.08) providing some evidence for mSHOX2 also being a predictive marker.
The longitudinal measurement of extracellular plasma mSHOX2 DNA yields information about the response to
cytotoxic treatment and allows an early assessment of treatment response for lung cancer patients. If confirmed in
a larger study this would be a valuable tool for selecting and guiding a cytotoxic treatment.
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Cancer D
P 166
Malignant potential in pancreatic neoplasm; new insights provided by circulating miR-223 in plasma
Mahito Miyamae1, Shuhei Komatsu1, Daisuke Ichikawa1, Tsutomu Kawaguchi1, Ryo Morimura1, Wataru
Okajima1, Takuma Ohashi1, Taisuke Imamura1, Jun Kiuchi1, Hisashi Ikoma1, Hiroki Taniguchi2, Eigo Otsuji1
1
2
Department of Surgery, Kyoto Second Red Cross Hospital
Background: Recent studies have identified that microRNAs are stably detectable in plasma/serum because of
their binding to specific proteins or being packaged in secretory vesicles and could be potential diagnostic and
therapeutic biomarkers in cancers.
Methods: We tested miR-223, which is reported to be highly expressed in pancreatic cancer (PCa), as a candidate
of novel plasma biomarker to detect PCa and predict malignant potential of intraductal papillary mucinous neoplasm
(IPMN).
Results: (1) miR-223 expression was significantly higher in PCa tissues (P = 0.0069) than in normal tissues.
(2) Plasma miR-223 concentrations were significantly higher in 71 PCa patients than 67 healthy volunteers (P <
0.0001). (3) Plasma miR-223 concentrations were significantly reduced in postoperative samples (P = 0.0297). (4)
Plasma level of miR-223 tended to discriminate the malignant potential between benign IPMN and malignant IPMN
(P = 0.0963), and the progressive extent of invasiveness between malignant IPMN and pancreatic invasive ductal
carcinoma (PIDC) (P = 0.0004). Multivariate logistic regression analysis revealed that a low plasma level of miR-223
was independent risk factors for PIDC [p=0.0012, Odds ratio 7.90 (95% CI: 2.06-41.2)]). (5) There was no significant
correlation between plasma miR-223 levels and the number of any blood cell types in the peripheral blood.
Conclusion: Plasma miR-223 might be a clinically useful biomarker for screening PCa, and predicting malignant
potential of IPMN and the invasiveness of PCa.
P 170
Plasma miR-141 and miR-205 as potential biomarkers of prostate cancer
Ivan Osipov1, Evgeny Morozkin2, Ivan Zaporozhchenko2, Anna Bondar3, Marat Zaripov4, Vladimir
Voytsitskiy4, Valentin Vlassov3, Pavel Laktionov2
1
Novosibirsk State University
2
3
4
Prostate cancer (PCa) is one of the most spread male malignancies worldwide. Mortality caused by this disease is
concerned with problems of early detection. Present non-invasive methods are not capable of reliably discriminating
between early stage cancer and benign prostate hyperplasia (BPH). Currently new diagnostic methods based on
molecular genetics of PCa are actively developed. The one class of promising molecular biomarkers is miRNA.
The aim of our study was to reveal differences in expression levels of specific miRNAs in prostate cancer or benign
prostate hyperplasia subjects compared with normal controls.
We selected five potential prostate cancer miRNA biomarkers (miR-19b, miR-21, miR-126, miR-141, miR-205),
86
whose expression was shown to be associated with PCa development, prognosis of tumor aggressiveness and
treatment effectiveness. Expression levels of selected miRNAs were measured by quantitative RT-PCR in plasma
of 47 healthy donors, 34 patients with BPH, and 48 PCa patients and normalized to miR-16 and miR-101.
We found that circulating miR-141 and miR-205 were significantly upregulated in plasma of prostate cancer patients.
Combination of these two miRNAs discriminated PCa patients and healthy donors were 72.9% and 83% sensitivity
and specificity. Further, we determined the influence of clinic-pathological characteristics on miRNA expression
level. Expression level of miR-141 significantly distinguished PCa patients with Gleason’s score 6 and 7 as well as
both of these groups from health donors. In addition, patients with Gleason’s score 6 significantly differed from BPH
patients. It was discovered that patients with early TNM stages of prostate cancer have dramatically upregulated
miR-141, which decreases with disease progression. Patients with T2 PCa can be discriminated from health donors
with sensitivity of 76.2% and specificity of 100%.
P 172
Tracking KRAS mutation in circulating cell free DNA as biomarker for anti-VEGF and EGFR colorectal
cancer therapies
Shiro Kitano1, Takeshi Yamada2, Takuma Iwai2, Masato Nakayama1, Uchida Eiji2
1
Toppan Printing Co., Ltd, Technical Research Institute
2
Department of Digestive Surgery, Nippon Medical School
Background: KRAS status in the primary tumor site is given as a predictive molecular biomarker of responsiveness
to EGFR targeting therapy in metastatic colorectal cancer (mCRC). Recently, previous evidence shows that bloodbased testing can track the emergence of drug resistant and provide an early warning of treatment failure, especially
on the anti-EGFR colorectal cancer therapy (e.g. cetuximab). Although a predictive marker of VEGF targeted therapy
have been reported by several research groups previously, decisive candidate factor is not yet. Here we report the
clinical validation study about prediction of the response for anti- EGFR and VEGF therapies in colorectal cancer
patients with resected primary tumor by KRAS status in the patient’s plasma.
Material and methods: For the analysis of circulating cell free DNA (ccfDNA), we had constructed an ultra sensitive
(max 0.1%) and quantitative genotyping method which is based on droplet digital PCR (ddPCR) system (BioRad).
Assays were set up using plasmids containing hot spots KRAS (G12A, G12C, G12D, G12R, G12S, G12V and G13D
mutations. ccfDNA samples were purified from plasma samples (n=24) of mCRC patients (n=22) which treated by
anti-EGFR (cetuximab or panitumumab) or anti-VEGF ( bevacizumab) and cytotoxic drug combination therapies.
Results: In the clinical study, the primary KRAS status showed 80.0% specificity and 81.8 % sensitivity in comparison
with plasma KRAS status. Against patients had KRAS wild-type in the both of primary tumor and plasma, cetuximab
was successful 100 percent (CR and SD) for drug response. In addition, in two cases which KRAS status was the
mutant type in primary tumor and it was wild type in plasma, bevacizumab with FOLFOX has long-term (over the 1
year) successful (SD).
Conclusions: In the real-time KRAS mutation monitoring using blood based testing, this test is a promising clinical
tool for noninvasive assessment of drug response, emerging resistance and early relapse for CRC patients.
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P 173
Circulating microRNA expression level during the lung cancer combined therapy: chemotherapy followed
by surgical resection
Anastasia Ponomaryova1, Evgeny Morozkin2, Elena Rykova3, Ivan Zaporozhchenko2, Tatyana Skvortsova3,
Aleksey Dobrodeev4, Alexander Zavyalov4, Sergei Tuzikov4, Nadezhda Cherdyntseva5, Valentin Vlassov3,
Pavel Laktionov2
1
Tomsk Cancer Research Institute of Sb Rams; National Research Tomsk Polytechnic University
2
3
4
Tomsk Cancer Research Institute
5
Tomsk Cancer Research Institute of Sb Rams; National Research Tomsk State University
Expression levels of cancer-associated microRNAs were reported to be changed in tumours and serum/plasma
samples from lung cancer patients. The purpose of this study was to estimate the value of 5 selected miRNAs
plasma levels as markers of response to the combined antitumor therapy and survival prognosis in lung cancer
patients. MiR-19b, miR-126, miR-25, miR-205, miR-125b have been evaluated by quantitative reverse transcription
PCR (qRT-PCR) versus control miR-16 in blood plasma samples from 23 lung cancer (LC) patients. Plasma samples
were obtained from LC patients before treatment (untreated - UT), within 30 days after completing two courses of
chemotherapy (post-chemotherapy - PC) and 15 days after surgery (post-operative - PO). Repeated Measures
ANOVA demonstrated the changes in miR-19b expression levels in patients’ plasma during the combined treatment
(decreased in PC, increased in PO) characterized by a significant quadratic trend (P=0,03). MiR-125b expression
levels increased during the combined treatment and demonstrated a significant linear trend (P=0,03). MiR-125b/
miR-19b ratio changed in PC and PO plasma samples with a significant linear trend (P=0,04). Individual analysis
in the groups of patients with partial tumor regression in response to chemotherapy (group 1) and patients with
tumor stabilization or progression (group 2) showed different trends for miR-19b, miR-125b and miR-125b/miR-19b
ratio between the groups. The Kaplan-Meier survival curves showed an association of miR-125b/miR-19b ratio
value with the survival time without the tumor relapse (P<0,1). Dynamic changes trends of miR-19b and miR-125
expression levels and miR-125b/miR-19b ratio in the blood plasma have shown a potentiality to discriminate types
of response to the combined antitumor therapy in lung cancer patients. Further in-depth investigation is needed to
establish a direct link between the miRNAs expression levels in blood plasma and therapy response.
Study has been supported by Russian Foundation for Basic Research (RFBR, grant No. 14-04-01881), Postdoctorate program in TPU
P 194
Clinical validation of the analysis of circulating DNA for theragnostics and multiparametric strategy for
cancer patients’ prognosis
Safia El Messaoudi1, Brice Pastor2, Cynhia Sanchez2, Stanislas Du Manoir2, Florent Mouliere3, Caroline
Mollevi2, Charles Theillet2, Brigitte Gillet4, Michelle Nouaille5, Denis Pezet4, Muriel Mathonnet5, Marc
Ychou2, Alain R. Thierry2
1
Ircm, Institut de Recherche En Cancérologie de Montpellier
2
Ircm, Institut de Recherche En Cancérologie de Montpellier, Insermu-1194, Universités de Montpellier,
Institut Du Cancer de Montpellier F-34298, France
3
Ircm, Institut de Recherche En Cancérologie de Montpellier, Insermu-1194, Universités de Montpellier,
88
Institut Du Cancer de Montpellier F-34298, France. ; Cancer Research UK Cambridge Institute, University
of Cambridge, Cambridge, UK
4
Centre Hospitalier Universitaire de Clermont-Ferrand, Service de Chirurgie Digestive Unité D’oncologie
Digestive, Umr Unité Inserm/Université D’auvergne U1071, Clermont-Ferrand, France
5
Centre Hospitalier Universitaire de Limoges, Service de Chirurgie Digestive, Centre D’investigation
Clinique, Inserm 0801, Limoges, France
Background: We previously showed the strong theranostic value of ccfDNA analysis in a blinded clinical study on a
cohort of 106 metastatic colorectal cancer (mCRC) patients. We wanted to go further by analyzing prognostic value
of multiparametric ccfDNA analysis and by following blinded clinical studies.
Methods: We analyzed overall survival (OS) on the previous 106 patient’s cohort using our Intplex method which
simultaneously determines total ccfDNA concentration and fragmentation, KRAS/BRAF/NRAS mutational status,
mutant ccfDNA concentration and mutation load. ). In order to evaluate application of ccfDNA analysis in a clinical
setting, we realized a second blinded prospective multicentric and just-in-time clinical study including 140 mCRC
patients comparing KRAS/NRAS/BRAF mutations determination by tissue and plasma carried out in the conditions
of standard management care.
Results: All ccfDNA parameters were of prognostic interest: high ccfDNA levels positively correlated with shorter
OS (p=0.0087, HR=1.94). Multivariate analysis revealed that it was a significant independent prognostic factor
(p=0.034, HR=1.73). High mutant ccfDNA levels as well as high mutation positively correlated with shorter OS
(p=0.0089, HR=2.7 and p=0.05, HR=2.78 respectively). In addition, high ccfDNA fragmentation was positively
related with decreased OS in the mutant cohort of patients (n=43, p=0.0052, HR=3.84) and appeared as a strong
independent prognostic factor (p=0.0072, HR=3.57) while it was not significant in the exclusive WT cohort of patients
(p=0.67). We revealed that ccfDNA analysis had higher prognostic value than CEA (p=0.48, HR=1.24). Our second
blinded clinical study confirms the power of ccfDNA as a liquid biopsy with a high concordance rate with tumor tissue
analysis and a median turnaround time of 2 days.
Conclusions: Our OS study revealed for the first time the strong prognostic value of multiparametric ccfDNA
analysis better than CEA. Our recent data assess the power of the liquid biopsy and will allow in a few months to
assess the prognostic value of such analysis.
P 203
Clinical implications of genomic alterations in the tumor and circulation of pancreatic cancer patients
Mark Sausen1, Jillian Phallen1, Vilmos Adleff1, Sian Jones2, Rebecca J. Leary1, Michael T. Barrett3, Valsamo
Anagnostou1, Sonya Parpart-Li2, Derek Murphy2, Qing Kay Li1, Carolyn A. Hruban1, Rob Scharpf1, James
R. White1, Peter J. O’Dwyer4, Peter J. Allen5, James R. Eshleman6, Craig B. Thompson7, David S. Klimstra8,
David C. Linehan9, Anirban Maitra1, Ralph H. Hruban6, Luis Diaz1, Daniel D. Von Hoff10, Julia S. Johansen11,
Jeffrey A. Drebin12, Victor E. Velculescu1
1
The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine
2
Personal Genome Diagnostics Inc.
3
The Translational Genomics Research Institute, Mayo Clinic Arizona
4
Department of Medicine, University of Pennsylvania Perelman School of Medicine
5
Department of Surgery, Memorial Sloan Kettering Cancer Center
6
The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine;
Department of Pathology, The Sol Goldman Pancreatic Cancer Research Center, Johns Hopkins
89
University School of Medicine
7
Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center
8
Department of Pathology, Memorial Sloan Kettering Cancer Center
9
School of Medicine and Dentistry, University of Rochester
10
The Translational Genomics Research Institute, Virginia Piper Cancer Center, Scottsdale Healthcare
11
Department of Oncology and Medicine, Herlev Hospital, University of Copenhagen
12
Department of Surgery, Perelman School of Medicine, University of Pennsylvania
Pancreatic adenocarcinoma has the worst mortality of any solid cancer. To evaluate the clinical implications of
genomic alterations in this tumor type, we performed whole-exome analyses of 24 tumors, targeted genomic
analyses of 77 tumors, and used non-invasive approaches to examine tumor-specific mutations in the circulation
of these patients. These analyses revealed somatic mutations in chromatin regulating genes MLL, MLL2, MLL3,
and ARID1A in 20% of patients that were associated with improved survival. We observed alterations in genes with
potential therapeutic utility in over a third of cases. Liquid biopsy analyses demonstrated that 43% of patients with
localized disease have detectable circulating tumor DNA (ctDNA) at diagnosis. Detection of ctDNA after resection
predicts clinical relapse and poor outcome, with recurrence by ctDNA detected 6.5 months earlier than with CT
imaging. These observations provide genetic predictors of outcome in pancreatic cancer and have implications for
new avenues of therapeutic intervention.
P 206
Multiplex KRAS G12/G13 mutation testing of 16ng of unamplified cell-free DNA from plasma of patients
with advanced cancers using Droplet Digital PCR
Helen J. Huang1, Dawne Shelton2, Siqing Fu1, Sarina Anne Piha-Paul1, Apostolia M. Tsimberidou3, Jennifer
J. Wheler1, Aung Naing1, David Hong1, Gerald S. Falchook4, Scott Kopetz5, Raja Luthra6, Bryan K. Kee5,
George Karlin-Neumann2, Funda Meric-Bernstam1, Filip Janku1
1
Investigational Cancer Therapeutics, Ut M. D. Anderson Cancer Center
2
Bio-Rad Digital Biology Center
3
Departments of Investigational Cancer Therapeutics, Ut M. D. Anderson Cancer Center
4
Sarah Cannon Research Institute at Healthone
5
Gastrointestinal Medical Oncology, Ut M. D. Anderson Cancer Center
6
Molecular Diagnostic Laboratory, Ut M. D. Anderson Cancer Center
Background: Novel, multiplex and accurate diagnostic systems using low amounts of DNA are needed for further
development of plasma cfDNA testing in personalized therapy.
Methods: cfDNA from plasma samples of 94 patients with advanced cancers who progressed on systemic therapy
was tested with a KRAS multiplex assay to detect 7 common mutations in the G12/G13 hotspot of exon 2 using the
QX200 Droplet Digital PCR™ platform (Bio-Rad). Results were compared to mutation analysis of archival primary/
metastatic tumor tissue obtained at different points of clinical care from a CLIA-certified laboratory, generally months
to years prior to blood collection. A subset of 22 patients with KRAS-mutant tumors were followed longitudinally in
plasma with the multiplex KRAS assay.
Results: Using only 16ng unamplified cfDNA, KRAS G12/G13 mutations were detected in 59% (55/94) of plasma
samples and in 69% (65/94) of archival tumor samples, resulting in 87% concordance (85% sensitivity, 93%
specificity). Discrepancies were significantly reduced with higher DNA input: only 5 discordant FFPE positives with
≤ 80ng cfDNA, and only 2 when high cfDNA input and single mutant assays were used (96% concordance, 97%
90
sensitivity). Of interest, 1 of 2 colorectal cancer patients with KRAS mutation in cfDNA but not in tumor, experienced
rapid disease progression after 1 cycle of cetuximab-based therapy. Correlations of these cfDNA results with other
clinical parameters will be presented along with results from longitudinal analysis of plasma samples.
Conclusions: Multiplex detecting of KRAS G12/G13 mutations in a low amount of unamplified plasma cfDNA using
the Bio-Rad QX200 system has high concordance and sensitivity and should be investigated further for testing of
KRAS mutations in cancer.
Basic Science / Technical Issues
P 21
Purification of Circulating Cell-Free DNA from plasma using the automated Large-Volume Extraction on
the QIAsymphony SP Instrument
Alexander Wolf1, Manuel Frietsch1, Sebastian Groemminger2, Wera Hofmann2, Matthias Sachse2, Jana
Fassunke3, Katharina Beller1
1
Qiagen GmbH, Research & Development
2
Lifecodexx AG
3
Institute of Pathology, University medical center Cologne
Introduction: Circulating, cell-free DNA (ccfDNA) is a key analyte for non-invasive prenatal diagnostics and cancer
biomarker analysis. Due to extremely low concentration and high degree of fragmentation, the extraction of ccfDNA
is technically challenging. Here, the efficiency of a new automated large volume ccfDNA extraction method was
evaluated against a manual reference method.
Methods: Plasma from EDTA and Streck Cell-Free DNA blood collection tubes from healthy donors or clinical
samples were used for the development of a new ccfDNA extraction protocol on the QIAsymphony SP instrument.
Quality and quantity of ccfDNA was analyzed (I) by an Agilent DNA Chip, (II) by qPCR, (III) by NGS in combination
with DNAseq targeted panels and (IV) by determination of fetal fraction by a methylation-sensitive DNA digest in
combination with qPCR. The QIAamp® Circulating NA Kit served as reference method.
Results: The novel protocol showed a median ccfDNA recovery of 125 % (n=12) compared to the QIAamp®
Circulating NA Kit. An Agilent High Sensitivity DNA Chip revealed a comparable size distribution of the extracted
ccfDNA. Linearity was shown for the automated extraction of ccfDNA from different plasma volumes up to 6 ml.
Application of the novel extraction protocol for cancer samples revealed a successful detection of mutations with a
comparable frequency to the reference method. Plasma from pregnant woman showed a highly efficient extraction
of fetal ccfDNA from maternal plasma which allowed successful NGS-based analysis of chromosomes 13, 18 and
21 of the fetuses.
Conclusions: Data from the development of a new ccfDNA extraction protocol on the QIAsymphony® SP revealed
highly promising results regarding the recovery of total ccfDNA as well as cancer and fetal ccfDNA compared to the
QIAamp® Circulating NA Kit. Concluding, the novel protocol enables automated ccfDNA recovery from up to 4 ml
plasma and up to 96 samples per QIAsymphony® SP run in 6 hours combined with high recovery efficiency.
The applications presented here are for research use only. Not for use in diagnostic procedures.
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P 47
Artificial analogues of circulating box C/D RNAs induce microRNA activation in human adenocarcinoma
cells
Grigory Stepanov1, Julia Filippova1, Anna Nushtaeva1, Elena Kuligina1, Olga Koval1, Vladimir Richter1,
Dmitry Semenov1
1
Fragments of small nucleolar RNAs (snoRNAs) were found among various non-coding RNAs circulating in human
blood. Currently, the function of such cell-free sno-derived-RNAs is not clearly defined. This work is aimed at
identifying regulatory pathways controlled by extracellular small nucleolar RNAs.
The series of box C/D snoRNA analogues were synthesized by in vitro transcription with T7 RNA polymerase.
Artificial RNAs hold canonical elements of human snoRNAs, namely U24 (SNORD24) and U25 (SNORD25), and
novel guide sequences, so these RNAs were targeted at modifying predetermined nucleotides of different types of
cellular RNAs.
In order to determine molecular targets and pathways affected by box C/D RNAs we performed whole-transcriptome
Illumina array analysis after transfection artificial box C/D RNAs into human adenocarcinoma cells MCF-7. The
genes relating to innate immune response and apoptotic cascades were found to be activated in cells transfected by
artificial snoRNAs compared with control cells. It was supposed that the secondary structure of box C/D RNAs was
the key feature that provides observed effect. Intriguingly, the transfection of MCF-7 cells with artificial C/D snoRNA
induced the transcription activation of several microRNAs, such as MIR574, MIR599, and MIR21. The increase in
the level of miR21 precursor into MCF-7 and MDA-MB-231 cells was confirmed with RT-PCR analysis.
Our data demonstrated that extracellular small nucleolar RNAs being accepted by human cells can function as
regulators of expression of vital genes including microRNAs genes.
The work was supported by Russian Foundation for Basic Research (grants № 13-04-01058 and 14-04-31468),
the Interdisciplinary Integration Project of SB RAS № 84 and Fellowship of the President of Russian Federation for
young researchers (2063.2015.4).
P 61
cirDNA size and termini in blood of healthy females and breast cancer patients
Svetlana Tamkovich1, Natalia Kirushina2, Valentin Vlassov3, Pavel Laktionov4
1
Institute of Chemical Biology and Fundamental Medicine, Novosibirsk State University
2
National Novosibirsk Regional Oncologic Dispensary
3
4
Circulation Pathology of Acadamician E.N. Meshalkin
Although circulating DNA (cirDNA) represent the valuable source of diagnostic material some basic features of
cirDNA remains to be weakly investigated. In this study we have investigated size of cirDNA in plasma and eluates
from blood cell surface of healthy female (HF, n = 40) and primary breast cancer patients (BCP, n = 8, T1-2N0M0) and
as well as termini of cirDNA molecules in both patients groups.
Blood was fractionated into plasma and cells, and cell-surface-bound DNA (csbDNA) fractions were obtained as
described earlier [Tamkovich, 2005]. CirDNA were isolated and quantified by multiplex Q-PCR [Bryzgunova, 2011].
Agilent 2100 Bioanalyser TM using High Sensitivity DNA Kit was used to characterize cirDNA. The termini of cirDNA
were examined by ligation using biotinilated ds-oligonucleotide adapters with random overhangs, subsequent
92
isolation of cirDNA by streptavidin sepharose and quantification of sepharose bound/unbound DNA by Q-PCR.
It was found that bulk normal plasma sample mainly contained DNA fragments ~ 171 bp and fragments 8022 and
13810 bp to a much smaller extent. Only high molecular weight DNA was found in csbDNA from HF (from 1522 to
14490 bp). cirDNA of BCP is mainly contained high molecular weight DNA fragments and fragments ~ 171 and ~
180 bp to a much smaller extent. csb-cirDNA from BCP demonstrate the same profile as that in HF. cirDNA from
HF demonstrate heterogeneity in its termini - molecules with different 5’-protruding, 3’- protruding and blunt-ends
were found. csbDNA from BCP mainly contains 5’-protruding ends. The data obtained demonstrate higher stability
of csbDNA and a prominent amount of high weight cirDNA in BCP which is likely generated by necrosis of tumor
surrounding tissues, but sources of high weight cirDNA remains to be investigated.
P 115
A New Blood Collection Tube and Extraction Method for the Analysis of circulating cell-free DNA (ccfDNA)
Andrea Ullius1, Thorsten Voss1, Joachim Bonnet2, Wera Hofmann2, Markus Stumm3, Nadine Dettmann1,
Katharina Pfaff1, Franziska Heese1, Daniel Grölz1
1
Qiagen GmbH, Hilden, Germany
2
Lifecodexx AG, Konstanz, Germany
3
Center of prenatal diagnosis and human genetics, Berlin, Germany
Background: Circulating cell-free DNA (ccfDNA) has become an emerging tool in non-invasive prenatal testing
(NIPT) and in cancer diagnostics. PreAnalytiX has developed the PAXgene® Blood ccfDNA System consisting of
a blood collection tube, which prevents the release of genomic DNA (gDNA) from blood cells during transport and
storage, and the QIAsymphony PAXgene Blood ccfDNA workflow for automated extraction. Here, we demonstrate
the performance of this system.
Methods: Whole blood was drawn into PAXgene Blood ccfDNA Tubes, Streck Cell-Free DNA BCT® or EDTA tubes
and stored for up to 10 days. ccfDNA was extracted from plasma using the QIAamp Circulating Nucleic Acid Kit or
the QIAsymphony PAXgene Blood ccfDNA workflow and the obtained ccfDNA was analyzed by quantitative realtime PCR. Fetal ccfDNA from pregnant donors was analyzed by Prena Test® (LifeCodexx AG). Additionally, gDNA
was purified from stored blood with the QIAamp DNA Mini kit and analyzed by gel electrophoresis.
Results: The fraction of ccfDNA in blood collected and stored for 7 days in EDTA tubes showed an up to 100-fold
increase of 18S rDNA fragments. By contrast, copy numbers of these fragments remained constant after blood
storage in PAXgene ccfDNA tubes. PrenaTest of ccfDNA from pregnant women revealed comparable values for
fetal fractions and identical outcomes of NGS between Streck BCT and PAXgene Blood ccfDNA Tubes. gDNA
extracted from blood stored in EDTA tubes displayed an apoptotic band pattern in gel electrophoresis while PAXgene
stabilized blood revealed no additional bands.
Conclusions: The PAXgene Blood ccfDNA Tube enables whole blood stabilization needed for NIPT. The fully
automated QIAsymphony PAXgene Blood ccfDNA workflow is an efficient method for extraction of ccfDNA.
For Research Use Only. Not for use in diagnostic procedures.
93
P 136
Comparison of microRNA content in plasma and urine suggests the existence of a transrenal passage of
selected microRNAs.
Eva Pazourkova1, Sarka Pospisilova2, Iveta Svobodova2, Ales Horinek2, Antonin Brisuda3, Viktor Soukup4,
Jan Hrbacek3, Otakar Capoun4, Jaroslav Mares5, Tomas Hanus4, Marek Babjuk3, Marie Korabecna2
1
Department of Nephrology, 1st Faculty of Medicine, Charles University and General Faculty Hospital in
Prague
2
Department of Biology and Human Genetics, 1st Faculty of Medicine, Charles University and General
Faculty Hospital in Prague
3
Department of Urology, 2nd Faculty of Medicine, Charles University and University Hospital Motol in
Prague
4
Department of Urology, 1st Faculty of Medicine, Charles University and General Faculty Hospital in
Prague
5
Department of Biology and Medical Genetics, 2nd Faculty of Medicine, Charles University in Prague
Background: MicroRNAs (miRNAs) are examined as potential biomarkers in body fluids. In the field of forensic
medicine, the lists of miRNAs specific for different body fluids were reported but urine was surprisingly not analyzed
in this context. We streamed to find the cell-free miRNAs characteristic for urine samples without any dependence
on the sex, age and health status of the examined individuals and we compared the list of such miRNAs with
miRNAs reported to be specific for plasma and venous blood (Silva et al. Forensic Science International: Genetics14
2015).
Material and method: We examined the urine samples from 70 individuals (45 males, 25 females, mean of age
65 years, range 20-84 years). 15 urine donors were healthy volunteers, 5 donors were patients with non-cancer
diseases, 50 urine donors were patients with different stages of bladder cancer. To examine the spectrum of miRNAs
in the cell-free fraction of urine, we used TaqMan Human miRNA Array Card A.
Results: We examined the presence of 384 miRNAs in each urine sample and found 31 microRNAs which were
constantly present in all examined samples. We compared miRNA found in urine with microRNAs reported as
specific for plasma samples (miR-135a, miR-139-3p, miR-182, miR-224, miR-299-5p, miR-330-5p, miR-369-3p,
miR-373, miR-508-3p, miR-518f, miR-519d, miR-551b,). Excluding miR-330-5p and miR-369-3p, we found all these
microRNAs in cell-free fractions of urine at least in some subjects. All 7 microRNAs (miR-16, miR-20a, miR-106a,
miR-126, miR-150, miR-185, miR-451) reported as specific for venous blood were also frequently found in urine
samples analyzed by us.
Conclusion: We provided the list of 31 miRNAs found constantly in urine. Comparison with the lists of miRNAs
reported as specific for plasma and venous blood suggests that some miRNA could be transferred from circulation
into urine. Further research in this area may elucidate the mechanisms of renal clearance of miRNAs.
The study was supported by the grant no. NT12417 of the Internal Grant Agency of the Ministry of Health of the
Czech Republic.
P 139
A quantitative assessment of circulating nucleic acids utilizing several housekeeping genes:
Measurements from four different cell lines
Janine Aucamp1, Abel Bronkhorst1, Piet Pretorius1
1
94
Quantitative real-time PCR (qPCR) is regularly used to quantify circulating nucleic acids (cirNAs) in order identify
biomarkers for various pathologies. The targeting of housekeeping genes as biomarkers, especially β globin, in cirNA
quantification is becoming quite common, but is it the most appropriate gene to target? In qPCR, housekeeping
genes are primarily used as internal controls as they are expected to be expressed in all cells regardless of the
tissue type, developmental stage, cell cycle state or external signal. However, this is not always the case as
studies have shown notable expression variation between healthy and diseased tissues, treated and untreated
cell lines, as well as interindividual expression. The prerequisites for internal control housekeeping genes are: (i)
adequate expression in the target tissues, and (ii) minimal expression variability between both the samples and
the experimental conditions used. This promotes the normalization of sample differences generated during sample
preparation. In terms of biomarker detection quite the opposite is required, as the goal is to search for variations
in gene expression to identify pathologies. However, the internal control prerequisites can be used to optimize
cirNA quantification and to study cirNA gene expression changes after administering external signals. This study
focuses on the utilization of housekeeping gene expression analysis in the optimization of cirNA quantification.
The GeNorm Reference Gene Selection Kit (Primerdesign) was used to identify the cirNA housekeeping genes of
multiple cancerous and non-cancerous cell lines that were the most adequately expressed with the least expression
variability between biological replicates. The kit was also used to compare housekeeping gene expression between
the cirNAs in cell culture medium and the total RNA of the cell lines in order to elucidate their relationship. Changes
in cirNA gene expression of these cell lines were also analyzed under various experimental conditions.
P 148
Circulating DNA-mediated immunoinhibiting involves Ku protein complexing
Anna Cherepanova1, Ivan Zaporozhchenko2, Valentin Vlassov1, Pavel Laktionov2
1
Insitute of Chemical Biology and Fundamental Medicine of Sb Ras
2
Previously, we have demonstrated that free and cell-surface-bound (csb) circulating DNA (cirDNA) inhibit poly(I:C)induced production of pro-inflammatory cytokines in human primary (fibroblasts and endotheliocytes) and
transformed (Hela, A431) cells. ODNs containing the nucleotide motifs frequently found in csbDNA also trigger
the same immunoinhibiting effects. Biological effects of cirDNA suggest its interaction with theeffector molecules
exposed on the cell surface or localized inside the cell.Using these specific ODNs and affinity modification/isolation
approach combined with subsequent MALDI-TOF the main cellular target was identified as Ku protein – heterodimer
of KU70 and KU80 (Cherepanova et al, Exp. Opin. Biol. Ther., 2012).The goal of this study was to reveal whether
Ku protein is involved in the inhibiting effect of ODN analogs of csbDNA.
Fluorescent microscopy using fluorescein-labeled ssODN and dsODN and rhodamine-labeled antibodies to Ku70
revealed that both ODN and cytoplasm form of Ku-protein localize mainly in perinuclear region of cytoplasm, however,
no credible co-localization centers have been identified, probably due to low concentration of Ku in cytoplasm as
compared to nuclei. Additionally, ssODN were found to penetrate cells more effectively than dsODN, which may
explain their stronger immunoinhibiting effect.
The level of Ku70 in Hela cells was reduced by siRNA, which were shown to inhibit expression of Ku70 by up to
70%. In Ku70 knock-down cells inhibiting effect of ODN is approximately twice lower as compared to control siRNA,
indicating that ODN binding to Ku protein is important for the inhibition. Whether Ku is directly involved in nucleic
acid pattern recognition or exerts auxiliary functions, mediating ODN localization or poly(I:C)-proteins interactions,
remains to be investigated.
95
P 168
DNA/protein content of circulating nucleoprotein complexes
Svetlana Tamkovich1, Oleg Tutanov1, Danil Serdukov1, Tatyana Duzhak2, Natalia Kirushina3, Valentin
Vlassov4, Pavel Laktionov5
1
Institute of Chemical Biology and Fundamental Medicine, Novosibirsk State University
2
Institute “international Tomografic Center”
3
National Novosibirsk Regional Oncologic Dispensary
4
5
Circulation Pathology of Acadamician E.N. Meshalkin
Cell-free DNA (cfDNA) was found to circulate in blood being packed in apoptotic bodies or nucleosomes. Except
from histones only few serum proteins (Bulter P, 1990) were shown to be potentially involved in cfDNA binding and
circulation although a lot of cellular and plasma proteins could bind DNA or histones. In the current study we have
investigated DNA and protein content of deoxyribonucleoprotein complexes (DNPC) circulating in blood of healthy
donors (HD) and breast cancer patients (BCP).
DNPC were isolated from blood plasma of HD and primary BCP by affinity chromatography using immobilized
polyclonal anti-histone antibodies. DNA isolated from NPC was analyzed by Agilent 2100 Bioanalyser TM using
High Sensitivity DNA Kit, proteins were identified using MALDI-TOF mass-spectrometry after 10-20% SDS-PAGE.
170-180 b.p. DNA was found to be main component of DNPC circulating in plasma of HD, whereas equal amount
of 170-180 b.p. and more than 6 k.b.p. DNA was found in DNPC from blood of BCP, 111 and 56 additional proteins
(excluding histones) were identified with a good score in DNPC of HD and BCP, correspondingly, however only
4 from these proteins were found in 30% of all samples. Seven proteins (G1/S‑specific cyclin-E2, Rho GTPaseactivating protein 30, NADP‑dependent malic enzyme, SHC SH2 domain‑binding protein 1‑like protein, protein
Mdm4, Rho guanine nucleotide exchange factor 9, Neuroplastin) previously described as tumor specific proteins
were found in DNPC of BCP and no one of them was found in HD. The data obtained demonstrate involvement
of number of cellular and extracellular matrix proteins in cfDNA circulation. The meaning of the proteins in cfDNA
circulation is unclear but their diagnostic potentialities or use for tumor-specific cfDNA isolation can be potentially
explored and employed.
P 175
Oxidized and GC-rich extracellular DNA have different effect on NF-kB and NRF2 signalling pathways in
hMSC, in HUVEC, in MCF7 and in HELF.
Vasilina Sergeeva1, Natalia Veiko1, Elizaveta Ershova1, Larisa Kameneva1, Tatiana Smirnova1, Anna
Alekseeva1, Svetlana Kostyuk1
1
Federal State Budgetary Institution “Research Centre for Medical Genetics”
Oxidized extracellular DNA causes rapid activation of NF-kB and NRF2 signalling pathways in human mesenchymal
stem cells: both transcription and expression of NF-kB (NF-kB1, RELA, MAP3K1, M4K4F) increases in 30 minutes,
but in 1 hour starts to decrease due to activation of NRF2 transcription. Addition of GC-rich DNA fragments stimulates
transcription of NF-kB within 3-24 hours, whereas transcription level of NRF2 only gets higher in 24 hours.
Exposure of HUVEC to both GC-rich DNA fragments and oxidized extracellular DNA enhances expression of NRF2
in 2-3 hours. GC-rich DNA fragments cause the same increase in expression of NF-kB, but the level of NF-kB is
approximately 2 times higher than of NRF2. But the addition of oxidized extracellular DNA increases expression of
96
NF-kB only in 24 hours when the level of NRF2 starts to decrease.
Both oxidized and GC-rich extracellular DNA cause increase of NRF2 expression in MCF7. The maximum of NRF2
concentration is observed in 2 hours upon addition. Unlike NRF2, NF-kB expression upon addition of CG-rich DNA
fragments differs from that upon addition of oxidized extracellular DNA. The former causes increase of NF-kB
expression in 24 hours which lasts for a long time, whereas with the latter peak concentration of mRNA NF-kB is
obseved in 3 hours.
In HELF cultivated in serum-free medium oxidized extracellular DNA inhibits transcriptional activity of NF-kB and
activates NRF2 pathway.
P 176
Non-invasive detection of tissue-specific cell death in ALS
Hai Zemmour1, Roni Werman1, Daniel Neiman1, Joshua Moss1, Judith Magenheim1, Marc Gotkine2,
Benjamin Glaser2, Ruth Shemer1, Yuval Dor1
1
The Hebrew University-Hadassah Medical School
2
Hadassah-Hebrew University Medical Center
Amyotrophic lateral sclerosis (ALS) is a lethal adult-onset neurodegenerative disorder characterized by irreversible
loss of motor neurons followed by muscle wasting. There are currently no circulating biomarkers that can detect
neuronal cell death in ALS. We have developed an approach to detect the tissue origins of cell-free circulating
DNA, based on quantification of tissue-specific DNA methylation signatures of circulating DNA. The method allows
monitoring the rate of cell death in a tissue of interest even when its DNA sequence is identical to that of the host. We
observe very low levels of neuronal- and muscle-derievd DNA in the blood of healthy individuals. A large proportion
of ALS patients show neuron- and muscle-derived circulating DNA in plasma. Sensitive and specific detection of
neuronal and muscle cell death in the circualtion of ALS patients may allow for early diagnosis, stratification of
patients by progression rate, monitoring of response to drugs and a greater understanding of disease dynamics.
P 199
Quantitative detection method of DNA without PCR
Yoichi Makino1, Masato Nakayama1
1
Toppan Technical Research Institute, Toppan Printing Co., LTD
Aim: Digital PCR and next generation sequencer enables noninvasive and sensitive detection of circulating tumor
DNA. However, these methods include PCR step, and miselongation of the DNA polymerase may cause a false
result. Therefore we have developed a technique for detection of single molecule DNA without PCR process. This
method can identify the difference of a single base within 15 minutes. And, this method is quantitative.
Methods: Reaction reagent and target DNA was placed in a glass plate device with a large number of micro wells.
Then, the oil was placed in to a device to form large number of droplets. At this time, the volume of one droplet is
less 100 fL. The device was heated for 15 minutes 62 ° on a hot plate, increase in the fluorescence intensity occurs
in microdroplets. After reaction, the numbers of bright droplets were counted by a fluorescence microscope. For the
detection of mutation rate, and using two kinds of fluorescent filter NIBA and mCherry. Count of droplets was carried
out automatically using the metamorph.
Results: Nunber of bright droplets was depends on the concentration of expected abundance of target DNA. And, It
was confirm that the method is capable of relative quantitive detection in the range of 0.1% to 50% for EGFR T790
97
mutant DNA. In addition, we have also successfully detected from DNA extracted from the cells.
Conclusions: We have established a method that can detect single nucleotide mutation quantitively in 15
minutes. Furthermore, the method does not require special equipment, which is detected by a simple hot plate and
fluorescence microscopy. This digital counting method has great possibilities of detecting various biomarkers such
as DNA, mRNA and miRNA in blood or tissue of cancer patients.
P 215
Circulating, exosomal microRNAs as biomarkers for cancer and their clinical relevance
Heidi Schwarzenbach1
1
In cancer, deregulated expression levels of microRNAs are associated with tumorigenesis, tumor progression,
metastases and drug resistance. Apart from their release by apoptotic and necrotic cells, microRNAs can also be
actively secreted into the blood circulation by exosomes. Exosomal microRNAs are thought to play an important
role in cell-to-cell communication. Based on their biological functions and the possibility of quantifying microRNAs
in patient blood in real-time, these small non-coding RNA molecules may be a new promising class of potential noninvasive biomarkers. Screening of these liquid biopsies may provide information on target molecules of microRNAs
and aberrant signaling pathways that can be blocked by a chosen targeted therapy. Consequently, therapyassociated modulations may facilitate treatment decisions. The present study shows the potential clinical use of
exosomal microRNAs as diagnostic and prognostic tumor markers. The emerging role of exosomes as a mediator
of cell-to-cell signaling, to transfer microRNAs between cells, is also discussed.
P 216
Screening for epigenetic tumor markers circulating in blood: pitfalls and way out
Evgeny Morozkin1, Elena Rykova2, Anna Bondar2, Tatyana Skvortsova2, Ivan Zaporozhchenko2, Alexander
Kurilshikov2, N.P. Krasnova3, E.S. Polovnikov3, O.A. Pashkovskaya3, E.A. Pokushalov3, Anastasia
Ponomaryova4, Nadezhda Cherdyntseva4, Pavel Laktionov1
1
Institute of Chemical Biology and Fundamental Medicine, Sb Ras; Research Institute of Blood Circulation
Pathology, Ministry of Health
2
3
Research Institute of Blood Circulation Pathology, Ministry of Health
4
Cancer Research Institute, Sb Rams
Epigenetic biomarkers predominantly tumor-specific aberrantly methylated DNA (amDNA) and microRNAs (miRNAs)
circulating as cell-free NA (cfNA) in blood represent markers with the highest potential for cancer diagnostics.
amDNA is more easily and efficiently detected in blood plasma/serum in presence of large excess of wild type
DNA compared with mutated DNA. miRNAs are much more stable than coding mRNA and are present in blood in
detectable amounts. However still only few cfNA-based diagnostics detecting epigenetic markers have come to the
market.
To overcome the obvious drawbacks of both cfNA two approaches are to be utilized:
• Biased comprising selection of markers valid for tumor tissues, elaboration of the demands for inclusion/
exclusion, development of PCR with absolute specificity and high sensitivity, increasing of cfNA concentration
in the analytical sample.
• Unbiased comprising search for potential markers in cfDNA/RNA using modern methods including MPS/
98
microarray with subsequent bioinformatics data analysis, formulation of the requirements to analytical systems
based on MPS data, development and validation of the diagnostics.
We used both approaches for development of lung and prostate cancer (LC, PC) diagnostics. Biased approach
based on sorting of list of markers provide sensitivity of LC diagnostics 89,2% and specificity 97%. Unbiased
approach included locus-specific MPS of 2 aberrantly methylated genes enable to obtain 95% specificity and 100%
sensitivity of PC diagnostics.
Selection of miRNA markers by miRCURY LNA microRNA PCR Serum/Plasma Panel (Exiqon Ltd) from primary lung
cancer versus healthy patients enable us to create 2 miRNA based qRT-TaqMan PCR diagnostics demonstrating
66% sensitivity and 89% specificity.
P 218
A novel technology for enrichment of cell free DNA. Factors of influence for the successful extraction
Heidi Schwarzenbach1, Bettina Steinbach1, Vipul Patel2, Magdalena Grunt2, Timo Hillebrand2
1
2
Aj Innsucreen GmbH
Circulating cell free DNA (ccfDNA) in the bloodstream derived from a tumor will play an important role in cancer
diagnostics and for cancer patient monitoring. Furthermore, circulating DNA plays a significant important role in
prenatal screening. We describe a novel and simple technology for efficient enrichment and extraction of ccfDNA
from up to 10 ml of sample. Important for isolation of ccfDNA are the pre-analytical treatments prior to an extraction.
We describe here factors which are from influence on the nucleic acids isolation, like the differences between serum
and plasma, the impact of storage time and temperature of the blood sample before extraction, the influence of
the blood collection system and other. Further we compare different methods of DNA concentration measurement
based on various principles.
99
Abstracts of Industrial Session
Droplet Digital PCR: Ally in Precision Cancer Diagnosis and Treatment
George Karlin-Neumann
Bio-Rad’s QX100/200 Droplet Digital PCRTM (ddPCRTM) system is extremely well-suited to the task of detecting
and quantifying known actionable biomarkers in cell-free DNA: it is not only highly sensitive and specific, providing
absolute quantification without need for a standard curve, but importantly, it is also agile, robust and economical. It
offers quick turnaround time (<1d) with simple analysis, requires minimal patient sample manipulation and displays
tolerance to PCR inhibitors. The large number of wet-lab validated rare mutation detection (RMD) and CNV assays
and multiplex RMD kits available, and the new automated droplet generator (AutoDG), enable any user to attain
expert levels of performance. It has become widely adopted in >1000 labs globally with cancer applications and
liquid biopsy studies in the forefront.
Recent studies with the QX100/200 system demonstrate that it can detect and quantify cell-free tumor DNA (ctDNA)
in the blood to accurately genotype advanced colorectal cancer (CRC) patients (Siravegna et al, 2015) and even
early-stage breast cancer patients with very low ctDNA levels (Beaver et al, 2014); to assess tumor response to
targeted anti-EGFR therapy in lung cancer and to detect disease progression due to primary or acquired resistance
markers (T790M) up to 4 months before radiographic detection (Oxnard et al, 2014); to help adjust therapy in
response to occult disease in melanoma (Abdel-Wahab et al, 2014) and tumor evolution in CRC (Siravegna et
al, 2015); and to address the challenges of tumor heterogeneity for the early detection of disease recurrence by
monitoring for the presence of personalized structural variants in CRC (Reinert et al, 2015) and breast cancer
(Olsson et al, 2015). Examples will be illustrated in the talk.
Nucleosomics®- translating epigenetic biomarkers into clinical diagnostics
M. Herzog*1, E. Josseaux1, D. Pamart1, Mark Eccleston1, J. Micallef1, H. Neilson2, R. Anderson3, R. Louis4
1
Belgian Volition, Centre Technologique, Namur, Belgium
2
Department of Surgical Gastroenterology 360,Hvidovre Hospital, University of Copenhagen, Hvidovre,
Denmark
3
Department of Surgery, Clinical Sciences Lund, Lund University, Skåne University Hospital, Lund,
Sweden
4
Pneumology Department, Centre Hospitalier Universitaire, Liège, Belgium
*Corresponding author: [email protected]
Background: Nucleosomics® combines cutting edge epigenetic profiling with a simple low cost immunoassay
technology to improve clinical diagnosis of cancer.
Circulating cell-free DNA of diagnostic relevance in cancer has been shown be similar in size to nucleosome bound
DNA. A proportion of circulating nucleosomes also contain tissue specific DNA mutations confirming their tumor
chromatin origin. In addition, genome-wide epigenetic histone modifications have been identified in cancer tissue
(histo-oncoproteins).
Volition has developed global (as opposed to gene specific) epigenetic profiling of cell free nucleosomes as a
potentially powerful clinical platform for accessible, affordable and accurate diagnosis of a range of cancers.
We present preliminary data from a major validation trial in colorectal cancer together with pilot data in prostate,
pancreatic and lung cancer studies.
Methodology: Specific nucleosome associated histone modifications, histone variants and DNA modification levels
in circulating nucleosomes were evaluated in patient and control serum by ELISA (NuQ®). Assay combinations with
100
optimal discriminations were identified using a linear discrimination algorithm.
Results: Data from the first phase of a 4800 cohort clinical trial have demonstrated test performance that exceeds
both traditional stool testing and blood based methylated Septin 9 in detection of colorectal cancer (84% sensitivity at
78% specificity) as well as polyps (60% sensitivity). Pilot results show equivalent levels of performance for sensitivity
and specificity in Prostate cancer (80%/70%), Lung cancer (77%/92%) and Pancreatic cancer (84/%/92%)
Conclusions: Whilst total levels of cell free nucleosomes can be elevated by inflammation, infectious disease as
well as cancer, thus limiting diagnostic utility, profiling of global levels of epigenetic modifications in nucleosomes
could provide disease specific diagnostic information.
“GATCLiquid: The molecular X-Ray of the 21st century”
Tobias Paprotka
GATC Biotech AG
As a pioneer in using circulating nucleic acids in plasma or serum (CNAPS) for diagnostic purposes, GATC Biotech
has played a key role in the commercialisation of CNAPS analysis for non-invasive prenatal testing. With years
of experience in extracting and sequencing CNAPS, GATC Biotech is well prepared to enter the field of oncology
diagnostics.
Currently, GATC Biotech is collaborating with renowned research institutes to help establish liquid biopsy solutions.
One such project, EpiFemCare, is devoted to establishing a blood-based test for early stage breast and ovarian
cancer detection. In another project together with the German Cancer Research Center, GATC Biotech is investigating
actionable mutations in CNAPS from cancer patients in order to guide treatment decisions.
Liquid biopsies have the potential to replace traditional invasive diagnostics methods that are often ineffective or
nearly impossible to perform. The development of GATC Biotech’s liquid biopsy diagnostics, GATCLiquid, is aimed
at improving treatment monitoring and early cancer diagnostics. By improving the outcome for cancer patients,
liquid biopsy can ultimately become the X-ray of the 21st century.
From Sample to Insight: QIAGEN´s Approach Pioneering the Liquid Biopsy Revolution
Michael Kazinski
Qiagen GmbH
Liquid Biopsy has the potential to transform healthcare and biomedical research. The presentation will give an
overview on QIAGEN’s approach to provide integrated solutions for isolation of circulating biomarkers and their
analysis in research and diagnostic applications. The presentation will cover QIAGEN’s solutions ranging from
sample isolation and stabilization, nucleic acid purification, PCR and NGS based analysis up to bioinformatics
analysis and interpretation. QIAGEN’s solutions have proven clinical utility and the presentation will give examples
on how minimally invasive approaches provide relevant insights for researchers and clinicians.
101
Personal Genome Diagnostics (PGDx) – turning The Promise of Personalized Medicine into Reality
N.N.
Personal Genome Diagnostics
New developments in cancer treatment including the development of personalized cancer therapy, non-invasive
cancer diagnosis and monitoring, and early detection are requiring the development and delivery of new NGS
based testing options.
PGDx was founded to turn the promise of personalized medicine into reality. PGDx research is focused on novel
technological approaches including the detection and analysis of circulating nucleic acids in plasma. The company is
deploying these solutions for both research and clinical needs. Now PGDx is developing and globally implementing
a product solution to enable both tissue and plasma analysis in molecular pathology and research laboratories
102
Author Index
Adleff, Vilmos 31, 89
Akers, Johnny 20, 42
Albus, Miriam 29, 78
Alekseeva, Anna 29, 32, 80, 96
Allen, Peter J. 31, 89
Allen, Stephanie 21, 45
Alvarez-Gómez, Sara 28, 78
Alyas, Sofia 23, 59
Amit, Hadas 30, 84
Anagnostou, Valsamo 31, 89
Anderson, R. 100
Anekella, Barathi 21, 46
Angenendt, Philipp 23, 60
Antonenko, Oksana 29, 79
Arita, Tomohiro 26-28, 68, 70-71, 73-74
Armisen-Garrido, Javier 30, 84
Asslaber, Daniela 21, 48
Aucamp, Janine 10, 20, 23, 32, 41, 56, 94
Auer, Martina 21, 49
Azad, Nilo 24, 63
Babjuk, Marek 31, 94
Bächmann, Sibylle 23, 60
Bagrowski, Michal 27, 69
Baird, Richard 26, 65
Barker, Colin 30, 84
Barlett, Bjarne 24, 63
Barrett, J. Carl 26, 65
Barrett, Michael T. 31, 89
Baryakin, Dmitri 26-27, 67, 70
Bedi, Atul 22, 51
Behren, Andreas 23, 58
Belic, Jelena 21, 49
Beller, Katharina 31, 91
Bellido-Diaz, Maria Luz 28, 74
Bender, Klaus 22, 54
Bernal, Carmen 28, 74
Bespalov, Vladimir 20, 42-43
Besse, Benjamin 29, 80
Beyer, Julia 30, 85
Bianco, Katherine 21, 46
Biggs, Heather 24, 62
Boeck, Stefan 23, 60
Bondar, Anna 15, 24, 30, 32, 61, 86, 98
Bonerigo, Sarah 24, 63
Bonnet, Joachim 31, 93
Bootz, Friedrich 24, 62
Bork, Ines 30, 85
Boustred, Christopher 20, 44
Brackenridge, Anna 22, 53
Bradford, James 30, 84
Brady, Ben 21, 48
Brahmer, Alexandra 29, 78
Brahmer, Julie 24, 63
Brait, Mariana 22, 51
Brenner, Eugeniy 26, 67
Brenton, James D. 24, 62
Brindle, Kevin 24, 61
Brisuda, Antonin 31, 94
Bronkhorst, Abel 10, 13-14, 20, 23, 32, 41, 56, 94
Browner, Ilene 24, 63
Bryan, Richard T. 23, 59
Bryzgalov, Leonid 29, 79
Bryzgunova, Olga 29, 82
Bustamante-Aragones, Ana 11, 21, 46
Caldas, Carlos 24, 62
Callahan, Jason 21, 48
Cameron, Daniel 23, 58
Capoun, Otakar 31, 94
Carr, T. Hedley 26, 65
Cebon, Jonathan 23, 58
Chang, Chia Lin 28, 76
Chang, Chia Yu 28, 76
Chang, Xiaofei 22, 51
Chen, Clark 20, 42
Chen, Wenhan 23, 56
Cheng, K.K. 23, 59
Cheng, Po Jen 28, 76
Cherdyntseva, Nadezhda 27, 30, 32, 72, 88, 98
Cherepanova, Anna 32, 95
Chikova, Elena 27, 70
Chitty, Lyn 20, 44
Chorostowska-Wynimko, Joanna 27, 72
Christoffel, Gabriele 14, 22, 55
Christophi, Christopher 23, 58
Cleary, Siobhan 21, 45
Clokie, Samuel 21, 45
Coin, Lachlan 23, 56
Cole, Trevor 21, 45
Constantin, Tudor 26, 65
Corbacho, Javier Garcia 26, 65
Court, Samantha 21, 45
Cox, Angela 30, 84
Cullinane, Carleen 21, 48
Davies, Patrick 22, 50
103
Dawson, Mark A. 21, 49
Dawson, Sarah-Jane 21, 24, 48-49, 62
de Vos, Luka 16, 24, 62
Deichmann, Eva Ricarda 22, 54
Demko, Zachary 26, 65
Deshmukh, Nayneeta 23, 59
Despotovic, Milenko 22, 50
Dettmann, Nadine 31, 93
Devall, Adam J. 23, 59
Diaz, Inga Medina 29, 81
Diaz, Luis 12-13, 24, 31, 38, 63, 89
Diehl, Frank 14, 23, 26, 29, 60, 67, 81
Dietrich, Dimo 13, 24, 30, 62, 85
Distler, J. 27, 69
Do, Hongdo 15, 23, 58
Dobrodeev, Aleksey 30, 88
Dobrovic, Alexander 15, 23, 58
Donas, Marta 21, 46
Dor, Yuval 22, 32, 54, 97
Dougherty, Brian 26, 65
Drebin, Jeffrey A. 31, 89
Drury, Suzanne 11, 20, 44
Du Manoir, Stanislas 30, 88
du Plessis, Lissinda 23, 56
Duzhak, Tatyana 32, 96
Eccleston, Mark 13-14, 100
Edgeworth, Bernice 30, 84
Edwards, Samantha 20, 44
Egle, Alexander 21, 48
Eiji, Uchida 30, 87
El Messaoudi, Safia 30, 88
Enderle, Daniel 22, 55
Ershova, Elizaveta 32, 96
Eshleman, James R. 31, 89
Etheridge, Alton 20, 43
Fahem, Abdelaziz 29, 80
Falchook, Gerald S. 31, 90
Fassunke, Jana 31, 91
Fendrich, Volker 22, 54
Ferguson, Kaltin 23, 56
Ferro, Marta 20, 45
Fertig, Elana 22, 51
Filippova, Julia 31, 92
Fischer, Bernd 28, 76
Fleischhacker, Michael 28, 30, 75, 85
Forshew, Tim 29, 80
Foster, Joseph M. 23, 59
104
Fredebohm, Johannes 26, 29, 67, 81
Friese, Bianca 29, 81
Frietsch, Manuel 31, 91
Ftouni, Sarah 21, 49
Fu, Siqing 31, 90
Fujita, Yuuji 28, 74
Fujiwara, Hitoshi 27, 70, 73
Galas, David 20, 43
Gale, Davina 24, 26, 29, 61-62, 65, 80
Ganesamoorthy, Devika 15, 23, 56
García-Fernández, Noelia 26, 28, 66, 74, 78
Garlick, Russell K. 21, 46
Geigl, Jochen 21, 49
Gelinas, Laura 22, 50
Gezer, Ugur 10, 12, 21, 47
Giacca, Mauro 22, 54
Gibson, Naomi 20, 44
Gillet, Brigitte 30, 88
Giraldez, Maria 10, 20, 43
Glaser, Benjamin 22, 32, 54, 97
Gnanapragasam, Vincent 29, 83
Godfrey, Tony 23, 57
Golan, Talia 22, 54
Gómez-Bravo, Miguel Angel 28, 74, 78
Gos, Monika 27, 72
Gotkine, Marc 32, 97
Greil, Richard 21, 48
Griffiths, Mike 21, 23, 45, 59
Grigorieva, Alina 29, 82
Groemminger, Sebastian 31, 91
Grölz, Daniel 31, 93
Grompe, Markus 22, 54
Grunt, Magdalena 32, 99
Grybel, Katarzyna 28, 76
Guerrero, Juan Miguel 20, 26, 28, 45, 66, 74, 78
Gürke, Jacqueline 28, 76
Gyorki, David 21, 48
Haas, Michael 23, 60
Hanus, Tomas 31, 94
Harkins, Seth 11, 21, 46
Harrington, Elizabeth 26, 65
Hatzimihalis, Athena 21, 48
Heese, Franziska 31, 93
Heinemann, Volker 23, 60
Heitzer, Ellen 12, 14-15, 21, 49
Heller, Michael 10, 13, 20, 22, 42, 52
Helmig, Susanne 10, 14, 22, 29, 54, 78
Herzog, M. 100
Hewitt, Julie 21, 45
Hicks, Rodney 21, 48
Hill, Matthew 26, 65
Hill, Melissa 20, 44
Hillebrand, Timo 32, 99
Hoang, Bin 26, 65
Hodgkin, Charlotte 24, 62
Hofmann, Wera 31, 91, 93
Holdenrieder, Stefan 6, 12, 14-15, 21, 23, 47, 60
Holtrup, Frank 26, 29, 67, 81
Hong, David 31, 90
Hoon, Dave 12, 15, 37
Hoque, Mohammad O. 22, 51
Horinek, Ales 28, 31, 75, 94
Howarth, Karen 29, 80
Hrbacek, Jan 31, 94
Hruban, Carolyn A. 31, 89
Hruban, Ralph H. 31, 89
Huang, Helen J. 31, 90
Hubert, Ayala 22, 54
Hudson, Christopher 23, 58
Hufnagl, Clemens 12, 21, 48
Hunter, Tane A. 21, 49
Ibsen, Stuart 10, 20, 22, 42, 52
Ichikawa, Daisuke 26-30, 68, 70-71, 73-74, 82, 86
Ikoma, Hisashi 26-27, 30, 68, 70, 86
Imamura, Taisuke 26-27, 29-30, 68, 70-71, 73, 82, 86
Iwai, Takuma 30, 87
Izumchenko, Evgeny 22, 51
Jackson, Jennifer 15, 23, 57
James, Nicholas D. 23, 59
Janku ,Filip 31, 90
Jenkins, Lucy 20, 44
Jiménez-Arriscado, Pilar 20, 26, 28, 45, 66, 74, 78
Johansen, Julia S. 31, 89
John, Thomas 23, 58
Jones, Linda 24, 62
Jones, Sian 22, 31, 51, 89
Josseaux, E. 100
Kabilov, Marsel 24, 61
Kagohara, Luciane T. 22, 51
Kameneva, Larisa 29, 32, 80, 96
Karalliedde, Janaka 28, 77
Karlin-Neumann, George 13, 31, 90, 100
Kasahara, Kazuo 26, 68
Kato, Mamoru 23, 59
Kawaguchi, Tsutomu 26-27, 29-30, 68, 73, 82, 86
Kazinski, Michael 13, 101
Kee, Bryan K. 31, 90
Kee, Damien 21, 48
Keskin, Metin 21, 47
Kimura, Hideharu 26, 68
Kirkizlar, Eser 26, 65
Kirushina, Natalia 31-32, 92, 96
Kitano, Shiro 30, 87
Kiuchi, Jun 26-27, 29-30, 68, 70-71, 73, 82, 86
Klimstra, David S. 31, 89
Knelangen, Julia M. 28, 76
Koba, Hayato 26, 68
Koch, Marina 21, 49
Komatsu, Shuhei 26-30, 68, 70-71, 73-74, 82, 86
König, T. 27, 69
Konigshofer, Yves 21, 46
Konishi, Hirotaka 26-28, 68, 70-71, 73-74
Kopetz, Scott 31, 90
Korabecna, Marie 28, 31, 75, 94
Kostyuk, Svetlana 29, 32, 80, 96
Kosuga, Toshiyuki 27, 71
Koval, Olga 31, 92
Kozlov, Vadim 26-27, 67, 70
Kozlov, Vladimir 29, 79
Krasnova, N.P. 32, 98
Kristiansen, Glen 24, 62
Krzyzanowski, Paul 23, 57
Ku, Alice T. 21, 46
Kubiak, Thomas 29, 78
Kuligina, Elena 26-27, 31, 67, 70, 92
Kupis, Wlodzimierz 27, 72
Kurilshikov, Alexander 24, 26, 32, 61, 67, 98
Kuriu, Yoshiaki 27, 70
Lacroix, Ludovic 29, 80
Laktionov, Pavel 24, 27, 29-32, 61, 72, 79, 82, 86, 88, 92,
95-96, 98
Lam, Enid 21, 49
Langfort, Renata 27, 72
Lavon, Iris 22, 54
Lawrence, Eloise 13, 22, 53
Lawson, Andrew 29, 80
Leary, Rebecca J. 31, 89
Lee, Da Xian 28, 76
Leisse, Annette 24, 62
Lekchnov, Evgeniy 29, 82
Li, Qing Kay 31, 89
105
Liebenberg, Volker 30, 85
Linehan, David C. 31, 89
Lo, Dennis 6, 11, 35
Lo, Kitty 20, 44
Löber, Ann-Kathrin 26, 29, 67, 81
Lorda-Sanchez, Isabel 21, 46
Louis, R. 100
Luthra, Raja 31, 90
MacDonald, Fiona 21, 45
Macher, Hada C. 11, 20, 26, 28, 45, 66, 74, 78
Magenheim, Judith 22, 32, 54, 97
Mair, Richard 24, 61
Maitra, Anirban 31, 89
Makino, Yoichi 32, 97
Maldonado, Erica 29, 81
Marass, Francesco 24, 26, 29, 62, 65, 83
Marchionni, Luigi 22, 51
Marciniak Wright, Jennifer 22, 52
Mares, Jaroslav 31, 94
Marinaki, Anthony 22, 28, 53, 77
Marziali, Andre 12, 22, 50
Mason, Sarah 20, 44
Massie, Charlie 29, 83
Mathonnet, Muriel 30, 88
McArthur, Grant 21, 48
McGowan, Barbara 22, 53
McKay, Fiona 20, 44
McMullan, Dominic J. 23, 59
Mehnert, Daniel 26, 29, 67, 81
Meißnitzer, Thomas 21, 48
Melchardt, Thomas 21, 48
Meric-Bernstam, Funda 31, 90
Micallef, J. 100
Mitchell, Paul 23, 58
Miyamae, Mahito 26-27, 29-30, 68, 70-71, 73, 82, 86
Moik, Martin 21, 48
Molania, Ramyar 23, 58
Molinero, Patrocinio 20, 28, 45, 74, 78
Mollevi, Caroline 30, 88
Morimura, Ryo 26-27, 30, 68, 70, 86
Morozkin, Evgeny 24, 27, 29-30, 32, 61, 72, 79, 82, 86, 88, 98
Morris, James 24, 26, 29, 62, 65, 83
Moss, Joshua 22, 32, 54, 97
Mouliere, Florent 13, 16, 24, 26, 30, 61, 65, 88
Murayama, Yasutoshi 27, 70
Murone, Carmel 23, 58
Murphy, Derek 31, 89
106
Naing, Aung 31, 90
Nakamura, Hiromi 23, 59
Nakanishi, Masayoshi 27, 70
Nakayama, Masato 30, 32, 87, 97
Neilson, H. 100
Neiman, Daniel 22, 32, 54, 97
Neureiter, Daniel 21, 48
Newton, Antony 13
Nishikawa, Shingo 26, 68
Noe, Johannes 14
Noerholm, Mikkel 22, 55
Noguerol, Pilar 20, 45
Nouaille, Michelle 30, 88
Nushtaeva, Anna 31, 92
O’Dwyer, Peter J. 31, 89
O’Sullivan, Brendan 23, 59
Ochiai, Toshiya 26, 68
Ohashi, Takuma 26-27, 29-30, 68, 70-71, 73, 82, 86
Okajima, Wataru 26-27, 29-30, 68, 70-71, 73, 82, 86
Okamoto, Kazuma 26-27, 68, 70-71, 73
Orlowski, Tadeusz 27, 72
Ormanns, Steffen 23, 60
Osipov, Ivan 30, 86
Otsuji, Eigo 26-30, 68, 70-71, 73-74, 82, 86
Oumie, Assa 23, 59
Özgür, Emre 21, 47
Pacey, Simon 26, 65
Palit, Tanuka 28, 77
Pamart, D. 100
Papenfuss, Tony 21, 23, 48, 58
Paprotka, Tobias 13-14, 101
Parkinson, Christine 24, 62
Parks, Michael 10-11, 21, 45
Parpart-Li, Sonya 16, 24, 31, 63, 89
Pashkovskaya, O.A. 32, 98
Pastor, Brice 30, 88
Patel, Keval 29, 83
Patel, Vipul 32, 99
Pazourkova, Eva 28, 31, 75, 94
Pel, Joel 22, 50
Pelham, Robert 26, 65
Pendzialek, Mareike 11, 28, 76
Perez, Esperanza 29, 80
Perikles, Simon 22, 29, 54, 78
Perlado-Marina, Sara 21, 46
Petrone, Pasquale 21, 49
Pezet, Denis 30, 88
Pfaff, Katharina 31, 93
Phallen, Jillian 31, 89
Piha-Paul, Sarina Anne 31, 90
Piskorz, Anna 24, 62
Plagnol, Vincent 24, 29, 62, 80
Podgornaya, Olga 20, 43
Pokushalov, E.A. 32, 98
Polovnikov, E.S. 32, 98
Ponomaryova, Anastasia 27, 30, 32, 72, 88, 98
Pospisilova, Sarka 28, 31, 75, 94
Pretorius, Piet 20, 23, 32, 41, 56, 94
Prinz, Ina 23, 60
Pugh, Michelle 29, 80
Puvirajasinghe, Clinda 20, 44
Radsak, Markus 22, 54
Raleigh, Jeanette 21, 48
Rehbein, Grit 28, 75
Remon, Jordi 29, 80
Richter, Vladimir 26-27, 31, 67, 70, 92
Risberg, Bente 24, 62
Robertson, Alan 23, 56
Rodriguez de Alba, Marta 21, 46
Rosenfeld, Nitzan 12, 15, 24, 26, 29, 39, 61-62, 65, 80, 83
Ross, Carina 23, 60
Roszkowski-Sliz, Kazimierz 27, 72
Rubio, Amalia 20, 26, 28, 45, 66, 74, 78
Rudzinski, Piotr 27, 72
Ruiz-ValdepenasMartindeAlmagro, Andrea 24, 62
Ryabchikova, Elena 29, 82
Ryan, Amy 24, 63
Rykova, Elena 13, 27, 29-30, 32, 72, 79, 88, 98
Sachse, Matthias 31, 91
Sanchez, Cynhia 30, 88
Sandhu, Shahneen 21, 48
Santos, Anne Navarrete 28, 76
Sausen, Mark 24, 31, 63, 89
Savelyeva, Anna 27, 70
Schäfer, Christian 14
Scharpf, Rob 31, 89
Schiffel, Florian 29, 81
Schindler, Maria 28, 76
Schlegel, A. 27, 69
Schlumpberger, Martin 22, 55
Schmeier-Jürchott, Anna 22, 54
Schmidt, Bernd 2, 4, 6, 28, 30, 75, 85
Schmitt, Karin 30, 84
Schmitz, Dagmar 11, 36
Schröck, Andreas 24, 62
Schwarzenbach, Heidi 6, 12-13, 22, 32, 53, 98-99
Semenov, Dmitry 26-27, 31, 67, 70, 92
Serdukov, Danil 32, 96
Sergeeva, Vasilina 32, 96
Seymour, John F. 21, 49
Shackleton, Mark 21, 48
Shelton, Dawne 31, 90
Shemer, Ruth 22, 32, 54, 97
Shibata, Tatsuhiro 23, 59
Shiozaki, Atsushi 26-27, 68, 70-71, 73
Shoda, Katsutoshi 28, 74
Sidransky, David 12, 22, 51
Sigurjonsson, Styrmir 26, 65
Simpson, Peter 23, 56
Sinha, Devbarna 12, 21, 49
Sizikov, Aleksey 29, 79
Skog, Johan 22, 55
Skvortsova, Tatyana 27, 29-30, 32, 72, 82, 88, 98
Smalley, Sarah 29, 80
Smirnova, Tatiana 29, 32, 80, 96
Sninsky, John 30, 84
Sone, Takashi 26, 68
Soukup, Viktor 31, 94
Spalding, Kirsty 22, 54
Speicher, Michael 21, 49
Spink, Karen G. 23, 59
Spitzer, Karolin 22, 55
Sprenger-Haussels, Markus 22, 55
Ståhlberg, Anders 23, 57
Stein, Lincoln 23, 57
Steinbach, Bettina 32, 99
Steininger, Philipp 21, 48
Stepanov, Grigory 31, 92
Stumm, Markus 31, 93
Suarez-Artacho, Gonzalo 28, 74, 78
Svobodova, Iveta 28, 31, 75, 94
Swaminathan, Ramasamyiyer 22, 28, 53, 77
Swenerton, Ryan 26, 65
Szpechcinski, Adam 15, 27, 72
Takai, Erina 15, 23, 59
Tam, Constantine 21, 49
Tamkovich, Svetlana 10, 31-32, 92, 96
Taniere, Phillipe 23, 59
Taniguchi, Hiroki 26-27, 30, 68, 70, 86
Taylor, Fiona 30, 84
Teare, M. Dawn 30, 84
107
Tetzner, R. 27, 69
Tewari, Muneesh 20, 43
Thapa, Bibhusal 23, 58
Theillet, Charles 30, 88
Thierry, Alain R. 10, 14, 30, 34, 88
Thompson, Craig B. 31, 89
Togneri, Fiona 15, 23, 59
Tomson, Farol L. 21, 46
Totoki, Yasushi 23, 59
Trujillo Tiebas, Maria José 21, 46
Trujillo, Elena 26, 28, 66, 78
Tsao, Simon 23, 58
Tsimberidou, Apostolia M. 31, 90
Tsui, Dana 16, 24, 29, 61-62, 83
Tsujiura, Masahiro 27, 71
Tug, Suzan 22, 29, 54, 78
Tutanov, Oleg 32, 96
Tuzikov, Sergei 30, 88
Ullius, Andrea 31, 93
Ulz, Peter 21, 49
Vaknin-Dembinsky, Adi 22, 54
Van Dyk, Etresia 23, 56
VanRahden, Vanessa 26, 67
Vasilyeva, Irina 10, 20, 42-43
Vasques, Fabiana Ramos 23, 59
Veiko, Natalia 29, 32, 80, 96
Velculescu, Victor E. 24, 31, 63, 89
Vergara, Ismael 21, 48
Vlassov, Valentin 24, 27, 29-32, 61, 72, 79, 82, 86, 88, 92, 95-96
Von Hoff, Daniel D. 31, 89
Voss, Thorsten 31, 93
Voytsitskiy, Vladimir 24, 30, 61, 86
Wagner, Eva 22, 54
Wallach, Elise 21, 49
Wang, Kai 20, 43
Ward, Douglas G. 23, 59
Watts, Colin 24, 61
Wayham, Nicholas 26, 65
Weiland, Johanna 29, 81
Weiss, G. 27, 69
Weiss, Jonathan 23, 58
Weiss, Lukas 21, 48
Wentzel, Jaco 23, 56
Werman, Roni 14, 22, 32, 54, 97
Westra, William H. 22, 51
Wheler, Jennifer J. 31, 90
White, James R. 31, 89
108
Wei, Shen 30, 84
Williams, Denise 21, 45
Witkowski, Tom 23, 58
Wojtowicz, Paula 23, 59
Wolf, Alexander 31, 91
Woll, Penella 30, 84
Wong, Stephen 12, 21, 48-49
Woodward, Robert 30, 84
Wright, Jennifer 20, 42
Yachida, Shinichi 23, 59
Yamada, Takeshi 30, 87
Ychou, Marc 30, 88
Yeh, Paul 21, 49
Yoneda, Taro 26, 68
Yörüker, Ebru E. 21, 47
Zahn, Daniela 29, 78
Zaleska, Jolanta 27, 72, 105
Zaporozhchenko, Ivan 27, 29-30, 32, 72, 82, 86, 88, 95, 98
Zaripov, Marat 24, 29-30, 61, 82, 86
Zavyalov, Alexander 30, 88
Zeegers, Maurice P. 23, 59
Zemmour, Hai 22, 32, 54, 97
Zick, Aviad 22, 54
Zimmermann, Bernhard 26, 65
Zimmermann, Tim 22, 54
Zinkin, Valery 20, 42

Scientific Program

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