ESTRO 36 Abstract Book

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S525 ESTRO 36 2017
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the 3D reconstructs to study radiation effects on the
immune system.
PO-0958 Radiogenomics: role of non-coding RNA genes
in increased radiotherapy sensitivity
L. Duran-Lozano1, V. Reyes2, M. Mollà2, M.J. Fuentes-
Raspall3, M. Altabas2 , T. Ramón y Cajal4, A. Barnadas4, O.
Diez1,5, J. Giralt2, S. Gutiérrez-Enríquez1
1 Vall d'Hebron Institute of Oncology-VHIO, Oncogenetics
Group, Barcelona, Spain
2 Vall d'Hebron University Hospital, Department of
Radiation Oncology, Barcelona, Spain
3 Hospital de la Santa Creu i Sant Pau, Department of
Radiation Oncology, Barcelona, Spain
4 Hospital de la Santa Creu i Sant Pau, Medical Oncology
Department, Barcelona, Spain
5 Vall d'Hebron University Hospital, Area of Clinical and
Molecular Genetics, Barcelona, Spain
Purpose or Objective
Breast cancer (BC) is the first cause of cancer-related
mortality of Spanish women and most common cancer in
women worldwide. It is frequently treated with
radiotherapy (RT), which can cause early and late side-
effects that impact negatively on quality-of-life of cancer
survivors. MicroRNAs and long non-coding RNAs (lncRNAs)
modulate key cellular pathways in response to radiation.
Single nucleotide polymorphisms (SNPs) in these two types
of non-coding RNAs can alter their function and
consequently modify the expression of genes that
regulate, affecting the respective biological activities. As
part of a long-term ongoing study, our aims were to test
genetic association of SNPs in microRNAs and lncRNAs with
late radiotherapy-induced toxicity and to characterize the
expression of lncRNAs in blood cells of BC treated
patients. Our final goal is to discover new genetic
endpoints to predict individuals with increased
susceptibility to radiotherapy side effects.
Material and Methods
DNA samples and clinical data were collected from 198
prospectively and 72 retrospectively recruited BC patients
treated with RT in two hospitals. All patients were
followed at least between two and six years after RT. 34
SNPs in microRNAs and lncRNA genes related to radiation
response were genotyped using iPLEX® Gold with
MassArray Agena Bioscience (Sequenom). RNA was
obtained from blood before and after radiotherapy of 19
BC patients from the prospective cohort. Eight lncRNAs
(FAS-AS1, MALAT1, TP53TG1, HOTAIR, PANDA, MEG3,
ANRIL and LINC00467) involved in radiation cell response
were assessed by RT-PCR, agarose gels and direct
sequencing. A semiquantitative capillary electrophoresis
of fluorescent amplicons was performed to estimate the
proportion of total transcripts.
Results
The first analysis showed an association of overall long
term toxicity after radiotherapy with rs4559081 A/A
genotype of LINC00336 (OR=3.91 95%CI = 1.34-11.37), and
grade ≥ 2 late radiation skin toxicity (fibrosis or
telangiectasias) with rs17762938 A/C or CC of PCAT1
(OR=2.63 95% CI=1.00-6.86) and rs2910164 C/G of miR-
146a (OR=0.27 95%CI = 0.008-0.94). The expression and
presence of different isoforms of all lncRNAs evaluated,
except ANRIL and HOTAIR, were observed in blood cells.
After radiotherapy the level of MEG3 and PANDA
expression increased.
Conclusion
The potential genetic association of the lncRNAs
LINC00336 and PCAT1, and microRNA miR-146a with
radiotherapy-induced late toxicity needs to be confirmed
in larger breast cancer cohorts. The expression of lncRNAs
can be a biomarker of radiotherapy response measurable
in blood.
Funding: This research was supported by a grant [FIS
05/2181] from ‘‘Fondo de Investigación Sanitaria,
Instituto de Salud Carlos III, Ministerio Español de
Economía y Competitividad”.
PO-0959 REQUITE: Radiation Induced Lymphocyte
Apoptosis assay as a predictor for radiotherapy side
effects
C. Talbot1, A. Appanvel2, A. Botma2, T. Rancati3, A.
Webb1, D. Azria4, T. Burr5, J. Chang-Claude2, C.
Herskind 6, D. De Ruysscher7, R. Elliott8, S. Gutiérrez
Enríquez 9, P. Lambin7, B. Rosenstein10, T. Rattay11, A.
Vega12, F. Wenz6, R. Valdagni3, C. West8
1University of Leicester, Department of Genetics,
Leicester, United Kingdom
2German Cancer Research Centre DKFZ, Genetic
Epidemiology Unit, Heidelberg, Germany
3Fondazione IRCCS Istituto Nazionale dei Tumori,
Prostate Cancer Program, Milan, Italy
4University of Montpellier, Institut du cancer de
Montpellier, Montpellier, France
5Source Bioscience, R&D, Nottingham, United Kingdom
6Heidelberg University, Department of Radiation
Oncology, Mannheim, Germany
7Maastricht University Medical Center, Department of
Radiation Oncology MAASTRO clinic, Maastricht, The
Netherlands
8University of Manchester, Institute of Cancer Sciences,
Manchester, United Kingdom
9Vall d’Hebron Institute of Oncology-VHIO, Radiation
Oncology Department, Barcelona, Spain
10Mount Sinai School of Medicine, Department of
Radiation Oncology, New York, USA
11University of Leicester, Department of Cancer Studies,
Leicester, United Kingdom
12Universidade de Santiago de Compostela, Centro de
Investigación Biomédica en Red de Enfermedades Raras
CIBERER, Santiago de Compostela, Spain
Purpose or Objective
Recently the first replicated genetic associations for
radiotherapy-induced adverse reactions were reported.
The European Union funded REQUITE consortium aims to
validate known predictors of adverse reactions to develop
clinically useful tools. One such predictor is low levels of
radiation induced lymphocyte apoptosis which has
previously been found in patients experiencing increased
rates of late radiation induced toxicity.
Material and Methods
REQUITE is a multi-centre, observational study. Enrolment
was open for two and a half years in nine centres (eight in
Europe and one in the United States), with another two
years of follow-up still ongoing. The primary endpoints are
change in breast appearance at two years (breast), rectal
bleeding at two years (prostate) and breathlessness at 12
months (lung). Work Package 4 involves validation of
biomarkers. This includes genetic polymorphisms and the
radiation induced lymphocyte apoptosis assay (RILA). The
RILA was carried out in three of the European centres using
a standardised protocol which had been verified with inter
lab testing; it assesses percentage radiation induced
apoptosis in lymphocytes, detected by flow cytometry, 48
hours after ex-vivo irradiation of whole blood.
Results
More than 4300 patients have been enrolled in REQUITE.
1322 samples have been analysed using the apoptosis
assay. The levels of apoptosis 48 hours after ex-vivo
irradiation increase over baseline in a range from 2.4% to
62.4%, confirming large inter-patient variability. In the
Leicester cohort mean RILA is higher in the prostate
patients compared to the breast patients (24.9% vs 20.3%;
p=0.004). Analysis of predictive value for acute toxicity is
being carried out.
Conclusion
Variation in percentage of lymphocyte apoptosis is in
keeping with previous studies. This large scale prospective
observational study will be the largest to date to assess