Belka, C.

Braselmann, H.

Gimenez Aznar, I.

Hess, J.

Jendrossek, V.

Klein, D.

Lauber, K.

Michna, A.

Orth, M.

Schneider, L.

Schötz, U.

Schüttrumpf, L.

Unger, K.

Zangen, V.

Zitzelsberger, H.

Elsevier - Publisher Connector 

OC-0441: Genomic amplification of FancA in HNSCC: mechanisms of radioresistance and clinical relevance 

ESTRO 35 2016                                                                                                                                                    S205 
______________________________________________________________________________________________________ 
or p16 negative cells, indicating a differential role of p16 
protein expression depending on its localization. Strikingly, 
cells expressing nuclear p16 (p16-NLS) -although showing a 
similar level of gH2AX induction- were characterized with 
lower number RAD51 foci formation compared to cells 
expressing cytoplasmic p16 (p16-NES), suggesting an impaired 
HRR. 
 
Conclusion: Cellular p16 localization is an important factor 
for stratification of HNSCC patients with nuclear p16 
expression showing a superior predictive value for 
radiotherapy response. 
 
OC-0440  
Impact of chemokine receptor CXCR4 and its ligand SDF1 
expression on loco-regional control in HNSCC 
A. Menegakis
1University Hospital Tübingen Eberhard Karls University 
Tübingen, Radiation oncology, Tübingen, Germany 
1,2, C. De Colle1,3, D. Moennich1,2, F. Fend4, P.S. 
Mauz5, S. Welz1, I. Tinhofer6,7, V. Budach6,7, E. Gkika8,9, M. 
Stuschke8,9, P. Balermpas10,11, C. Roedel10,11, M. Avlar12,13, A.L. 
Grosu12,14, A. Abdollahi15,16,17,18,19, J. Debus15,17,18,19,20, C. 
Bayer21, C. Belka21,22, S. Pigorsch21,23, S.E. Combs21,23, M. 
Krause24,25,26, M. Baumann24,25,26, D. Zips1,2 
2German Cancer Research Center DKFZ- Heidelberg and 
German Cancer Consortium DKTK, Partner site Tuebingen, 
Tuebingen, Germany 
3Azienda Ospedaliero-Universitaria- Citta' della Salute e 
della Scienza di Torino- University of Turin, Radiation 
Oncology, Turin, Italy 
4University Hospital Tübingen Eberhard Karls University 
Tübingen, Pathology, Tübingen, Germany 
5University Hospital Tübingen Eberhard Karls University 
Tübingen, Otorhinolaryngology, Tübingen, Germany 
6German Cancer Research Center DKFZ- Heidelberg and 
German Cancer Consortium DKTK, Partner site Berlin, Berlin, 
Germany 
7Charité University Hospital- Berlin, Radiooncology and 
Radiotherapy, Berlin, Germany 
8German Cancer Research Center DKFZ- Heidelberg and 
German Cancer Consortium DKTK, Partner site Essen, Essen, 
Germany 
9Medical Faculty- University of Duisburg-Essen, 
Radiotherapy, Essen, Germany 
10German Cancer Research Center DKFZ- Heidelberg and 
German Cancer Consortium DKTK, Partner site Frankfurt, 
Frankfurt, Germany 
11Goethe-University Frankfurt, Radiotherapy and Oncology, 
Frankfurt, Germany 
12German Cancer Research Center DKFZ- Heidelberg and 
German Cancer Consortium DKTK, Partner site Freiburg, 
Freiburg, Germany 
13Clinical Study Section- University of Freiburg, Radiation 
Oncology, Freiburg, Germany 
14University of Freiburg, Radiation Oncology, Freiburg, 
Germany 
15German Cancer Research Center DKFZ- Heidelberg and 
German Cancer Consortium DKTK, Partner site Heidelberg, 
Heidelberg, Germany 
16University of Heidelberg Medical School and German Cancer 
Research Center DKFZ, Translational Radiation Oncology, 
Heidelberg, Germany 
17University of Heidelberg Medical School and German Cancer 
Research Center DKFZ, National Center for Tumor Diseases 
NCT, Heidelberg, Germany 
18Heidelberg Ion Therapy Center HIT- University of 
Heidelberg Medical School, Radiation Oncology, Heidelberg, 
Germany 
19National Center for Radiation Research in Oncology NCRO- 
University of Heidelberg Medical School and German Cancer 
Research Center DKFZ, Heidelberg Institute of Radiation 
Oncology HIRO, Heidelberg, Germany 
20University of Heidelberg Medical School and German Cancer 
Research Center DKFZ, Clinical Cooperation Unit Radiation 
Oncology, Heidelberg, Germany 
21German Cancer Research Center DKFZ- Heidelberg and 
German Cancer Consortium DKTK, Partner site Munich, 
Munich, Germany 
22Ludwig-Maximilians-Universität, Radiotherapy and 
Radiation Oncology, Munich, Germany 
23TechnischeUniversität München, Radiation Oncology, 
Munich, Germany 
24German Cancer Research Center DKFZ- Heidelberg and 
German Cancer Consortium DKTK, Partner site Dresden, 
Dresden, Germany 
25National Center for Radiation Research in Oncology- 
Faculty of Medicine and University Hospital Carl Gustav 
Carus- TechnischeUniversität Dresden- Helmholtz-Zentrum 
Dresden – Rossendorf, OncoRay, Dresden, Germany 
26Faculty of Medicine and University Hospital Carl Gustav 
Carus- TechnischeUniversität Dresden, Radiation Oncology, 
Dresden, Germany 
 
Purpose or Objective: To retrospectively assess the 
prognostic value of the potential biomarkers, i.e. chemokine 
receptor CXCR4, its ligand CXCL12 (SDF1), and nuclear EGFR 
expression in a cohort of 201 patients with locally advanced 
HNSCC. Patients were treated between 2005 and 2011 in 8 
German cancer centers, as part of a multicenter biomarker 
study of the German Cancer Consortium Radiation Oncology 
Group (DKTK-ROG). Experimental data and first clinical 
observations suggest that activation of CXCR4 and SDF1 
signaling pathway and nuclear location of EGFR are 
implicated in tumour cell proliferation, cellular survival, 
tumour progression, worse overall survival, metastasis and 
enhanced treatment resistance in different tumour types. 
 
Material and Methods: Patients with locally advanced SCC of 
the oral cavity, oropharynx and hypopharynx were treated 
with resection and adjuvant RT and Cisplatin-based CT. 
Tissue micro-arrays (TMAs) were generated from surgical 
specimens and evaluated for the expression of the 
biomarkers by immunofluorescence with a semi-quantative 
method, based on their cellular location (membranous, 
intracellular, nuclear), extent of expression on TMA area and 
staining intensity. The results of the biomarker analysis along 
with the clinical parameters were then correlated with the 
clinical outcome. 
 
Results: In univariate analysis, tumours with either SDF1 or 
CXCR4 intracellular overexpression displayed a significant 
negative correlation with loco-regional control (LCR) (HR: 
2.52, p=0.01 and HR: 1.96, p=0.05 respectively). No 
correlation was observed for the nuclear expression of EGFR 
(HR: 0.85, p= 0.67), membranous expression of SDF1 (p=0.73) 
or CXCR4 (p=0.38). Tumours with intracellular co-expression 
of both SDF1 and CXCR4 were significantly correlated with 
poor LRC (HR: 2.72, p=0.01). Previously published data from 
the same cohort, showed that absence of p16 (negative HPV 
status) was correlating with poor LRC. Importantly, increased 
expression of SDF1 or co-expression with CXCR4 could 
identify a group of patients with significantly worse outcome 
within the HPV negative group (p=0.01). Multivariate cox 
regression analysis including HPV status, tumour localisation, 
tumour volume and the respective biomarkers indicated a 
significant independent role of SDF1 (HR: 2.20, p=0.04) and 
co-expression with CXCR4 (HR: 2.19, p=0.05) on LRC. 
 
Conclusion: In summary, pre-treatment overexpression of 
CXCR4/SDF1 is an independent negative prognostic factor for 
the outcome of patients with locally advanced HNSCC who 
receive surgery and standard RT-CT. Further investigation in 
a cohort of patient receiving primary RT-CT and a 
prospective validation study is currently ongoing. 
SDF1/CXCR4 appears to be a promising biomarker for 
treatment individualization, in particular in HPV negative 
advanced HNSCC patients and supports strategies using 
drugable targets against this pathway to enhance efficacy of 
standard treatment. 
 
OC-0441  
Genomic amplification of FancA in HNSCC: mechanisms of 
radioresistance and clinical relevance 
J. Hess1,2, I. Gimenez Aznar1, A. Michna1, D. Klein3, U. 
Schötz4, M. Orth4, L. Schneider1,2, H. Braselmann1, L. 
S206                                                                                     ESTRO 35 2016 
 _____________________________________________________________________________________________________ 
Schüttrumpf4, V. Jendrossek3, C. Belka2,4, V. Zangen1, K. 
Unger1,2, H. Zitzelsberger1,2, K. Lauber2,4 
1Research Unit Radiation Cytogenetics, Helmholtz Zentrum 
Muenchen - German Research Center for Environmental 
Health, Neuherberg, Germany 
2Clinical Cooperation Group ‘Personalized Radiotherapy of 
Head and Neck Cancer’, Helmholtz Zentrum Muenchen - 
German Research Center for Environmental Health, 
Neuherberg, Germany 
3Department of Molecular Cell Biology, Institute of Cell 
Biology Cancer Research- Medical Faculty- University of 
Duisburg-Essen, Essen, Germany 
4Molecular Oncology, Department of Radiation Oncology- 
Ludwig-Maximilians-Universität München, Munich, Germany 
 
Purpose or Objective: Radio(chemo)therapy is a crucial 
treatment modality for head and neck squamous cell 
carcinoma (HNSCC). Radiotherapy resistance is a major 
reason for therapy failure and, therefore, predictive 
biomarkers for therapy response are urgently needed. DNA 
gains on chromosome 16q23-24 have been shown to be 
associated with genomic amplification of the FancA gene and 
to correlate with reduced progression-free survival of HNSCC 
patients after radiotherapy. This study aimed to analyze the 
effects of the potential predictive marker FancA on radiation 
sensitivity in vitro, to characterize the underlying molecular 
mechanisms, and to evaluate the clinical relevance in HNSCC. 
 
Material and Methods: We generated FancA overexpressing 
human oral keratinocytes (OKF6/FancA) and analyzed several 
endpoints upon irradiation. To identify signaling pathways 
involved in FancA-mediated resistance, global transcriptome 
analyses were performed after irradiation with 4 Gy or sham-
irradiation followed by pathway enrichment analysis and 
reconstruction of function interaction networks. The clinical 
relevance of the cytogenetic marker 16q23-24, the FancA 
gene and our in vitro results was analyzed in data of 113 
radiotherapy-treated patients from The Cancer Genome Atlas 
(TCGA) HNSCC cohort (Nature, 2015). 
 
Results: Overexpression of FancA resulted in enhanced 
survival after in vitro irradiation. Moreover, FancA 
overexpressing cells demonstrated accelerated DNA damage 
repair mechanisms paralleled by increased repair fidelity: 
enhanced p53 and p21 response, accelerated kinetics in the 
disappearance of γ-H2AX DNA damage repair foci, faster 
pATM translocation, reduced accumulation of chromosomal 
translocations, but no increase in FancD2 mono-
ubiquitinylation. Global mRNA expression analyses identified 
interferon signaling as a major candidate pathway, which was 
affected by FancA overexpression. Functional interaction 
networks of genes deregulated upon irradiation pointed to 
pathways exclusively involved in FancA-mediated 
radioresistance including the senescence-associated secretory 
phenotype (SASP). Increased levels of basal and irradiation-
induced cellular senescence accompanied by enforced SASP 
formation further support their potential involvement in 
FancA-mediated radiation resistance. The clinical relevance 
of our findings was validated in the data of 113 radiotherapy-
treated patients of the TCGA HNSCC cohort demonstrating 
the association of chromosomal gains on 16q24.3 with 
increased FancA mRNA expression levels and impaired overall 
survival. Furthermore, the translation of our in vitro model 
derived results into the HNSCC patient specimens revealed 
similar gene expression changes linked to FancA 
overexpression. 
 
Conclusion: Our data suggest an important role for FancA in 
cellular mechanisms of radioresistance in HNSCC. 
 
OC-0442  
Does miR-210 predict benefit from hypoxia modification in 
BCON randomised bladder cancer patients? 
C. West
1The University of Manchester, Christie Hospital, 
Manchester, United Kingdom 
1, J. Irlam-Jones2, A. Eustance2, H. Denley3, P. 
Hoskin4, A. Choudhury5 
2The University of Manchester, Translational Radiobiology 
Group, Manchester, United Kingdom 
3Central Manchester University Hospitals NHS Foundation 
Trust, Department of Histopathology, Manchester, United 
Kingdom 
4Mount Vernon Hospital, Cancer Centre, Northwood, United 
Kingdom 
5The Christie Hospital NHS Foundation Trust, Department of 
Clinical Oncology, Manchester, United Kingdom 
 
Purpose or Objective: The addition of hypoxia modifiers 
carbogen and nicotinamide (CON) to radiotherapy (RT) 
improved overall survival in bladder cancer patients enrolled 
in the BCON phase III clinical trial. We investigated whether 
the expression of miR-210 in the BCON patient samples 
reflects hypoxia and predicts benefit from hypoxia-
modification. 
 
Material and Methods: The retrospective study involved 183 
T1-T4b patients: 86 received RT+CON and 97 received RT 
alone. Formalin-fixed samples taken prior to radiotherapy 
were available and RNA extracted. Customised TaqMan plates 
were used to assess miR-210 expression using quantitative 
real-time PCR. Patients were classified as low miR-210 
(<median expression) or high miR-210 (≥ median). Data on 
other hypoxia biomarkers were available for comparison. 
 
Results: Patients with high miR-210 had a trend towards 
improved five-year OS with RT+CON (53.2%) compared with 
RT alone (37.8%; HR 1.68, 95% CI 0.95-2.95, P=0.08). No 
benefit was seen with low miR-210 (HR 1.02, 95% CI 0.58-
1.79, P=0.97). High expression of miR-210 was also associated 
with high HIF-1α protein (P=0.0008), CA9 protein (P=0.004), 
Glut-1 protein (P=0.02), expression of a 26-gene hypoxia 
signature (P=0.01), tumour necrosis (P=0.04) and concurrent 
pTis (P=0.03). 
 
Conclusion: High miR-210 expression may reflect tumour 
hypoxia and should be investigated further as a potential 
biomarker to identify bladder cancer patients who would 
benefit from hypoxia-modifying therapies. 
 
OC-0443  
Radiotherapy sensitivity in breast cancer is influenced by 
the DNA cytosine deaminase APOBEC3B 
P.N. Span
1Radboud University Medical Center, Department of 
Radiation Oncology, Nijmegen, The Netherlands 
1, A. Post1, J.W.M. Martens2, R.S. Harris3 
2Erasmus MC Cancer Institute, Department of Medical 
Oncology and Cancer Genomics Netherlands, Rotterdam, The 
Netherlands 
3University of Minnesota- Masonic Cancer Center, 
Department of Biochemistry- Molecular Biology- and 
Biophysics, Minneapolis, USA 
 
Purpose or Objective: The DNA cytosine deaminase APOBEC3 
proteins catalyze deamination of cytidines in single-stranded 
DNA, providing innate protection against retroviral 
replication. Recent studies have implicated APOBEC3B as a 
major source of mutation in breast cancer, suggesting a role 
for these enzymes in tumor initiation and/or progression. 
APOBEC3B expression levels were earlier found to correlate 
with poor outcomes for patients with estrogen receptor 
positive breast cancer, especially after Tamoxifen. Given its 
role in mutagenesis, we set out to assess whether APOBEC3B 
associates with radiosensitivity in breast cancer. 
 
Material and Methods: MCF7 breast cancer cells were 
cultured radioresistant by daily 2 Gy treatments or 
tamoxifen-resistant by continuous culturing in up to 10 uM 4-
OH-tamoxifen. The effect of irradiation on expression of 
APOBECs was assessed by RNAseq and qPCR in radiosensitive 
and radioresistant MCF7, and by qPCR in radioresistant MDA-
MB231 cells. Furthermore, we studied a retrospective cohort 
of 535 non-systemically treated breast cancer patients. The 
predictive power of APOBEC3B was assessed in patients that 
did or did not receive radiotherapy as part of their primary 
therapy. Next, we suppressed endogenous APOBEC3B in MCF-


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OC-0441: Genomic amplification of FancA in HNSCC: mechanisms of radioresistance and clinical relevance

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