Bellone, M.

Benigni, F.

Briganti, A.

Calandrino, R.

Capelli, A.

Cozzarini, C.

Di Muzio, N.

Fiorino, C.

Mondino, A.

Perani, L.

Sacco, V.

Sini, C.

Spinelli, A.

Venturini, M.

Viale, A.

Elsevier - Publisher Connector 

S968                                                                                                                                                  ESTRO 35 2016 
_____________________________________________________________________________________________________ 
 
Purpose or Objective: The standard treatment regimen of 
patients with primary glioblastoma multiforme (PGBM) 
consists of neurosurgery, radio- and chemotherapy. Despite 
this multimodal treatment the overall survival of patients 
with PGBM is still approximately 15 months.  
The stress-inducible heat shock protein 70 (Hsp70) 
contributes to tumor cell survival and is associated with poor 
prognosis, metastasis and therapy resistance. Therefore, the 
aim of this study is to analyze Hsp70 in PGBM tumor samples 
as a future prognostic biomarker and possible therapy target. 
 
Material and Methods: Formalin fixed paraffin embedded 
(FFPE) sections of 44 human PGBM patients (isocitrate 
dehydrogenase wildtype) were analyzed by 
immunohistochemistry for Hsp70 (cmHsp70.1, IgG1, 
multimmune GmbH, Munich, Germany). Taking the intensity 
of Hsp70 staining into account, quantitative expression 
analysis of tumor cells with stained cytoplasm was 
performed. Two categories of Hsp70 staining were defined: 
Up to 40% and more than 40% positive tumor cells within the 
tumor regions. The Hsp70 immunoreactivity was correlated 
with the survival of the patients using the Cox regression 
analysis. 
 
Results: Preliminary data show that the median survival of 
PGBM patients can be predicted by the Hsp70 
immunoreactivity of the tumor cells. Regression analysis 
showed that patients with Hsp70 expression of more than 40% 
have a higher risk of disease progression with a hazard ratio 
of 2.59 (p= 0.045). 
 
 
 
Hsp70 expression in FFPE IHC section (Hsp70 positive tumor 
cells are brown) 
 
Conclusion: These data provide the first evidence that Hsp70 
expression in FFPE sections of PGBM patients is associated 
with disease progression. Moreover, measuring Hsp70 in FFPE 
sections of PGBM patients before radiotherapy treatment may 
be used as biomarker for the success of the therapy. The 
independency of Hsp70 expression and O6-methylguanin-DNA 
methyltransferase (MGMT) is currently under investigation. 
 
EP-2052  
Expression of molecular biomarkers in wound drainage 
fluids: a pilot study in head and neck cancer 
M. Sottili
1University of Florence, Experimental and Clinical Biomedical 
Sciences, Firenze, Italy 
1, M. Mangoni1, P. Bonomo1, A. Deganello2, A. 
Javarone2, T. Gualtieri2, I. Desideri1, M. Loi1, I. Meattini1, F. 
Paiar1, L. Livi1 
2University of Florence, Department of Surgery and 
Translational Medicine, Firenze, Italy 
 
Purpose or Objective: In recent years, it has been suggested 
that wound drainage fluids (WDF) of patients operated for 
head and neck squamous cell carcinoma (HNSCC) may be 
characterized by molecular biomarkers with potential 
prognostic and predictive value. The detection of adverse 
features in the early perioperative setting could possibly lead 
to a refinement of current adjuvant treatments in high-risk 
patients. The purpose of our study is to report on the 
feasibility and preliminary results of a pilot prospective study 
on WDF analysis in HNSCC. 
 
Material and Methods: 14 consecutive surgically resected 
HNSCCs were studied. WDF were collected 1 day and 3 days 
after surgery from the cancer operative bed (COB). In 5 
patients, WDF was collected also from free flap donor site 
(FFDS). WDF were centrifuged for 15 min at 3500 rpm, then 
divided in aliquots and stored at -80°C until analysis. The aim 
of the present study was to evaluate the expression of factors 
involved in tumor growth and progression 1 day and 3 days 
after surgery. EGF, VEGF, SDF-1 and osteopontin levels were 
measured in WDF using commercially available enzyme-linked 
immunosorbent assay (ELISA) kits. Each sample was analyzed 
in duplicates and then averaged for a mean value. Quality 
control pools of low, normal, or high concentrations for all 
parameters were included in each assay. The obtained results 
were expressed as pg/ml (EGF, VEGF, SDF-1 ) or ng/ml 
(osteopontin).  
 
Results: A mean of 67 ml of WDF from COB and 51 ml from 
FFDS at day 1, and 42 ml from COB and 20 ml from FFDS at 
day 3 were collected for each patient. EGF expression was 
significantly reduced from day 1 to day 3 after surgery both 
in COB (140.7±10.55 vs. 45.12±13.35 pg/ml, p<0.001) and in 
FFDS (157.1±4.08 vs. 95.59±32.89 pg/ml, p<0.05). VEGF 
expression increased from 1 to 3 day both in COB 
(1277.74±64.54 vs. 1616.81±151.4 pg/ml, p<0.05) and in FFDS 
(1227.51±19.39 vs. 1400.25±77.66 pg/ml, p<0.05). The 
expression of markers of invasiveness and metastasis 
increased from day 1 to day 3: osteopontin expression 
significantly increased from day 1 to day 3 both in COB 
(9.97±0.68 vs. 16.87±0.56 ng/ml, p<0.001) and in FFDS 
(9.51±1.23 vs. 15.83±1.08 ng/ml, p<0.01). SDF-1 expression 
increased from day 1 to day 3 in COB (646.8±65.39 vs. 
1084.22±148.8 pg/ml, p<0.05). No differences in SDF-1 
expression were detected in FFDS. 
 
Conclusion: Preliminary data from pilot study evidenced that 
microenvironment induced by surgery favors residual tumor 
cell proliferation and progression. Growth factor expression is 
higher early after surgery (24 hours); on the contrary, 
expression of markers of invasiveness and metastasis 
increases from day 1 to day 3 after surgery. The few samples 
of WDF from FFDS do not allow to evidence differences of 
biomarkers expression between COB and FFDS. 
 
EP-2053  
In-vivo imaging of rat leukocytes redistribution after pelvic 
irradiation 
F. Benigni
1San Raffaele Scientific Institute, URI/Urology, Milan, Italy 
1, C. Cozzarini2, C. Sini3, A. Spinelli4, M. Venturini5, 
L. Perani6, V. Sacco2, A. Viale2, A. Capelli2, A. Mondino7, A. 
Briganti1, M. Bellone8, C. Fiorino9, R. Calandrino9, N. Di 
Muzio2 
2San Raffaele Scientific Institute, Radiotherapy, Milan, Italy 
3Fondazione Centro San Raffaele, Medical Physics, Milan, 
Italy 
4San Raffaele Scientific Institute, Experimental Imaging 
Center- Medical Physics, Milan, Italy 
5San Raffaele Scientific Institute, Radiology, Milan, Italy 
6San Raffaele Scientific Institute, Experimental Imaging 
Center, Milan, Italy 
7San Raffaele Scientific Institute, Lymphocyte Activation 
Unit- Immunology- Transplantation and Infectious Disease 
Division, Milan, Italy 
8San Raffaele Scientific Institute, Unit of Cellular 
Immunology, Milan, Italy 
9San Raffaele Scientific Institute, Medical Physics, Milan, 
Italy 
 
Purpose or Objective: Hematologic toxicity and in particular 
decrease in the peripheral blood leukocyte and lymphocyte 
count is an important side effect of pelvic radiation therapy. 
The aim of this study was to investigate the kinetic of the 
redistribution of circulating leukocytes after pelvic 
irradiation in a animal model with in vivo non-invasive 
imaging modality. 
ESTRO 35 2016                                                                                                                                                    S969 
________________________________________________________________________________ 
Material and Methods: A rat model was used to investigate a 
possible selective accumulation of circulating lymphocites to 
specific anatomical districts after radiation treatment 
focused to the urinary bladder. Eight Fisher rats were 
adoptively transferred with 4x107 VivoTag-750-labelled 
syngeneic primary splenocytes at two hours before the 
bladder irradiation. Two of eight rats were used as controls. 
Animals were transurethrally catheterized to allow contrast 
agent instillation. A kV cone beam computed tomography 
(CBCT) was acquired for each rat, to precisely deliver 6 MeV 
monofraction photon field. Rats were divided into three 
groups (n=2/group) receiving different levels of dose: 15, 20 
and 25 Gy. A bolus thickness equal to 1cm was positioned on 
the rat skin surface in the pelvic region. Ultrasound images of 
the pelvic region were acquired at baseline, at 2, 4 and 6 
days after irradiation to monitor thickness variations of the 
bladder wall tissue. In vivo fluorescent imaging was used to 
evaluated accumulation sites of labelled leukocytes. 
 
Results: A significant increase in the bladder wall thickness 
was found 4 days after irradiation in animals treated with a 
dose equal to 25 Gy. A fluorescent signal, secondary to 
labelled splenocytes accumulation, emerged in the liver and 
lymph nodes of all adoptively transferred rats, 2 and 6 days 
after irradiation, as expected. A modest specific signal (30% 
increase) at the bladder level resulted only in two animals 
receiving the higher dose (Figure 1.a), when compared to the 
non-irradiated (Figure 1.b). No specific fluorescent signal was 
detected at the bladder levels in animals treated with 20 and 
15 Gy. 
 
 
 
Conclusion: The relocalization of peripheral leukocytes in 
the damaged tissue depends on the radiation dosage and it 
may be evaluated by means of a non-invasive imaging 
technique. Further analyses are currently ongoing. 
 
EP-2054  
Expression of DNA-PK in squamous cell lung cancer has 
gender differences and depends on smoking 
J. Jaal1, L. Mägi1, T. Jõgi2, M. Kase3, A. Minajeva1, V. Markus2, 
T. Vooder4, R. Roosipuu5, J. Jaal
1University of Tartu, Faculty of Medicine, Tartu, Estonia 
2 
2Tartu University Hospital, Dept of Radiotherapy and 
Oncological Therapy, Tartu, Estonia 
3East-Tallinn Central Hospital, Cancer Centre, Tallinn, 
Estonia 
4Centre for Thoracic Diseases, Dept of Thoracic Surgery, 
Ruhr, Germany 
5Tartu University Hospital, Dept of Pathology, Tartu, Estonia 
 
Purpose or Objective: Lung cancer is one of the most 
frequent and deadly types of cancer in Europe. Several 
aspects of non-small cell lung cancer (nsclc) in men and 
women continue to indicate potential male-female 
differences. Among these, higher treatment responses to 
current therapies in women are supposed, since women have 
better prognosis in any stage of the disease. In most stages of 
nsclc cytotoxic anti-cancer therapy (radiotherapy, 
chemotherapy) is used. It is known that treatment efficacy of 
cytotoxic anti-cancer therapy depends on tumor DNA-repair. 
Therefore, the aim of this study was to evaluate gender 
differences in the expression of DNA repair enzyme DNA 
protein kinase (DNA-PK). 
Material and Methods: Surgically excised nsclc tissues 
(n=111, 50 adenocarcinomas, 61 squamous cell carcinomas) 
were examined for DNA-PK expression. After 
immunohistochemistry, the staining intensity of DNA-PK was 
quantified using an arbitrary score ranging from 0 (no 
staining) to 3 (strong signal). Also, the proportion (%) of DNA-
PK positive (DNA-PK+) tumor cells was determined. All 
parameters were examined by 2 independent researchers in 
10 randomly chosen microscopic fields (magnification x40). 
 
Results: Immunohistochemical parameters were examined by 
2 independent researchers whose results were in good 
accordance (p<0.0005). Staining intensities of DNA-PK and 
the proportion of DNA-PK+ tumor cells varied, being in the 
whole nsclc group 2.4±0.4 (mean±SD) and 86.3±9.1% 
respectively. There were no significant gender differences in 
adenocarcinoma. However, we detected significant 
differences among nsclc patients with squamous cell 
carcinoma. Both, DNA-PK staining intensity and the 
proportion of DNA-PK+ tumor cells were significantly higher 
in men than in women, 2.5±0.3 and 86.3±8.8% vs 2.1±0.6 and 
79.6±11.9% respectively (DNA-PK intensity: p<0.01; DNA-PK+ 
proportion: p=0,03). Additionally, we found that in squamous 
cell carcinoma, the expression of DNA-PK depends on smoking 
and pack-years. There was a correlation between pack-years 
and DNA-PK intensity (p=0.04), as well as between pack-years 
and the proportion of DNA-PK+ tumor cells (p=0.04). 
 
Conclusion: Expression of DNA-PK in squamous cell lung 
cancer has gender differences and depends on smoking. 
Significantly lower expression of tumor DNA-PK was found in 
women with this histological subtype of nsclc. Latter might 
be one of the reasons why cytotoxic anti-cancer therapy is 
more efficacious in women than in men. In further studies, 
the combination of DNA repair inhibitors and cytotoxic anti-
cancer therapy should be tested. 
 
EP-2055  
Fibro-inflammatory circulating proteins as biomarkers for 
response in locally advanced rectal cancer 
P. Bulens
1University Hospital Leuven, Radiation Oncology, Leuven, 
Belgium 
1,2, A. Debucquoy2, I. Joye1,2, O. De Wever3, A. 
Wolthuis4, A. D'Hoore4, E. Van Cutsem5, V. Vandecaveye6, X. 
Sagaert7, C. Deroose8, O. Gevaert2,9, K. Haustermans1,2 
2University of Leuven, Oncology, Leuven, Belgium 


EP-2053: In-vivo imaging of rat leukocytes redistribution after pelvic irradiation 

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EP-2053: In-vivo imaging of rat leukocytes redistribution after pelvic irradiation

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