A. Yaromina

PO-106 Unique and overlapping role of HIF proteins in radiation response and tumour cell metabolism

Introduction Hypoxia is a common feature of human solid tumours and caused by limited diffusion and structural aberrations of tumour vasculature. Tumour hypoxia in cancer is linked to worse outcome and treatment resistance. Hypoxic cells are also characterised by changes in their energy requirements and radiation response. Many of these adaptations are driven by the hypoxia-inducible transcription factors or HIFs. Mammalian cells encode for three HIF1–3 proteins which are O2 regulated. The similarities and differences between HIF proteins in regulating tumour cell metabolism and radiation response are still understudied. Material and methods Using gene-editing with the CRISPR/CAS9 system we generated isogenic series of cells lacking HIF1, HIF2 or both HIF1,2 in the H1299 non-small cell lung cancer cell line. We analysed the differences in hypoxia response on downstream target gene activation. In parallel, we analysed the upregulation of HIF protein expression under hypoxic conditions for each cellular model. Proliferative capacity and hypoxic tolerance were assessed for each cell line. We determined the metabolic consequences of silencing HIF by measuring oxygen consumption rate, extracellular acidification rate, extracellular pH and lipid droplet accumulation. We determined the radiation response of each cellular model under both normoxic and hypoxic conditions using clonogenic survival assays, gamma-H2AX staining and cell cycle analysis. Results and discussions We found that HIF2 compensates for the depletion of HIF1, but HIF1 is not upregulated upon HIF2 depletion. Both HIF1 and HIF2 proteins appear to be crucial for the upregulation of downstream HIF target genes such as CAIX, GLUT1 or TWIST. HIF depleted cells appear to be less glycolytic and at the same time trigger mitochondrial activity. The radiation response of the different cellular models shows a specific profile in terms of clonogenic survival and double-strand break formation in both normoxic and hypoxic conditions. Conclusion Taken together these genetically-modified cell models may help us to further define the radiation response of hypoxic tumour cells for therapeutic interventions.

Prognostic Role of Hypoxia-Inducible Factor-2α Tumor Cell Expression in Cancer Patients: A Meta-Analysis

Hypoxia-inducible factor-2α (HIF-2α) plays an important role in tumor progression and metastasis. A number of studies have evaluated the correlation between HIF-2α overexpression and clinical outcome in cancer patients but yielded inconsistent results. To comprehensively and quantitatively summarize the evidence on the capability of HIF-2α to predict the prognosis of cancer patients with solid tumors, a meta-analysis was carried out. Renal cell carcinoma (CC-RCC) was separately analyzed due to an alternative mechanism of regulation. Systematic literature searches were performed in PubMed and Embase databases for relevant original articles until February 2018. Forty-nine studies with 6,052 patients were included in this study. The pooled hazard ratios (HRs) with corresponding confidence intervals were calculated to assess the prognostic value of HIF-2α protein expression in tumor cells. The meta-analysis revealed strong significant negative associations between HIF-2α expression and five endpoints: overall survival [HRu2009=u20091.69, 95% confidence interval (95% CI) 1.39–2.06], disease-free survival (HRu2009=u20091.87, 95% CI 1.2–2.92), disease-specific survival (HRu2009=u20091.57, 95% CI 1.06–2.34), metastasis-free survival (HRu2009=u20092.67, 95% CI 1.32–5.38), and progression-free survival (HRu2009=u20092.18, 95% CI 1.25–3.78). Subgroup analyses revealed similar associations in the majority of tumor sites. Overall, these data demonstrate a negative prognostic role of HIF-2α in patients suffering from different types of solid tumors.

Synergistic effects of NOTCH/γ-secretase inhibition on chemotherapeutics and radiation combination treatments in non-small cell lung cancer cells

Background: Lung cancer is the leading cause of cancer death worldwide. More effective treatments are needed to increase durable responses and prolong patient survival. Standard of care treatment for patients with non-operable stage III-IV NSCLC is concurrent chemotherapy and radiation. An activated NOTCH signaling pathway is associated with poor outcome and treatment resistance in non-small cell lung cancer (NSCLC). NOTCH/γ-secretase inhibitors have been effective in controlling tumor growth in preclinical models but the therapeutic benefit of these inhibitors as monotherapy in patients has been limited so far. Because NOTCH signaling has been implicated in treatment resistance, we hypothesized that by combining NOTCH inhibitors with chemotherapy and radiotherapy this could result in an increased therapeutic effect. A direct comparison of the effects of NOTCH inhibition when combined with current treatment combinations for NSCLC is lacking. Methods: Using monolayer growth assays, we screened 101 FDA-approved drugs from the Cancer Therapy Evaluation Program alone, or combined with radiation, in the H1299 and H460 NSCLC cell lines to identify potent treatment interactions. Subsequently, using multicellular three-dimensional tumor spheroid assays, we tested a selection of drugs used in clinical practice for NSCLC patients, and combined these with a small molecule inhibitor, currently being tested in clinical trials, of the NOTCH pathway (BMS-906024) alone, or in combination with radiation, and measured specific spheroid growth delay (SSGD). Statistical significance was determined by one-way ANOVA with post-hoc Bonferroni correction, and synergism was assessed using two-way ANOVA. Results: Monolayer assays in H1299 and H460 suggest that 21 vs. 5% were synergistic, and 17 vs. 11% were additive chemoradiation interactions, respectively. In H1299 tumor spheroids, significant SSGD was obtained for cisplatin, etoposide, and crizotinib, which increased significantly after the addition of the NOTCH inhibitor BMS-906024 (but not for paclitaxel and pemetrexed), and especially in triple combination with radiation. Synergistic interactions were observed when BMS-906024 was combined with chemoradiation (cisplatin, paclitaxel, docetaxel, and crizotinib). Similar results were observed for H460 spheroids using paclitaxel or crizotinib in dual combination treatment with NOTCH inhibition and triple with radiation. Conclusions: Our findings point to novel synergistic combinations of NOTCH inhibition and chemoradiation that should be tested in NSCLC in vivo models for their ability to achieve an improved therapeutic ratio.

OC-0382: A novel concept to tumour targeting: inverse dose-painting or targeting the "Low uptake drug volume"

Results: Composite perfusion changes were associated with dose. Statistically significant dose-dependent reduction in regional perfusion was observed at 3, 6 and 12 months FU. Comparison of dose-response curves based on their slopes showed a dose-dependent reduction in perfusion at all time intervals (R2=0.8-0.9) except 1 month (R2=0.4). Relative perfusion loss per dose bin was 4% at 1 month, 14% at 3 months, 13% at 6 months and 21% at 12 months FU (Figure 1).

OC-0234: Radiotherapy and L19-IL2: perfect match for an abscopal effect with long-lasting memory

ESTRO 35 2016 _____________________________________________________________________________________________________ and/or increase the likelihood of radiation-induced toxicities. Prospective trials have shown that RTQA variations have a significant impact on the primary study end-point and could bias the analysis of the trial results[6]. A large prospective phase III (i.e. TROG 02.02) trial showed indisputably that poor radiotherapy resulted in suboptimal patient’s outcomes. Moreover, the impact of poor quality radiotherapy delivery exceeded greatly the benefit of chemotherapy, thus biasing the primary end-point of this study. This large Australian trial provided a contemporary benchmark that future studies will need to exceed. Other specific consideration for RTQA in trials includes, but is not limited to, education of the accruing sites in RT-trial guidelines, promotion of consistency between centers and estimation of inter-patient and interinstitutional variations. Additionally, global cooperation is essential in the environment of common and rare cancers alike, in order to be able to create sufficiently large patient data sets within a reasonable recruitment period. This cooperation is not without issues and recently the need to have harmonized RTQA procedures has been strongly advocated by the Global Harmonisation Group. Ensuring RT compliance with protocol guidelines involves however gradually more resources-intensive procedures which are also labor intensive and are not cost-neutral. This will consequentially have a significant impact on the overall study budget. There are suggestion that QA programs are however cost-effective. This financial investment is of paramount importance, as non-adherence to protocol-specified RT requirements in prospective trials is very frequent. The European Organisation for the Research and Treatment of Cancer (EORTC) Radiation Oncology Group started to implement RTQA strategies in the 1980s, including on how to write a protocol for RT trials, defining RTQA procedures (such as benchmark case, dummy run and complex treatment dosimetry checks), assuring prospective individual case review feasibility and implementing an electronic dataexchange platform.