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Highlights • Nitroglycerin didn’t improve overall survival of NSCLC patients. • The toxicity of combining nitroglycerin with standard treatment was mild. • Increased uptake of HX4 showed negative prognostic significance in NSCLC patients. • Tumor perfusion after nitroglycerin treatment did not correlate with outcome., Background Nitroglycerin is proposed as an agent to reduce tumour hypoxia by improving tumour perfusion. We investigated the potential of nitroglycerin as a radio-sensitizer in non-small cell lung cancer (NSCLC) and the potential of functional imaging for patient selection. Material and methods Trial NCT01210378 is a single arm phase II trial, designed to detect 15% improvement in 2-year overall survival (primary endpoint) in stage IB-IV NSCLC patients treated with radical (chemo-) radiotherapy and a Transiderm-Nitro 5 patch during radiotherapy. Patients underwent dynamic contrast-enhanced CTs (DCE-CT) and HX4 (hypoxia) PET/CTs before and after nitroglycerin. Secondary endpoints were progression-free survival, toxicity and the prognostic value of tumour perfusion/hypoxia at baseline and after nitroglycerin. Results The trial stopped after a futility analysis after 42 patients. At median follow-up of 41 months, two-year and median OS were 58% (95% CI: 44–78%) and 38 months (95% CI: 22–54 months), respectively. Nitroglycerin could not reduce tumour hypoxia. DCE-CT parameters did not correlate with OS, whereas hypoxic tumours had a worse OS (p = 0.029). Changes in high-uptake fraction of HX4 and tumour blood flow were negatively correlated (r = -0.650, p = 0.022). The heterogeneity in treatment modalities and patient characteristics combined with a small sample size made further subgroup analysis of survival results impossible. Toxicity related to nitroglyerin was limited to headache (17%) and hypotension (2.4%). Conclusion Nitroglycerin did not improve OS of NSCLC patients treated with (chemo-)radiotherapy. A general ability of nitroglycerin to reduce hypoxia was not shown.

Highlights • TH-302 combined with RT enhances tumor growth delay in esophageal cancer models. • This combination does not increase normal tissue toxicity on the short and long term. • The increased therapeutic index (1.38) paves the way for future clinical trials., Background and purpose Esophageal cancer incidence is increasing and is rarely curable. Hypoxic tumor areas cause resistance to conventional therapies, making them susceptible for treatment with hypoxia-activated prodrugs (HAPs). We investigated in vivo whether the HAP evofosfamide (TH-302) could increase the therapeutic ratio by sensitizing esophageal carcinomas to radiotherapy without increasing normal tissue toxicity. Materials and methods To assess therapeutic efficacy, growth of xenografted esophageal squamous cell (OE21) or adeno (OE19) carcinomas was monitored after treatment with TH-302 (50 mg/kg, QD5) and irradiation (sham or 10 Gy). Short- and long-term toxicity was assessed in a gut mucosa and lung fibrosis irradiation model, sensitive to acute and late radiation injury respectively. Mice were injected with TH-302 (50 mg/kg, QD5) and the abdominal area (sham, 8 or 10 Gy) or the upper part of the right lung (sham, 20 Gy) was irradiated. Damage to normal tissues was assessed 84 hours later by histology and blood plasma citrulline levels (gut) and for up to 1 year by non-invasive micro CT imaging (lung). Results The combination treatment of TH-302 with radiotherapy resulted in significant tumor growth delay in OE19 (P = 0.02) and OE21 (P = 0.03) carcinomas, compared to radiotherapy only. Irradiation resulted in a dose-dependent decrease of crypt survival (P

Highlights • Hypoxia-activated prodrugs have yielded promising results up to phase II trials. • Implementation of hypoxia-activated prodrugs in the clinic has not been successful. • Phase III clinical trials lack patient stratification based on tumor hypoxia status. • Stratification will decrease the number of patients needed and increase success. • Improvements in hypoxia-activated prodrug design can also increase success rates., Hypoxia-activated prodrugs (HAPs) are designed to specifically target the hypoxic cells of tumors, which are an important cause of treatment resistance to conventional therapies. Despite promising preclinical and clinical phase I and II results, the most important of which are described in this review, the implementation of hypoxia-activated prodrugs in the clinic has, so far, not been successful. The lack of stratification of patients based on tumor hypoxia status, which can vary widely, is sufficient to account for the failure of phase III trials. To fully exploit the potential of hypoxia-activated prodrugs, hypoxia stratification of patients is needed. Here, we propose a biomarker-stratified enriched Phase III study design in which only biomarker-positive (i.e. hypoxia-positive) patients are randomized between standard treatment and the combination of standard treatment with a hypoxia-activated prodrug. This implies the necessity of a Phase II study in which the biomarker or a combination of biomarkers will be evaluated. The total number of patients needed for both clinical studies will be far lower than in currently used randomize-all designs. In addition, we elaborate on the improvements in HAP design that are feasible to increase the treatment success rates.

Background and purpose: We tested a novel treatment approach combining (1) targeting radioresistant hypoxic tumour cells with the hypoxia-activated prodrug TH-302 and (2) inverse radiation dose painting to boost selectively non-hypoxic tumour sub-volumes having no/low drug uptake.Material and methods: F-18-HX4 hypoxia tracer uptake measured with a clinical PET/CT scanner was used as a surrogate of TH-302 activity in rhabdomyosarcomas growing in immunoc6mpetent rats. Low or high drug uptake volume (LDUV/HDUV) was defined as 40% of the GTV with the lowest or highest F-18-HX4 uptake, respectively. Two hours post TH-302/saline administration, animals received either single dose radiotherapy (RT) uniformly (15 or 18.5 Gy) or a dose-painted non-uniform radiation (15 Gy) with 50% higher dose to LDUV or HDUV (18.5 Gy). Treatment plans were created using Eclipse treatment planning system and radiation was delivered using VMAT. Tumour response was quantified as time to reach 3 times starting tumour volume.Results: Non-unifOrm RT boosting tumour sub-volume with low TH-302 uptake (LDUV) was superior to the same dose escalation to HDUV (p Conclusions: The results support targeted dose escalation to non-hypoxic tumour sub-volume with no/low activity of hypoxia-activated prodrugs. This strategy applies on average a lower radiation dose and is as effective as uniform dose escalation to the entire tumour. It could be applied to other type of drugs provided that their distribution can be imaged. 2017 The Author(s). Published by Elsevier Ireland Ltd.

Objective: Lung cancer is the deadliest cancer worldwide. To increase treatment potential for lung cancer, pre-clinical models that allow testing and follow up of clinically relevant treatment modalities are essential. Therefore, we developed a single-nodule-based orthotopic non-small cell lung cancer tumor model which can be monitored using multimodal non-invasive imaging to select the optimal image-guided radiation treatment plan.Methods: An orthotopic non-small cell lung cancer model in NMRI-nude mice was established to investigate the complementary information acquired from 80 kVp microcone-beam CT (micro-CBCT) and bioluminescence imaging (BLI) using different angles and filter settings. Different micro-CBCT-based radiation-delivery plans were evaluated based on their dose-volume histogram metrics of tumor and organs at risk to select the optimal treatment plan.Results: H1299 cell suspensions injected directly into the lung render exponentially growing single tumor nodules whose CBCT-based volume quantification strongly correlated with BLI-integrated intensity. Parallel-opposed single angle beam plans through a single lung are preferred for smaller tumors, whereas for larger tumors, plans that spread the radiation dose across healthy tissues are favored.Conclusions: Closely mimicking a clinical setting for lung cancer with highly advanced preclinical radiation treatment planning is possible in mice developing orthotopic lung tumors.Advances in knowledge: BLI and CBCT imaging of orthotopic lung tumors provide complementary information in a temporal manner. The optimal radiotherapy plan is tumor volume-dependent.

Background and purpose The central hypothesis of "radiomics" is that imaging features reflect tumor phenotype and genotype. Until now only correlative studies have been performed. The main objective of our study is to determine whether a causal relationship exists between genetic changes and image features. The secondary objective is to assess whether the combination with radiotherapy (RT) influences these image features. Material and methods HCT116 doxycycline (dox) inducible GADD34 cells were grown as xenografts in the flanks of NMRI-nu mice. GADD34 overexpression decreases hypoxic fraction. Radiomics analyses were performed on computed tomography images obtained at 40kVp and again at 80kVp for validation, before radiotherapy at a volume of 200mm 3 , 4days post RT (10Gy) and 500mm 3 . To select reproducible features test–retest experiments were performed at baseline. Results Gene induction and/or irradiation translated into significant changes in radiomics features. Post irradiation, 17 features for 40kVp and 9 features for 80kVp differed significantly between dox+ and dox− combined with RT. 8 and 4 of these features remained consistent for 40 and 80kVp, respectively. Conclusion Radiomics is able to identify early effects of changed gene expression combined with radiation treatment in tumors with similar volumes which are not visible to human eye.

Purpose: To establish a clinically applicable protocol for quantification of residual gamma H2AX foci in ex vivo irradiated tumour samples and to apply this method in a proof-of-concept feasibility study to patient-derived tumour specimens. Material and methods: Evaluation of gamma H2AX foci formation and disappearance in excised FaDu tumour specimens after (a) different incubation times in culture medium, 4 Gy irradiation and fixation after 24 h (cell recovery), (b) 10 h medium incubation, 4 Gy irradiation and fixation after various time points (double strand break repair kinetics), and (c) 10 h medium incubation, irradiation with graded single radiation doses and fixation after 24 h (dose-response). The optimised protocol was applied to patient-derived samples of seminoma, prostate cancer and glioblastoma multiforme. Results: Post excision or biopsy, tumour tissues showed stable radiation-induced gamma H2AX foci values in oxic cells after >6 h of recovery in medium. Kinetics of foci disappearance indicated a plateau of residual foci after >12 h following ex vivo irradiation. Fitting the dose-response of residual gamma H2AX foci yielded slopes comparable with in situ irradiation of FaDu tumours. Significant differences in the slopes of ex vivo irradiated patient-derived tumour samples were found. Conclusion: A novel clinically applicable method to quantify residual gamma H2AX foci in ex vivo irradiated tumour samples was established. The first clinical results suggest that this method allows to distinguish between radiosensitive and radioresistant tumour types. These findings support further translational evaluation of this assay to individualise radiation therapy.

Background and purpose Recently, we have shown that radiotherapy (RT) combined with the immunocytokine L19-IL2 can induce long-lasting antitumour effects, dependent on ED-B expression and infiltration of cytotoxic T cells. On the other hand, in certain tumours, IL2 treatment can trigger a natural killer cell (NK) immune response. The aim of this study is to investigate the therapeutic effect of our combination therapy in the ED-B positive F9 teratocarcinoma model, lacking MHCI expression and known to be dependent on NK immune responses. Material and methods In syngeneic F9 tumour bearing 129/FvHsd mice tumour growth delay was evaluated after local tumour irradiation (10 Gy) combined with systemic administration of L19-IL2. Immunological responses were investigated using flow cytometry. Results Tumour growth delay of L19-IL2 can be further improved by a single dose of RT administered before immunotherapy, but not during immunotherapy. Furthermore, treatment of L19-IL2 favours a NK response and lacks cytotoxic T cell tumour infiltrating immune cells, which may be explained by the absence of MHCI expression. Conclusion An additive effect can be detected when the NK dependent F9 tumour model is treated with radiotherapy and L19-IL2 and therefore this combination could be useful in the absence of tumoural MHCI expression.

Purpose To apply our previously published residual ex vivo γH2AX foci method to patient-derived tumour specimens covering a spectrum of tumour-types with known differences in radiation response. In addition, the data were used to simulate different experimental scenarios to simplify the method. Materials and methods Evaluation of residual γH2AX foci in well-oxygenated tumour areas of ex vivo irradiated patient-derived tumour specimens with graded single doses was performed. Immediately after surgical resection, the samples were cultivated for 24h in culture medium prior to irradiation and fixed 24h post-irradiation for γH2AX foci evaluation. Specimens from a total of 25 patients (including 7 previously published) with 10 different tumour types were included. Results Linear dose response of residual γH2AX foci was observed in all specimens with highly variable slopes among different tumour types ranging from 0.69 (95% CI: 1.14–0.24) to 3.26 (95% CI: 4.13–2.62) for chondrosarcomas (radioresistant) and classical seminomas (radiosensitive) respectively. Simulations suggest that omitting dose levels might simplify the assay without compromising robustness. Conclusion Here we confirm clinical feasibility of the assay. The slopes of the residual foci number are well in line with the expected differences in radio-responsiveness of different tumour types implying that intrinsic radiation sensitivity contributes to tumour radiation response. Thus, this assay has a promising potential for individualized radiation therapy and prospective validation is warranted.

In his commentary, Claus Garbe described our study on the combination of radiotherapy (RT) and the immunocytokine L19-IL2 in the F9 fibrosarcoma model (1) and discussed our work in a checkpoint inhibitor related context. The author is positioning interleukin-2 (IL2) based immunotherapies in the so-called ‘age of the checkpoint inhibition’ and concluded that the role of IL2 and/or L19-IL2 should preferentially be examined in patients not responding to checkpoint blockade, since these patients are not able to develop specific cytotoxic T cell responses. Therefore, IL2 based immunotherapeutic approaches might circumvent this problem by activating the innate immune response against the tumor (2).

Objective To investigate the effect of radiation dose-escalation on local control in hypoxic versus non-hypoxic hypoxic tumours defined using [ 18 F]fluoromisonidazole ([ 18 F]FMISO) PET. Materials and methods FaDu human squamous cell carcinomas (hSCCs) growing subcutaneously in nude mice were subjected to [ 18 F]FMISO PET before irradiation with single doses of 25 or 35Gy under normal blood flow conditions. [ 18 F]FMISO hypoxic volume (HV) and maximum standardised uptake value (SUV max ) were used to quantify tracer uptake. The animals were followed up for at least 120days after irradiation. The endpoints were permanent local tumour control and time to local recurrence. Results HV varied between 38 and 291mm 3 (median 105mm 3 ). Non-hypoxic tumours (HV below median) showed significantly better local control after single dose irradiation than hypoxic tumours (HV above median) ( p =0.046). The effect of dose was significant and not different in non-hypoxic and in hypoxic tumours (HR=0.82 [95% CI 0.71; 0.93], p =0.002 and HR=0.86 [0.78; 0.95], p =0.001, respectively). Dose escalation resulted in an incremental increase of local tumour control from low-dose hypoxic, over low-dose non-hypoxic and high-dose hypoxic to high-dose non-hypoxic tumours. SUV max did not reveal significant association with local control at any dose level. Conclusions The negative effect of [ 18 F]FMISO HV on permanent local tumour control supports the prognostic value of the pre-treatment [ 18 F]FMISO HV. Making the assumption that variable [ 18 F]FMISO uptake in different FaDu tumours which all have the same genetic background may serve as an experimental model of intratumoural heterogeneity, the data support the concept of dose-escalation with inhomogeneous dose distribution based on pre-treatment [ 18 F]FMISO uptake. This result needs to be confirmed in other tumour models and using fractionated radiotherapy schedules.

Background and purpose Carbonic anhydrase IX (CAIX) plays an important role in pH regulation processes critical for tumor cell growth and metastasis. We hypothesize that a dual targeting bioreductive nitroimidazole based anti-CAIX sulfamide drug (DH348) will reduce tumor growth and sensitize tumors to irradiation in a CAIX dependent manner. Material and methods The effect of the dual targeting anti-CAIX (DH348) and its single targeting control drugs on extracellular acidification and radiosensitivity was examined in HT-29 colorectal carcinoma cells. Tumor growth and time to reach 4× start volume (T4×SV) was monitored for animals receiving DH348 (10 mg/kg) combined with tumor single dose irradiation (10 Gy). Results In vitro, DH348 reduced hypoxia-induced extracellular acidosis, but did not change hypoxic radiosensitivity. In vivo, DH348 monotherapy decreased tumor growth rate and sensitized tumors to radiation (enhancement ratio 1.50) without systemic toxicity only for CAIX expressing tumors. Conclusions A newly designed nitroimidazole and sulfamide dual targeting drug reduces hypoxic extracellular acidification, slows down tumor growth at nontoxic doses and sensitizes tumors to irradiation all in a CAIX dependent manner, suggesting no “off-target” effects. Our data therefore indicate the potential utility of a dual drug approach as a new strategy for tumor-specific targeting.

Purpose Expert review summarizing the overcome tumor cell hypoxia by treatment modification in radiation oncology. Methods An extensive literature search regarding various means of treatment modification was performed and key papers on those modifications were included in this review article. Results Based on the identified key papers the means to overcome hypoxia in radiation oncology were summarized in this review article, e.g., increasing levels of oxygen, combining radiotherapy with agents counteracting hypoxia, or modifying radiation treatment itself. Conclusions This review summarizes the results of preclinical and clinical studies counteracting hypoxia and highlights the measures that have found their way into clinical practice.

Purpose Previous studies have shown that the plasminogen activator inhibitor type-1 (PAI-1) and vascular endothelial growth factor (VEGF) are regulated by hypoxia and irradiation and are involved in neoangiogenesis. The aim of this study was to determine in vivo whether changes in PAI-1 and VEGF during fractionated irradiation could predict for radiation resistance. Methods and Materials Six xenografted tumor lines from human squamous cell carcinomas (HSCC) of the head and neck were irradiated with 0, 3, 5, 10, and 15 daily fractions of 2 Gy. The PAI-1 and VEGF antigen levels in tumor lysates were determined by enzyme-linked immunosorbent assay kits. The amounts of PAI-1 and VEGF were compared with the dose to cure 50% of tumors (TCD 50 ). Colocalization of PAI-1, pimonidazole (hypoxia), CD31 (endothelium), and Hoechst 33342 (perfusion) was examined by immunofluorescence. Results Human PAI-1 and VEGF (hVEGF) expression levels were induced by fractionated irradiation in UT-SCC-15, UT-SCC-14, and UT-SCC-5 tumors, and mouse VEGF (msVEGF) was induced only in UT-SCC-5 tumors. High hVEGF levels were significantly associated with radiation sensitivity after 5 fractions ( P =.021), and high msVEGF levels were significantly associated with radiation resistance after 10 fractions ( P =.007). PAI-1 staining was observed in the extracellular matrix, the cytoplasm of fibroblast-like stroma cells, and individual tumor cells at all doses of irradiation. Colocalization studies showed PAI-1 staining close to microvessels. Conclusions These results indicate that the concentration of tumor-specific and host-specific VEGF during fractionated irradiation could provide considerably divergent information for the outcome of radiation therapy.

Purpose There is conflicting evidence for whether the expression of epidermal growth factor receptor in human tumors can be used as a marker of radioresponse. Therefore, this association was studied in a systematic manner using squamous cell carcinoma (SCC) cell lines grown as cell cultures and xenografts. Methods and Materials The study was performed with 24 tumor cell lines of different tumor types, including 10 SCC lines, which were also investigated as xenografts on nude mice. Egfr gene dose and the length of CA-repeats in intron 1 were determined by polymerase chain reaction, protein expression in vitro by Western blot and in vivo by enzyme-linked immunosorbent assay, and radiosensitivity in vitro by colony formation. Data were correlated with previously published tumor control dose 50% data after fractionated irradiation of xenografts of the 10 SCC. Results EGFR protein expression varies considerably, with most tumor cell lines showing moderate and only few showing pronounced upregulation. EGFR upregulation could only be attributed to massive gene amplification in the latter. In the case of little or no amplification, in vitro EGFR expression correlated with both cellular and tumor radioresponse. In vivo EGFR expression did not show this correlation. Conclusions Local tumor control after the fractionated irradiation of tumors with little or no gene amplification seems to be dependent on in vitro EGFR via its effect on cellular radiosensitivity.

Objective To investigate the relationships between radiobiological hypoxic fraction (rHF), pimonidazole hypoxic fraction (pHF) as well as other histological parameters of the tumour microenvironment, and local tumour control after fractionated irradiation in human squamous cell carcinomas (hSCCs). Material and methods Ten different hSCC cell lines were transplanted into nude mice and rHF was calculated from local tumour control rates after single dose irradiation under normal or clamped blood flow conditions. In parallel, tumours were irradiated with 30 fractions within 6weeks. Radiation response was quantified as dose required to cure 50% of tumours (TCD 50 ). Unirradiated tumours were excised for histological evaluation including relative hypoxic area (pHF), relative vascular area (RVA), and fraction of perfused vessels (PF). Results A weak but significant positive correlation between rHF ( R 2 =0.6, p =0.014) and TCD 50 after fractionated irradiation was found. The pHF did not correlate with rHF but was significantly associated with the TCD 50 after single dose clamp ( R 2 =0.8, p =0.003) and showed a trend for an association with TCD 50 after fractionated irradiation ( R 2 =0.4, p =0.067). Relative vascular area and fraction of perfused vessels did not show an association with rHF or TCD 50 after fractionated irradiation. Conclusions Our data suggest that radiobiological hypoxia contributes to the response after fractionated irradiation but that also other radiobiological mechanisms are involved. In the present study, pimonidazole labelling does not reflect rHF and has a limited value to predict local tumour control after fractionated irradiation. The association between pHF and TCD 50 after single dose clamp warrants further investigation.

Purpose We aimed to establish a core needle biopsy technique to investigate the impact on outcome of irradiation of the microenvironment in individual experimental tumours. Methods Nude mice bearing FaDu, UT-SCC-5, UT-SCC-14, and UT-SCC-15 tumours ( n =67) were injected with pimonidazole hypoxia and Hoechst 33342 perfusion markers. One core needle biopsy was taken from the central part of the tumour under anaesthesia and the rest of the tumour was excised after marking the position of the needle. Relative hypoxic area (pHF), relative vascular area (RVA), fraction of perfused vessels (PF), and necrotic fraction (NF) were compared in the biopsies and the adjacent whole tumour sections. In a TCD 50 (dose to cure 50% of tumours) assay, 223 UT-SCC-5 tumours were irradiated with 30 fractions over 6weeks either with or without a core biopsy before the start of radiotherapy. Results The correlations between histological parameters measured in the biopsies and the adjacent tumour sections were dependent on the tumour line. All the four parameters showed weak although significant correlations only in UT-SCC-5. PF was the only parameter which showed a weak but significant correlation in all the four tumour lines. The needle biopsy procedure did not significantly impact on TCD 50 after fractionated irradiation in UT-SCC-5: 98Gy [92; 106] versus 105Gy [96; 117] ( p =0.12). pHF, RVA, PF, and NF measured in the pre-treatment biopsy did not predict the outcome of fractionated irradiation within the UT-SCC-5 tumour line. Conclusion A single pre-treatment core needle biopsy may provide valid results for parameters of the tumour micromilieu, however the accuracy is limited by significant intratumoural heterogeneity in the parameters and sampling error. The needle biopsy procedure does not significantly affect local tumour control rates after fractioned irradiation and may therefore be integrated for longitudinal studies in radiobiological experiments. Pre-treatment histological parameters measured in the biopsy did not correlate with the outcome of fractionated irradiation within the UT-SCC-5 tumour line.

To evaluate the effect of lovastatin alone or combined with radiation on U87MG and FaDu cells in vitro and U87MG tumors in vivo. Cell number, p21WAF1 expression, apoptosis, reproductive cell death, and cell-cycle distribution were investigated after incubation of U87MG and FaDu cells in vitro. The effect of lovastatin (50 mg/kg/day) on tumor growth and on tumor growth delay after single-dose irradiation with 20 Gy was investigated using U87MG tumors in nude mice. Lovastatin dose dependently decreased cell number and proliferation of U87MG and FaDu cells. The proportion of cells in G0/G1 phase, apoptosis and p21 protein expression increased after lovastatin alone or combined with 4-Gy irradiation in both cell lines. Effects of lovastatin on cell cycle and cell number were more pronounced in U87MG compared to FaDu. No radiosensitization of clonogenic cells by lovastatin could be demonstrated in both cells lines, but the colony-forming ability after lovastatin alone was decreased in FaDu cells. In vivo, lovastatin decreased tumor volume over time but did not increase growth delay after irradiation of U87MG tumors with 20 Gy. The data support effects of lovastatin on proliferation, apoptosis and colony-forming ability in vitro and tumor volume in vivo. At the drug concentration achievable, lovastatin did not improve the effects of radiation on U87MG tumors in vivo.

Purpose To study the effects of BAY-84-7296, a novel orally bioavailable inhibitor of mitochondrial complex I and hypoxia-inducible factor 1 (HIF-1) activity, on hypoxia, microenvironment, and radiation response of tumors. Methods and Materials UT-SCC-5 and UT-SCC-14 human squamous cell carcinomas were transplanted subcutaneously in nude mice. When tumors reached 4 mm in diameter BAY-84-7296 (Bayer Pharma AG) or carrier was daily administered to the animals. At 7 mm tumors were either excised for Western blot and immunohistologic investigations or were irradiated with single doses. After irradiation animals were randomized to receive BAY-84-7296 maintenance or carrier. Local tumor control was evaluated 150 days after irradiation, and the dose to control 50% of tumors (TCD50) was calculated. Results BAY-84-7296 decreased nuclear HIF-1α expression. Daily administration of inhibitor for approximately 2 weeks resulted in a marked decrease of pimonidazole hypoxic fraction in UT-SCC-5 (0.5% vs 21%, P

Objective: To investigate local tumour control after dose-escalation based on [18F]2-fluoro-2-deoxy-D-glucose (FDG) positron emission tomography (PET) obtained before and early during fractionated irradiation. Materials and methods: 85 mice bearing FaDu xenografts underwent FDG-PET twice: first immediately prior to the first 2-Gy fraction of irradiation (PET1_0) and second after 18 degrees Gy (PET2_18). After these 9 fractions, animals were randomly allocated to: (1) continuation of 2-Gy fractions (cumulative dose of 60 degrees Gy; n = 31), (2) dose-escalation with 3-Gy fractions (cumulative EQD2-dose 86.25 degrees Gy [alpha/beta-value:10]; n = 25), or (3) with 4-Gy fractions (cumulative EQD2-dose 116 degrees Gy; n = 29). The effects of SUV(max)0 degrees Gy, SUV(max)18 degrees Gy, and dose on local tumour control were analysed in two ways. First, the Cox proportional hazards model was used with two covariates: continuous SUVmax values and dose. Second, the Kaplan-Meier method was used, with tumours classified according to SUVmax greater than or less than (1) median maximum standardized uptake value (SUVmax) at PET1_0 and PET2_18, or (2) the cut-off value 2.5. Results: The multivariate Cox analysis revealed a significant negative association between higher SUVmax determined before start of treatment and local control (HR = 1.59, [95% CI 1.04, 2.42], p = 0.031), whereas higher dose had a significant positive effect (HR = 0.95, [0.93, 0.98], p

Background and purpose Waiting times before radiotherapy may reduce tumour control probability due to proliferation of tumour cells. The aim of the experiment was to test whether the growth inhibiting effect of epidermal growth factor receptor (EGFR)-inhibitors after surgery or tumour transplantation results in a lower tumour mass at time of irradiation and can thereby improve local tumour control. Materials and methods The EGFR-tyrosine kinase inhibitor BIBX1382BS was applied over 14days starting from microscopically non-in-sano-resection of FaDu tumours or from tumour transplantation, followed by irradiation (5f/5d). Endpoint was local tumour control. In addition, vital tumour areas, pimonidazole hypoxic fraction, BrdU labelling index, and colony forming ability in vitro were tested in control tumours and after BIBX1382BS treatment (starting from transplantation). Results The tumour volume at start of irradiation was significantly lower in the BIBX1382BS treated tumours as compared to the control groups by factors of 11 (post-surgery setting) and 2.7 (transplantation setting). However, the reduced volume did not translate into improved local control after irradiation. The TCD 50 values after surgery were 25.4Gy [95% CI 18; 33Gy] in the control group and 30.5Gy [24; 37] in the BIBX1382BS group ( p =0.25). Treatment after transplantation resulted in TCD 50 values of 41.1Gy [35; 47] in the control group and 41.1Gy [33; 49] in the BIBX1382BS group ( p =1). While the proportion of S-phase cells decreased after BIBX1382BS treatment, no differences were observed between the pimonidazole hypoxic fractions and in vitro colony forming ability. Conclusions EGFR-TK inhibition with BIBX1382BS over 14days between macroscopically complete tumour resection or tumour transplantation and start of radiotherapy significantly reduced tumour volume but did not improve local tumour control. One possible explanation is that the EGFR-TK inhibitor has a higher activity in nontumourigenic cancer cells compared to cancer stem cells. This hypothesis, along with the observation that tumours of similar size were significantly more radiosensitive after surgery than without surgery, warrants further investigation.

Tumour hypoxia and its molecular responses have been shown to be associated with poor prognosis. Detection of hypoxia, preferably in a non-invasive manner, could therefore predict treatment outcome and serve as a tool to individualize treatment. This review gives an overview of recent literature on hypoxia imaging markers currently used in clinical trials. Furthermore, recent progress made in targeting hypoxia (hypoxia-activated prodrugs) or hypoxia response (carbonic anhydrase IX inhibitors) is summarized. Last, window-of-opportunity trials implementing non-invasive imaging are proposed as an important tool to prove anti-tumour efficacy of experimental drugs early during drug development.

Advancements made over the past decades in both molecular imaging and radiotherapy planning and delivery have enabled studies that explore the efficacy of heterogeneous radiation treatment ("dose painting") of solid cancers based on biological information provided by different imaging modalities. In addition to clinical trials, preclinical studies may help contribute to identifying promising dose painting strategies. The goal of this current study was twofold: to develop a reproducible positioning and set-up verification protocol for a rat tumor model to be imaged and treated on a clinical platform, and to assess the dosimetric accuracy of dose planning and delivery for both uniform and positron emission tomography-computed tomography (PET-CT) based heterogeneous dose distributions. We employed a syngeneic rat rhabdomyosarcoma model, which was irradiated by volumetric modulated arc therapy (VMAT) with uniform or heterogeneous 6 MV photon dose distributions. Mean dose to the gross tumor volume (GTV) as a whole was kept at 12 Gy for all treatment arms. For the nonuniform plans, the dose was redistributed to treat the 30% of the GTV representing the biological target volume (BTV) with a dose 40% higher than the rest of the GTV (GTV - BTV) (~15 Gy was delivered to the BTV vs. ~10.7 Gy was delivered to the GTV - BTV). Cone beam computed tomography (CBCT) images acquired for each rat prior to irradiation were used to correctly reposition the tumor and calculate the delivered 3D dose. Film quality assurance was performed using a water-equivalent rat phantom. A comparison between CT or CBCT doses and film measurements resulted in passing rates98% with a gamma criterion of 3%/2 mm using 2D dose images. Moreover, between the CT and CBCT calculated doses for both uniform and heterogeneous plans, we observed maximum differences of2% for mean dose to the tumor and mean dose to the biological target volumes. In conclusion, we have developed a robust method for dose painting in a rat tumor model on a clinical platform, with a high accuracy achieved in the delivery of complex dose distributions. Our work demonstrates the technical feasibility of this approach and enables future investigations on the therapeutic effect of preclinical dose painting strategies using a state-of-the-art clinical platform.

Background and purpose The present study was performed to test the hypothesis that lactate accumulation correlates with the radioresistance of malignant tumors due to the radical scavenging capacity of lactate or metabolic intermediates of glycolysis, such as pyruvate. Materials and methods Five human head and neck squamous cell carcinoma cell lines (HNSCCs) xenografted in nude mice were treated with a clinically relevant irradiation protocol with 30 fractions within 6 weeks. The radiation dose necessary to locally control 50% of the tumors (TCD 50 ) ranged from 47.4 to 129.8Gy. Concentrations of glucose, lactate, and ATP in viable tumor regions as potential indicators of glycolytic activity were assessed with structure-associated quantitative bioluminescence imaging. Results Mean lactate concentrations of the different tumor cell lines were in the range of 7.3–25.9μmol/g. TCD 50 values were positively correlated with tumor lactate levels ( R =0.9824, p =0.0028). Conclusions The data obtained support the hypothesis that tissue lactate content correlates with radioresistance in solid human tumors. Furthermore, the results suggest that tumor lactate content determined non-invasively by proton magnetic resonance spectroscopy imaging may be used to predict for radioresistance of malignancies in the clinic; the data also imply that transient inhibition of glycolysis during treatment might possibly sensitize tumors to irradiation.

To investigate the influence on local control after fractionated radiotherapy of hypoxia measured in unirradiated tumours using the hypoxic marker Pimonidazole, using multivariate approaches.Five human squamous cell carcinoma lines (FaDu, UT-SCC-15, UT-SCC-14, XF354, and UT-SCC-5) were transplanted subcutaneously into the right hind-leg of NMRI nude mice. Histological material was collected from 60 unirradiated tumours after injection of Pimonidazole. The relative hypoxic area within the viable tumour area (Pimonidazole hypoxic fraction, pHF) was determined in seven serial 10 microm cross-sections per tumour by fluorescence microscopy and computerized image analysis. Local tumour control was evaluated in a total of 399 irradiated tumours at 120 days after 30 fractions given within 6 weeks with total doses between 30 and 115 Gy.Tumour lines showed pronounced heterogeneity in both pHF and TCD50. Mean pHF values varied between 5% and 37%, TCD50 values between 47 and 130 Gy. A Cox Proportional Hazards model of time to recurrence with two covariates, dose and pHF, yielded significant contributions of both parameters on local control (p0.005) but violated the proportional hazards assumption, suggesting that other factors also influence tumour control. Introduction of histological grade as an example of a confounding factor into the model improved the fit significantly. Local control rates decreased with increasing pHF and this effect was more pronounced at higher doses.This study confirms that tumour hypoxia measured using Pimonidazole in untreated tumours is a significant determinant of local control after fractionated irradiation. The data support the use of multivariate approaches for the evaluation of a single prognostic biomarker such as Pimonidazole, and more generally, suggest that they are required to establish accurate prognostic factors for tumour response.

The advent of immunotherapy is currently revolutionizing the field of oncology, where different drugs are used to stimulate different steps in a failing cancer immune response chain. This review gives a basic overview of the immune response against cancer, as well as the historical and current evidence on the interaction of radiotherapy with the immune system and the different forms of immunotherapy. Furthermore the review elaborates on the many open questions on how to exploit this interaction to the full extent in clinical practice.

SummaryImage-guided radiotherapy (IGRT) represents a novel method to precisely deliver radiation to tumours while sparing surrounding normal tissues. Integration of biological imaging using PET or MRI appears to be a promising concept to improve radiotherapy (Bio-IGRT). For this it is essential that biological imaging provides radiobiologically relevant information. Preclinical and clinical investigations into validation of PET tracers and MR methods in the context of curative radiotherapy and of concepts for biology-based escalation of radiation dose as well as other therapeutic interventions are an important task for further cancer research.

Polo-like kinase 1 (PLK1) plays an important role in mitotic progression, is frequently overexpressed and associated with a poor prognosis of cancer patients, thus providing a promising target in anticancer treatment. Aim of the current project was to evaluate the effect of the novel PLK1 inhibitor BI 6727 in combination with irradiation.In vitro proliferation and radiation cell survival assays as well as in vivo local tumour control assays after single treatment and combined radiation and drug application were carried out using the squamous cell carcinoma models A431 and FaDu. In addition, cell cycle phases were monitored in vitro and in vivo.BI 6727 showed a dose-dependent antiproliferative effect and an increase in the mitotic fraction. BI 6727 alone reduced clonogenic cell survival, while radiosensitivity in vitro (SF2) and in vivo (single-dose TCD(50) under clamped hypoxia) was not affected. In contrast, local tumour control was significantly improved after application of BI 6727 simultaneously to fractionated irradiation (A431: TCD(50) = 60.5 Gy [95% C.I. 57; 63] after IR alone and30 Gy after combined treatment; FaDu: 49.5 Gy [43; 56 Gy] versus 32.9 Gy [26; 40]).Despite the lack of direct cellular radiosensitisation, PLK1 inhibition with BI 6727 during fractionated irradiation significantly improves local tumour control when compared to irradiation alone. This result is likely explained by a considerable effect on cell cycle and an independent cytotoxic potential of BI 6727.