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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.

Carbonic anhydrase IX (CAIX) is a tumor-specific protein that is upregulated during hypoxic conditions where it is involved in maintaining the pH balance. CAIX causes extracellular acidification, thereby limiting the uptake of weak basic chemotherapeutic agents, such as doxorubicin, and decreasing its efficacy. The aim of this study was to determine if doxorubicin efficacy can be increased when combined with the selective sulfamate CAIX inhibitor S4. The effect of S4 on doxorubicin efficacy was tested in vitro using cell viability assays with MDA-MB-231, FaDu, HT29 -CAIX high and HT29 -CAIX low cell lines. In addition, the efficacy of this combination therapy was investigated in tumor xenografts of the same cell lines. The addition of S4 in vitro increased the efficacy of doxorubicin in the MDA-MB-231 during hypoxic exposure (IC50 is 0.25 versus 0.14 mu M, p = 0.0003). Similar results were observed for HT29-CAIX high with S4 during normoxia (IC50 is 0.20 versus 0.08 mu M, p

To compare a dedicated simulation model for hypoxia PET against tumor microsections stained for different parameters of the tumor microenvironment. The model can readily be adapted to a variety of conditions, such as different human head and neck squamous cell carcinoma (HNSCC) xenograft tumors.Nine different HNSCC tumor models were transplanted subcutaneously into nude mice. Tumors were excised and immunoflourescently labeled with pimonidazole, Hoechst 33342, and CD31, providing information on hypoxia, perfusion, and vessel distribution, respectively. Hoechst and CD31 images were used to generate maps of perfused blood vessels on which tissue oxygenation and the accumulation of the hypoxia tracer FMISO were mathematically simulated. The model includes a Michaelis-Menten relation to describe the oxygen consumption inside tissue. The maximum oxygen consumption rate M0 was chosen as the parameter for a tumor-specific optimization as it strongly influences tracer distribution. M0 was optimized on each tumor slice to reach optimum correlations between FMISO concentration 4 h postinjection and pimonidazole staining intensity.After optimization, high pixel-based correlations up to R(2) = 0.85 were found for individual tissue sections. Experimental pimonidazole images and FMISO simulations showed good visual agreement, confirming the validity of the approach. Median correlations per tumor model varied significantly (p0.05), with R(2) ranging from 0.20 to 0.54. The optimum maximum oxygen consumption rate M0 differed significantly (p0.05) between tumor models, ranging from 2.4 to 5.2 mm Hg/s.It is feasible to simulate FMISO distributions that match the pimonidazole retention patterns observed in vivo. Good agreement was obtained for multiple tumor models by optimizing the oxygen consumption rate, M0, whose optimum value differed significantly between tumor models.

PURPOSE: To compare a dedicated simulation model for hypoxia PET against tumor microsections stained for different parameters of the tumor microenvironment. The model can readily be adapted to a variety of conditions, such as different human head and neck squamous cell carcinoma (HNSCC) xenograft tumors. METHODS: Nine different HNSCC tumor models were transplanted subcutaneously into nude mice. Tumors were excised and immunoflourescently labeled with pimonidazole, Hoechst 33342, and CD31, providing information on hypoxia, perfusion, and vessel distribution, respectively. Hoechst and CD31 images were used to generate maps of perfused blood vessels on which tissue oxygenation and the accumulation of the hypoxia tracer FMISO were mathematically simulated. The model includes a Michaelis-Menten relation to describe the oxygen consumption inside tissue. The maximum oxygen consumption rate M0 was chosen as the parameter for a tumor-specific optimization as it strongly influences tracer distribution. M0 was optimized on each tumor slice to reach optimum correlations between FMISO concentration 4 h postinjection and pimonidazole staining intensity. RESULTS: After optimization, high pixel-based correlations up to R(2) = 0.85 were found for individual tissue sections. Experimental pimonidazole images and FMISO simulations showed good visual agreement, confirming the validity of the approach. Median correlations per tumor model varied significantly (p < 0.05), with R(2) ranging from 0.20 to 0.54. The optimum maximum oxygen consumption rate M0 differed significantly (p < 0.05) between tumor models, ranging from 2.4 to 5.2 mm Hg/s. CONCLUSIONS: It is feasible to simulate FMISO distributions that match the pimonidazole retention patterns observed in vivo. Good agreement was obtained for multiple tumor models by optimizing the oxygen consumption rate, M0, whose optimum value differed significantly between tumor models.

Hypoxia is a characteristic of many solid tumors and an adverse prognostic factor for treatment outcome. Hypoxia increases the expression of carbonic anhydrase IX (CAIX), an enzyme that is predominantly found on tumor cells and is involved in maintaining the cellular pH balance. Many clinical studies investigated the prognostic value of CAIX expression, but most have been inconclusive, partly due to small numbers of patients included. The present meta-analysis was therefore performed utilizing the results of all clinical studies to determine the prognostic value of CAIX expression in solid tumors. Renal cell carcinoma was excluded from this meta-analysis due to an alternative mechanism of upregulation. 958 papers were identified from a literature search performed in PubMed and Embase. These papers were independently evaluated by two reviewers and 147 studies were included in the analysis. The meta-analysis revealed strong significant associations between CAIX expression and all endpoints: overall survival [hazard ratio (HR) = 1.76, 95% confidence interval (95% CI) 1.58-1.98], disease-free survival (HR = 1.87, 95% CI 1.62-2.16), locoregional control (HR = 1.54, 95% CI 1.22-1.93), disease-specific survival (HR = 1.78, 95% CI 1.41-2.25), metastasis-free survival (HR = 1.82, 95% CI 1.33-2.50), and progression-free survival (HR = 1.58, 95% CI 1.27-1.96). Subgroup analyses revealed similar associations in the majority of tumor sites and types. In conclusion, these results show that patients having tumors with high CAIX expression have higher risk of locoregional failure, disease progression, and higher risk to develop metastases, independent of tumor type or site. The results of this meta-analysis further support the development of a clinical test to determine patient prognosis based on CAIX expression and may have important implications for the development of new treatment strategies.

Matrix metalloproteinase-2 (MMP2) is important in tumorigenesis, angiogenesis and tumor invasion. In this study, we investigated if the Cy5-tagged small immuno protein targeting the catalytic domain of human MMP2 (aMMP2-SIP) detects MMP2 in tumors non-invasively. For this purpose, we generated MMP2 expressing (empty vector EV) and knock-down (KD) HT1080, U373 and U87 cells, which were injected subcutaneously in the lateral flank of NMRI-nu mice. Optical imaging (Optix MX2) performed at 0.5, 2, 4, 8, 24 and 48 hour post injection (h.p.i.) of Cy5 tagged aMMP2-SIP, indicated significantly lower tumor to background ratios at both 24 (P = 0.0090) and 48 h.p.i. (P, Scientific Reports, 6, ISSN:2045-2322

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.

Cancer stem cells (CSC) have the unique ability to cause tumor recurrences if they survive treatment. Radiotherapy has curative potential because it has been functionally shown to sufficiently inactivate CSCs. It is well known that CSCs mediate the radiation resistance of tumors by tumor-specific factors, such as the pretreatment number of CSCs and repopulation or reoxygenation during fractionated radiotherapy. CSCs appear to have a higher intrinsic radioresistance than non-CSCs, a factor that is especially important for the development of predictive biomarkers that, if this finding holds true, can only be successfully established if they are stem-cell specific. Recent clinical data imply that stem-cell–related surface markers may be directly used as predictors for the radiocurability of tumors with comparable risk factors, such as histology and size. Future studies need to address the question of which additional markers need to be considered if more heterogeneous patient collectives are investigated. With the goal of developing a direct targeting approach, investigators are currently evaluating several drugs that are intended to target CSCs by inhibiting stem-cell–related signal transduction pathways. We need to preclinically test such drugs as combined-modality therapies in combination with radiotherapy to evaluate their curative potential, and optimize them by increasing their specificity to CSCs over normal tissue stem cells to avoid increased radiation toxicity. Clin Cancer Res; 17(23); 7224–9. ©2011 AACR.

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.

Hypoxia is a characteristic of many solid tumors and is defined as a low level or absence of oxygen due to an insufficient vascularization of the tumor or a transient blockage of blood vessels. Hypoxia activates a survival response within the tumor cells driving cancer progression and is associated with poor prognosis. Hypoxia-activated prodrugs (HAPs) are anti-neoplastic agents that can solely be activated in hypoxic areas allowing the targeted delivery of cytotoxic compounds in the hypoxic tumor niches. Convert Pharmaceuticals is developing CP-506, a novel generation of HAP presenting a strong bystander effect. A key step in the activation of CP-506 is its conversion by one-electron reductases to an oxygen-sensing intermediate. Under normoxic conditions, the intermediate is rapidly re-oxidized to the pro-drug. However, under severe hypoxic conditions, further reduction steps lead to the generation of the active cytotoxic metabolite, CP-506M. In an in vitro cytotoxicity assay, CP-506 significantly inhibited the viability of several tumor cell lines specifically under anoxic conditions. In spheroid models, CP-506 demonstrates a strong bystander effect i.e. the ability of the active metabolite to locally diffuse beyond the hypoxic core and induce tumor cell death. CP-506 was administered intraperitoneally at a dose of 600 mg/kg once a day for 5 consecutive days and showed a significant inhibition of tumor growth in four tumor xenograft models (triple negative breast cancer, lung and pancreatic cancers). In all tumor models, CP-506 treatment significantly increased the survival of treated mice assessed by the time to reach four times the start volume as a surrogate endpoint for survival. Different dosing regimens for CP-506 were investigated and all demonstrated a significant tumor growth inhibition compared to the vehicle group. The most profound anti-tumor effect was observed for the repeated cycles of daily consecutive administrations. Finally, no cumulative toxicity was observed for any of the repeated dosing schedules as indicated by bodyweight change. The successful development of CP-506 will require combination with treatments targeting well-oxygenated cells and the careful selection of patients predicted to benefit from a HAP treatment. To this end, we are currently identifying key parameters (presence of tumor hypoxia, expression of reductases and DNA repair capacity) in the activation of CP-506 to define predictive gene signatures in liquid biopsies enabling patient selection. Our current pre-clinical data strongly suggest that CP-506 is a potent, highly selective HAP with a favorable pharmacokinetic profile and the addition of predictive gene signatures or imaging biomarkers for patient selection holds great promise for the treatment of cancer. Citation Format: sophie thiolloy, Sofie Deschoemaeker, Nicolas Ongenae, Julie Gilissen, Ludwig Dubois, Ala Yaromina, Amir Ashoorzadeh, Jeff Smaill, Adam Patterson, Philippe Lambin, Arne Heyerick. CP-506, a next generation hypoxia-activated prodrug, as promising novel anti-cancer therapeutic [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4959.

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.

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.

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.

Purpose: Radiotherapy modifies the tumor microenvironment and causes the release of tumor antigens, which can enhance the effect of immunotherapy. L19 targets the extra domain B (ED-B) of fibronectin, a marker for tumor neoangiogenesis, and can be used as immunocytokine when coupled to IL2. We hypothesize that radiotherapy in combination with L19-IL2 provides an enhanced antitumor effect, which is dependent on ED-B expression. Experimental Design: Mice were injected with syngeneic C51 colon carcinoma, Lewis lung carcinoma (LLC), or 4T1 mammary carcinoma cells. Tumor growth delay, underlying immunologic parameters, and treatment toxicity were evaluated after single-dose local tumor irradiation and systemic administration of L19-IL2 or equimolar controls. Results: ED-B expression was high, intermediate, and low for C51, LLC, and 4T1, respectively. The combination therapy showed (i) a long-lasting synergistic effect for the C51 model with 75% of tumors being cured, (ii) an additive effect for the LLC model, and (iii) no effect for the 4T1 model. The combination treatment resulted in a significantly increased cytotoxic (CD8+) T-cell population for both C51 and LLC. Depletion of CD8+ T cells abolished the benefit of the combination therapy. Conclusions: These data provide the first evidence for an increased therapeutic potential by combining radiotherapy with L19-IL2 in ED-B–positive tumors. This new opportunity in cancer treatment will be investigated in a phase I clinical study for patients with an oligometastatic solid tumor (NCT02086721). An animation summarizing our results is available at https://www.youtube.com/watch?v=xHbwQuCTkRc. Clin Cancer Res; 21(5); 1151–60. ©2014 AACR.

Purpose: We tested therapeutic efficacy of two dose painting strategies of applying higher radiation dose to tumor subvolumes with high FDG uptake (biologic target volume, BTV): dose escalation and dose redistribution. We also investigated whether tumor response was determined by the highest dose in BTV or the lowest dose in gross tumor volume (GTV). Experimental Design: FDG uptake was evaluated in rat rhabdomyosarcomas prior to irradiation. BTV was defined as 30% of GTV with the highest (BTVhot) or lowest (BTVcold) uptake. To test efficacy of dose escalation, tumor response (time to reach two times starting tumor volume, TGTV2) to Hot Boost irradiation (40% higher dose to BTVhot) was compared with Cold Boost (40% higher dose to BTVcold), while mean dose to GTV remained 12 Gy. To test efficacy of dose redistribution, TGTV2 after Hot Boost was compared with uniform irradiation with the same mean dose (8 or 12 Gy). Results: TGTV2 after 12 Gy delivered heterogeneously (Hot and Cold Boost) or uniformly were not significantly different: 20.2, 19.5, and 20.6 days, respectively. Dose redistribution (Hot Boost) with 8 Gy resulted in faster tumor regrowth as compared with uniform irradiation (13.3 vs. 17.1 days; P = 0.026). Further increase in dose gradient to 60% led to a more pronounced decrease in TGTV2 (10.9 days; P < 0.0001). Conclusions: Dose escalation effect was independent of FDG uptake in target tumor volume, while dose redistribution was detrimental in this tumor model for dose levels applied here. Our data are consistent with the hypothesis that tumor response depends on the minimum intratumoral dose. Clin Cancer Res; 21(24); 5511–8. ©2015 AACR.

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.

Purpose: Conventional anticancer treatments are often impaired by the presence of hypoxia. TH-302 selectively targets hypoxic tumor regions, where it is converted into a cytotoxic agent. This study assessed the efficacy of the combination treatment of TH-302 and radiotherapy in two preclinical tumor models. The effect of oxygen modification on the combination treatment was evaluated and the effect of TH-302 on the hypoxic fraction (HF) was monitored using [18F]HX4-PET imaging and pimonidazole IHC stainings. Experimental Design: Rhabdomyosarcoma R1 and H460 NSCLC tumor-bearing animals were treated with TH-302 and radiotherapy (8 Gy, single dose). The tumor oxygenation status was altered by exposing animals to carbogen (95% oxygen) and nicotinamide, 21% or 7% oxygen breathing during the course of the treatment. Tumor growth and treatment toxicity were monitored until the tumor reached four times its start volume (T4×SV). Results: Both tumor models showed a growth delay after TH-302 treatment, which further increased when combined with radiotherapy (enhancement ratio rhabdomyosarcoma 1.23; H460 1.49). TH-302 decreases the HF in both models, consistent with its hypoxia-targeting mechanism of action. Treatment efficacy was dependent on tumor oxygenation; increasing the tumor oxygen status abolished the effect of TH-302, whereas enhancing the HF enlarged TH-302′s therapeutic effect. An association was observed in rhabdomyosarcoma tumors between the pretreatment HF as measured by [18F]HX4-PET imaging and the T4×SV. Conclusions: The combination of TH-302 and radiotherapy is promising and warrants clinical testing, preferably guided by the companion biomarker [18F]HX4 hypoxia PET imaging for patient selection. Clin Cancer Res; 21(13); 2984–92. ©2015 AACR.

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.

Hypoxia is a common characteristic of solid tumors and is associated with poor prognosis. Positron emission tomography (PET) can visualize tumor hypoxia in a non-invasive, 3-dimensional manner and can be used to acquire information longitudinally. Multiple 2-nitroimidazole based PET tracers are developed, validated and quantified in the search for the ideal hypoxia tracer and several tracers have shown to reliably represent tumor hypoxia. Furthermore, multiple studies describe the prognostic value of hypoxia PET imaging and the ability to monitor hypoxia during treatment. These applications can be of great potential and their role in treatment planning and modification needs to be further assessed with respect to personalized chemoradiation therapy. In this review we focus on the tracers that were positively validated in preclinical and clinical studies and report accurate quantification and visualization of hypoxia. The characteristics of these tracers are summarized for both preclinical and clinical studies. Furthermore, the clinical applications of hypoxia PET imaging are addressed with a focus on the ability to reliably monitor tumor hypoxia during treatment and the prognostic potential. Also the feasibility studies for hypoxia guided intensity modulated radiation therapy and the patient stratification for hypoxia targeted drugs are assessed.

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.