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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

BackgroundPoorly immunogenic tumors are hardly responsive to immunotherapies such as immune checkpoint blockade (ICB) and are, therefore, a therapeutic challenge. Combination with other immunotherapies and/or immunogenic therapies, such as radiotherapy (RT), could make these tumors more immune responsive. We have previously shown that the immunocytokine L19–IL2 combined with single-dose RT resulted in 75% tumor remission and a 20% curative abscopal effect in the T cell-inflamed C51 colon carcinoma model. This treatment schedule was associated with the upregulation of inhibitory immune checkpoint (IC) molecules on tumor-infiltrating T cells, leading to only tumor growth delay in the poorly immunogenic Lewis lung carcinoma (LLC) model.MethodsWe aimed to trigger curative therapeutic responses in three tumor models (LLC, C51 and CT26) by “pushing the accelerator” of tumor immunity with L19–IL2 and/or “releasing the brakes” with ICB, such as antibodies directed against cytotoxic T lymphocyte associated protein 4 (CTLA-4), programmed death 1 (PD-1) or its ligand (PD-L1), combined with single-dose RT (10 Gy or 5 Gy). Primary tumor endpoint was defined as time to reach four times the size of tumor volume at start of treatment (4T×SV). Multivariate analysis of 4T×SV was performed using the Cox proportional hazards model comparing each treatment group with controls. Causal involvement of T and natural killer (NK) cells in the anti-tumor effect was assessed by in vivo depletion of T, NK or both cell populations. Immune profiling was performed using flow cytometry on single cell suspensions from spleens, bone marrow, tumors and blood.ResultsCombining RT, anti-PD-L1 and L19–IL2 cured 38% of LLC tumors, which was both CD8+ T and NK cell dependent. LLC tumors were resistant to RT +anti-PD-L1 likely explained by the upregulation of other IC molecules and increased T regulatory cell tumor infiltration. RT+L19–IL2 outperformed RT+ICB in C51 tumors; effects were comparable in CT26 tumors. Triple combinations were not superior to RT+L19–IL2 in both these models.ConclusionsThis study demonstrated that combinatorial strategies rationally designed on biological effects can turn immunotherapy-resistant tumors into immunologically responsive tumors. This hypothesis is currently being tested in the international multicentric randomized phase 2 trial: ImmunoSABR (NCT03705403).

Simple Summary Immunotherapy provides the unprecedented opportunity to prolong the survival of cancer patients and even cure patients with previously untreatable malignancies. Preclinical and clinical studies show that standard photon-based radiotherapy and immunotherapy can synergize in order to promote both local and systemic anti-tumor immunity and that there is still ample room for improvement. Charged particle radiation is thought to have greater immunogenic potential compared to photon radiotherapy due to more lethal unrepaired damage, higher ionization density and thus more complex clustered DNA lesions. In this review, several factors determining the success of radiotherapy combined with immunotherapies, such as composition of the tumor, radiotherapy scheme and schedule, radiation dose, the type of radiation, are addressed. Furthermore, the theoretical basis, first pieces of evidences and new insights supporting a favorable immunogenicity profile of charged particle radiation are examined, including a depiction of best of knowledge for the immune-related responses triggered by charged particles and prospective clinical trials. Abstract Radiotherapy (RT) has been shown to interfere with inflammatory signals and to enhance tumor immunogenicity via, e.g., immunogenic cell death, thereby potentially augmenting the therapeutic efficacy of immunotherapy. Conventional RT consists predominantly of high energy photon beams. Hypofractionated RT regimens administered, e.g., by stereotactic body radiation therapy (SBRT), are increasingly investigated in combination with cancer immunotherapy within clinical trials. Despite intensive preclinical studies, the optimal dose per fraction and dose schemes for elaboration of RT induced immunogenic potential remain inconclusive. Compared to the scenario of combined immune checkpoint inhibition (ICI) and RT, multimodal therapies utilizing other immunotherapy principles such as adoptive transfer of immune cells, vaccination strategies, targeted immune-cytokines and agonists are underrepresented in both preclinical and clinical settings. Despite the clinical success of ICI and RT combination, e.g., prolonging overall survival in locally advanced lung cancer, curative outcomes are still not achieved for most cancer entities studied. Charged particle RT (PRT) has gained interest as it may enhance tumor immunogenicity compared to conventional RT due to its unique biological and physical properties. However, whether PRT in combination with immune therapy will elicit superior antitumor effects both locally and systemically needs to be further investigated. In this review, the immunological effects of RT in the tumor microenvironment are summarized to understand their implications for immunotherapy combinations. Attention will be given to the various immunotherapeutic interventions that have been co-administered with RT so far. Furthermore, the theoretical basis and first evidences supporting a favorable immunogenicity profile of PRT will be examined.

Background About 50% of non-small cell lung cancer (NSCLC) patients have metastatic disease at initial diagnosis, which limits their treatment options and, consequently, the 5-year survival rate (15%). Immune checkpoint inhibitors (ICI), either alone or in combination with chemotherapy, have become standard of care (SOC) for most good performance status patients. However, most patients will not obtain long-term benefit and new treatment strategies are therefore needed. We previously demonstrated clinical safety of the tumour-selective immunocytokine L19-IL2, consisting of the anti-ED-B scFv L19 antibody coupled to IL2, combined with stereotactic ablative radiotherapy (SABR). Methods This investigator-initiated, multicentric, randomised controlled open-label phase II clinical trial will test the hypothesis that the combination of SABR and L19-IL2 increases progression free survival (PFS) in patients with limited metastatic NSCLC. One hundred twenty-six patients will be stratified according to their metastatic load (oligo-metastatic: ≤5 or poly-metastatic: 6 to 10) and randomised to the experimental-arm (E-arm) or the control-arm (C-arm). The C-arm will receive SOC, according to the local protocol. E-arm oligo-metastatic patients will receive SABR to all lesions followed by L19-IL2 therapy; radiotherapy for poly-metastatic patients consists of irradiation of one (symptomatic) to a maximum of 5 lesions (including ICI in both arms if this is the SOC). The accrual period will be 2.5-years, starting after the first centre is initiated and active. Primary endpoint is PFS at 1.5-years based on blinded radiological review, and secondary endpoints are overall survival, toxicity, quality of life and abscopal response. Associative biomarker studies, immune monitoring, CT-based radiomics, stool collection, iRECIST and tumour growth rate will be performed. Discussion The combination of SABR with or without ICI and the immunocytokine L19-IL2 will be tested as 1st, 2nd or 3rd line treatment in stage IV NSCLC patients in 14 centres located in 6 countries. This bimodal and trimodal treatment approach is based on the direct cytotoxic effect of radiotherapy, the tumour selective immunocytokine L19-IL2, the abscopal effect observed distant from the irradiated metastatic site(s) and the memory effect. The first results are expected end 2023. Trial registration ImmunoSABR Protocol Code: NL67629.068.18; EudraCT: 2018–002583-11; Clinicaltrials.gov: NCT03705403; ISRCTN ID: ISRCTN49817477; Date of registration: 03-April-2019.

Hypoxia—a common feature of the majority of solid tumors—is a negative prognostic factor, as it is associated with invasion, metastasis and therapy resistance. To date, a variety of methods are available for the assessment of tumor hypoxia, including the use of positron emission tomography (PET). A plethora of hypoxia PET tracers, each with its own strengths and limitations, has been developed and successfully validated, thereby providing useful prognostic or predictive information. The current review focusses on [18F]-HX4, a promising next-generation hypoxia PET tracer. After a brief history of its development, we discuss and compare its characteristics with other hypoxia PET tracers and provide an update on its progression into the clinic. Lastly, we address the potential applications of assessing tumor hypoxia using [18F]-HX4, with a focus on improving patient-tailored therapies.

T cell immunotherapies have shown great promise in patients with advanced cancer disease, revolutionizing treatment. T cell cytotoxicity is crucial in its efficacy, therefore developing ex vivo methods testing tumor and T cell interactions is pivotal. Increasing efforts have been made in developing co-culture assays with sophisticated materials and platforms aiming to mimic the tumor microenvironment (TME), but its complexity makes it difficult to develop the ideal model. In this study, we developed a simple co-culture assay, reproducible in any lab, but respecting the multicellular nature of the TME. Our goal is to combine in a single assay well-established techniques such as a luciferase assay for target cell viability analysis, a CD107a degranulation assay, and multicolor flow cytometry for the detection of cytokines and cytotoxicity markers. Cell suspensions of whole spleens and tumors containing splenic or tumor-infiltrating effector T cells of mice bearing Lewis lung carcinoma (LLC) or CT26 colon carcinoma tumors treated with radiation alone or in combination with immunotherapies were used for co-culture. LLC and CT26 cell lines transduced with the firefly luciferase gene were used as target cells. We demonstrated that splenocytes and tumor-infiltrating T cells derived from mice treated with combination therapy were able to kill approximately 50% of target cells after 48 h of co-culture. This effect was tumor cell-specific and dependent on CD8

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.

About 50% of non-small cell lung cancer (NSCLC) patients have metastatic disease at initial diagnosis, which limits their treatment options and, consequently, the 5-year survival rate (15%). Immune checkpoint inhibitors (ICI), either alone or in combination with chemotherapy, have become standard of care (SOC) for most good performance status patients. However, most patients will not obtain long-term benefit, and new treatment strategies are therefore still needed. We previously demonstrated clinical safety of the tumour-selective immunocytokine L19-IL2, consisting of the anti-EDB scFv L19 antibody coupled to IL2, combined with stereotactic ablative radiotherapy (SABR). Within this phase II ImmunoSABR trial, the combination of SABR with or without ICI and the immunocytokine L19-IL2 will be tested as 1st, 2nd or 3rd line treatment in stage IV NSCLC patients. This bi-modal and triple treatment approach is based on the direct cytotoxic effect of radiotherapy, the tumour selective immunocytokine L19-IL2, the abscopal effect observed distant from the irradiated metastatic site(s), and the memory effect. This investigator initiated, multicentric, randomised controlled open-label phase II clinical trial (NCT03705403) will test the hypothesis that the combination of SABR and L19-IL2 increases the progression free survival (PFS) in patients with limited metastatic NSCLC. Patients will be stratified according to their metastatic load (oligo-metastatic: up to 5, or poly-metastatic: 6 to 10 metastases). Patients will be randomised by minimisation to the experimental (E-arm) or the control arm (C-arm). The C-arm will receive SOC, according to the local protocol. E-arm oligo-metastatic patients will receive SABR to all lesions followed by L19-IL2 therapy; radiotherapy for poly-metastatic patients consists of irradiation of at least one (symptomatic) to a maximum of 5 lesions (including ICI in both arms if this is the SOC). ImmunoSABR consists of 14 participating centres located in 6 countries. The accrual period will be 2.5 years, starting after the first centre is initiated and active. Primary endpoint is PFS at 1.5 years based on blinded radiological review, and secondary endpoints are overall survival, toxicity, quality of life and abscopal response. Associative biomarker studies, blood and tumour cell immune monitoring, CT-based radiomics, stool collection, iRECIST, and tumour growth rate will be performed. The first results are expected end 2023.

Hypoxia-activated prodrugs (HAPs) present a conceptually elegant approach to not only overcome, but better yet, exploit intra-tumoural hypoxia. Despite being successful in vitro and in vivo, HAPs are yet to achieve successful results in clinical settings. It has been hypothesised that this lack of clinical success can, in part, be explained by the insufficiently stringent clinical screening selection of determining which tumours are suitable for HAP treatments. Taking a mathematical modelling approach, we investigate how tumour properties and HAP-radiation scheduling influence treatment outcomes in simulated tumours. The following key results are demonstrated in silico: (i) HAP and ionising radiation (IR) monotherapies may attack tumours in dissimilar, and complementary, ways. (ii) HAP-IR scheduling may impact treatment efficacy. (iii) HAPs may function as IR treatment intensifiers. (iv) The spatio-temporal intra-tumoural oxygen landscape may impact HAP efficacy. Our in silico framework is based on an on-lattice, hybrid, multiscale cellular automaton spanning three spatial dimensions. The mathematical model for tumour spheroid growth is parameterised by multicellular tumour spheroid (MCTS) data., Author summary When cancer patients present with solid tumours, the tumours often contain regions that are oxygen-deprived or, in other words, hypoxic. Hypoxic tumour regions are more resistant to conventional anti-cancer therapies, such as chemotherapy and radiotherapy, and therefore tumour hypoxia may complicate treatments. Hypoxia-activated prodrugs constitute a conceptually elegant approach to not only overcome, but better yet, exploit tumour hypoxia. Hypoxia-activated prodrugs are drugs that act as Trojan horses, they are theoretically harmless vehicles that are converted into warheads when they reach their targets: hypoxic tumour regions. Despite being conceptually clever and successful in experimental settings, hypoxia-activated prodrugs are yet to achieve successful results in clinical trials. It has been hypothesised that this lack of clinical success can, in part, be explained by an insufficiently stringent clinical screening selection of determining which tumours are suitable for hypoxia-activated prodrug treatments. In this article, we investigate how simulated tumours with different oxygen landscapes respond to anti-cancer treatments that include hypoxia-activated prodrugs, either alone or in combination with radiotherapy. Our simulation framework is based on a mathematical model that describes how individual cancer cells in a tumour divide and respond to treatments. We demonstrate that the efficacy of hypoxia-activated prodrugs depends on both the treatment scheduling and the oxygen landscapes of the simulated tumours.

A paradigm shift from current population based medicine to personalized and participative medicine is underway. This transition is being supported by the development of clinical decision support systems based on prediction models of treatment outcome. In radiation oncology, these models 'learn' using advanced and innovative information technologies (ideally in a distributed fashion - please watch the animation: http://youtu.be/ ZDJFOxpwqEA) from all available/appropriate medical data (clinical, treatment, imaging, biological/genetic, etc.) to achieve the highest possible accuracy with respect to prediction of tumor response and normal tissue toxicity. In this position paper, we deliver an overview of the factors that are associated with outcome in radiation oncology and discuss the methodology behind the development of accurate prediction models, which is a multifaceted process. Subsequent to initial development/validation and clinical introduction, decision support systems should be constantly re-evaluated (through quality assurance procedures) in different patient datasets in order to refine and re-optimize the models, ensuring the continuous utility of the models. In the reasonably near future, decision support systems will be fully integrated within the clinic, with data and knowledge being shared in a standardized, dynamic, and potentially global manner enabling truly personalized and participative medicine. (C) 2016 Published by Elsevier B.V.

The hypoxia-inducible transcription factors (HIF)-1/2&alpha, are the main oxygen sensors which regulate the adaptation to intratumoral hypoxia. The aim of this study was to assess the role of the HIF proteins in regulating the radiation response of a non-small cell lung cancer (NSCLC) in vitro model. To directly assess the unique and overlapping functions of HIF-1&alpha, and HIF-2&alpha, we use CRISPR gene-editing to generate isogenic H1299 non-small cell lung carcinoma cells lacking HIF-1&alpha, HIF-2&alpha, or both. We found that in HIF1 knockout cells, HIF-2&alpha, was strongly induced by hypoxia compared to wild type but the reverse was not seen in HIF2 knockout cells. Cells lacking HIF-1&alpha, were more radiation resistant than HIF2 knockout and wildtype cells upon hypoxia, which was associated with a reduced recruitment of &gamma, H2AX foci directly after irradiation and not due to differences in proliferation. Conversely, double-HIF1/2 knockout cells were most radiation sensitive and had increased &gamma, H2AX recruitment and cell cycle delay. Compensatory HIF-2&alpha, activity in HIF1 knockout cells is the main cause of this radioprotective effect. Under hypoxia, HIF1 knockout cells uniquely had a strong increase in lactate production and decrease in extracellular pH. Using genetically identical HIF-&alpha, isoform-deficient cells we identified a strong radiosensitizing of HIF1, but not of HIF2, which was associated with a reduced extracellular pH and reduced glycolysis.

Tumour hypoxia has been associated with increased resistance to various cancer treatments, particularly radiation therapy. Conversely, tumour hypoxia is a validated and ideal target for guided cancer drug delivery. For this reason, hypoxia-activated prodrugs (HAPs) have been developed, which remain inactive in the body until in the presence of tissue hypoxia, allowing for an activation tendency in hypoxic regions. We present here an experimentally motivated mathematical model predicting the effectiveness of HAPs in a variety of clinical settings. We first examined HAP effectiveness as a function of the amount of tumour hypoxia and showed that the drugs have a larger impact on tumours with high levels of hypoxia. We then combined HAP treatment with radiation to examine the effects of combination therapies. Our results showed radiation-HAP combination therapies to be more effective against highly hypoxic tumours. The analysis of combination therapies was extended to consider schedule sequencing of the combination treatments. These results suggested that administering HAPs before radiation was most effective in reducing total cell number. Finally, a sensitivity analysis of the drug-related parameters was done to examine the effect of drug diffusivity and enzyme abundance on the overall effectiveness of the drug. Altogether, the results highlight the importance of the knowledge of tumour hypoxia levels before administration of HAPs in order to ensure positive results.

Recently, we have shown that the administration of the tumour-targeted antibody-based immunocytokine L19-IL2 after radiotherapy (RT) resulted in synergistic anti-tumour effect. Here we show that RT and L19-IL2 can activate a curative abscopal effect, with a long-lasting immunological memory. Ionizing radiation (single dose of 15Gy, 5 × 2Gy or 5 × 5Gy) was delivered to primary C51 colon tumour-bearing immunocompetent mice in combination with L19-IL2 and response of secondary non-irradiated C51 or CT26 colon tumours was evaluated. 15Gy + L19-IL2 triggered a curative (20%) abscopal effect, which was T cell dependent. Moreover, 10Gy + L19-IL2 treated and cured mice were re-injected after 150 days with C51 tumour cells and tumour uptake was assessed. Age-matched controls (matrigel injected mice treated with 10Gy + L19-IL2, mice cured after treatment with surgery + L19-IL2 and mice cured after high dose RT 40Gy + vehicle) were included. Several immunological parameters in blood, tumours, lymph nodes and spleens were investigated. Treatment with 10Gy + L19-IL2 resulted in long-lasting immunological memory, associated with CD44+CD127+ expression on circulating T cells. This combination treatment can induce long-lasting curative abscopal responses, and therefore it has also great potential for treatment of metastatic disease. Preclinical findings have led to the initiation of a phase I clinical trial (NCT02086721) in our institute investigating stereotactic ablative radiotherapy with L19-IL2 in patients with oligometastatic solid tumours. ISSN:2162-4011 ISSN:2162-402X

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.

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.

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.

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: 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. Material and methods: 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. Results: 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 TCD50 under clamped hypoxia) was not affected. In contrast, local tumour control was significantly improved after application of BI 6727 simultaneously to fractionated irradiation (A431: TCD50 = 60.5 Gy [95% C.I. 57; 63] after IR alone and

Purpose: Evaluation of micromilieu-dependent quantified gamma H2AX foci as a potential predictive biomarker in well-oxygenated tumour areas in 9 HNSCC xenograft models in vivo. Materials & methods: GammaH2AX foci were quantified in perfused tumour areas 30 min (initial gamma H2AX foci) and 24 h (residual gamma H2AX foci) after exposure to a single dose of 4 Gy. The initial and residual normalised gamma H2AX foci were correlated with TCD50 after single dose irradiation under clamped blood flow (SDclamp) or a fractionated irradiation setting under ambient blood flow (fx). Results: A significant negative correlation between initial and residual normalised gamma H2AX foci and TCD50 SDclamp and TCD50 fx for 9 HNSCC tumour xenograft models in vivo was found. Residual normalised gamma H2AX foci showed higher intertumoural variability and their correlation with TCD50 was more robust. Conclusions: For the first time a significant negative correlation between gamma H2AX foci and local tumour control after irradiation has been demonstrated. Our results underline the potential of residual gamma H2AX foci as a predictive biomarker for local tumour control after radiotherapy

Radiobiological and cell biological knowledge is increasingly used to further improve local tumour control or to reduce normal tissue damage after radiotherapy. Important research areas are evolving which need to be addressed jointly by nuclear medicine and radiation oncology. For this differences of the biological distribution of diagnostic and therapeutic nuclides compared with the more homogenous dose-distribution of external beam radiotherapy have to be taken into consideration. Examples for interdisciplinary biology-based cancer research in radiation oncology and nuclear medicine include bioimaging of radiobiological parameters characterizing radioresistance, bioimage-guided adaptive radiotherapy, and the combination of radiotherapy with molecular targeted drugs.

Functional imaging for radiotherapy is expected to provide diagnostic as well as prognostic information, to monitor treatment, to help stratification of patients for specific therapeutic interventions and to guide dose-painting. During the last years radiotracer-based functional imaging with positron emission tomography (PET), mainly using the glucose analogue [18F]2-fluoro-2-deoxy-D-glucose ([18F]FDG), has been widely implemented in radiotherapy for a more accurate staging and an improved target volume definition in a variety of tumor types [1, 2, 4]. While the technology for integration of functional imaging into clinical radiotherapy is increasingly available, the biological implications for radiation response are poorly understood [6]. Importantly, biomarker development needs to account for the specific parameters known to determine the results of curative radiotherapy. Clinical as well as preclinical studies are necessary to exploit the potential of functional imaging to improve outcome after radiotherapy. In the following sections recent findings from experimental studies using xenotransplanted tumors in nude mice carried out in our laboratories are briefly summarized.

Recently, we have shown that the administration of the tumour-targeted antibody-based immunocytokine L19-IL2 after radiotherapy (RT) resulted in synergistic anti-tumour effect. Here we show that RT and L19-IL2 can activate a curative abscopal effect, with a long-lasting immunological memory. Ionizing radiation (single dose of 15Gy, 5 × 2Gy or 5 × 5Gy) was delivered to primary C51 colon tumour-bearing immunocompetent mice in combination with L19-IL2 and response of secondary non-irradiated C51 or CT26 colon tumours was evaluated. 15Gy + L19-IL2 triggered a curative (20%) abscopal effect, which was T cell dependent. Moreover, 10Gy + L19-IL2 treated and cured mice were re-injected after 150 days with C51 tumour cells and tumour uptake was assessed. Age-matched controls (matrigel injected mice treated with 10Gy + L19-IL2, mice cured after treatment with surgery + L19-IL2 and mice cured after high dose RT 40Gy + vehicle) were included. Several immunological parameters in blood, tumours, lymph nodes and spleens were investigated. Treatment with 10Gy + L19-IL2 resulted in long-lasting immunological memory, associated with CD44+CD127+ expression on circulating T cells. This combination treatment can induce long-lasting curative abscopal responses, and therefore it has also great potential for treatment of metastatic disease. Preclinical findings have led to the initiation of a phase I clinical trial (NCT02086721) in our institute investigating stereotactic ablative radiotherapy with L19-IL2 in patients with oligometastatic solid tumours., OncoImmunology, 7 (14), ISSN:2162-4011, ISSN:2162-402X

Background About 50% of non-small cell lung cancer (NSCLC) patients have metastatic disease at initial diagnosis, which limits their treatment options and, consequently, the 5-year survival rate (15%). Immune checkpoint inhibitors (ICI), either alone or in combination with chemotherapy, have become standard of care (SOC) for most good performance status patients. However, most patients will not obtain long-term benefit and new treatment strategies are therefore needed. We previously demonstrated clinical safety of the tumour-selective immunocytokine L19-IL2, consisting of the anti-ED-B scFv L19 antibody coupled to IL2, combined with stereotactic ablative radiotherapy (SABR). Methods This investigator-initiated, multicentric, randomised controlled open-label phase II clinical trial will test the hypothesis that the combination of SABR and L19-IL2 increases progression free survival (PFS) in patients with limited metastatic NSCLC. One hundred twenty-six patients will be stratified according to their metastatic load (oligo-metastatic: ≤5 or poly-metastatic: 6 to 10) and randomised to the experimental-arm (E-arm) or the control-arm (C-arm). The C-arm will receive SOC, according to the local protocol. E-arm oligo-metastatic patients will receive SABR to all lesions followed by L19-IL2 therapy; radiotherapy for poly-metastatic patients consists of irradiation of one (symptomatic) to a maximum of 5 lesions (including ICI in both arms if this is the SOC). The accrual period will be 2.5-years, starting after the first centre is initiated and active. Primary endpoint is PFS at 1.5-years based on blinded radiological review, and secondary endpoints are overall survival, toxicity, quality of life and abscopal response. Associative biomarker studies, immune monitoring, CT-based radiomics, stool collection, iRECIST and tumour growth rate will be performed. Discussion The combination of SABR with or without ICI and the immunocytokine L19-IL2 will be tested as 1st, 2nd or 3rd line treatment in stage IV NSCLC patients in 14 centres located in 6 countries. This bimodal and trimodal treatment approach is based on the direct cytotoxic effect of radiotherapy, the tumour selective immunocytokine L19-IL2, the abscopal effect observed distant from the irradiated metastatic site(s) and the memory effect. The first results are expected end 2023., BMC Cancer, 20 (1), ISSN:1471-2407