Purpose: Predominant causes of head and neck cancer recurrence after radiotherapy are rapid repopulation, hypoxia, fraction of cancer stem cells, and intrinsic radioresistance. Currently, intrinsic radioresistance can only be assessed by ex vivo colony assays. Besides being time-consuming, colony assays do not identify causes of intrinsic resistance. We aimed to identify a biomarker for intrinsic radioresistance to be used before start of treatment and to reveal biologic processes that could be targeted to overcome intrinsic resistance.Experimental Design: We analyzed both microRNA and mRNA expression in a large panel of head and neck squamous cell carcinoma (HNSCC) cell lines. Expression was measured on both irradiated and unirradiated samples. Results were validated using modified cell lines and a series of patients with laryngeal cancer.Results: miRs, mRNAs, and gene sets that correlated with resistance could be identified from expression data of unirradiated cells. The presence of epithelial-to-mesenchymal transition (EMT) and low expression of miRs involved in the inhibition of EMT were important radioresistance determinants. This finding was validated in two independent cell line pairs, in which the induction of EMT reduced radiosensitivity. Moreover, low expression of the most important miR (miR-203) was shown to correlate with local disease recurrence after radiotherapy in a series of patients with laryngeal cancer.Conclusions: These findings indicate that EMT and low expression of EMT-inhibiting miRs, especially miR-203, measured in pretreatment material, causes intrinsic radioresistance of HNSCC, which could enable identification and treatment modification of radioresistant tumors. Clin Cancer Res; 21(24); 5630–8. ©2015 AACR.

Hypoxia is a common feature of solid tumors and is associated with increased tumor progression, resistance to therapy and increased metastasis. Hence, tumor hypoxia is a prognostic factor independent of treatment modality. To survive hypoxia, cells activate macroautophagy/autophagy. Paradoxically, in several cancer types, mutations or loss of essential autophagy genes have been reported that are associated with earlier onset of tumor growth. However, to our knowledge, the phenotypic and therapeutic consequences of autophagy deficiency have remained unexplored. In this study, we determined autophagy-defects in head and neck squamous cell carcinoma (HNSCC) and observed that expression of ATG12 (autophagy related 12) was lost in 25%-40% of HNSCC. In line, ATG12 loss is associated with absence of hypoxia, as determined by pimonidazole immunohistochemistry. Hence, ATG12 loss is associated with improved prognosis after therapy in two independent HNSCC cohorts and 7 additional cancer types. In vivo, ATG12 targeting resulted in decreased hypoxia tolerance, increased necrosis and sensitivity of the tumor to therapy, but in vitro ATG12-deficient cells displayed enhanced survival in nutrient-rich culture medium. Besides oxygen, delivery of glucose was hampered in hypoxic regions in vivo, which increases the reliance of cells on other carbon sources (e.g., L-glutamine). We observed decreased intracellular L-glutamine levels in ATG12-deficient cells during hypoxia and increased cell killing after L-glutamine depletion, indicating a central role for ATG12 in maintaining L-glutamine homeostasis. Our results demonstrate that ATG12^low tumors represent a phenotypically different subtype that, due to the lowered hypoxia tolerance, display a favorable outcome after therapy. Abbreviations: ARCON:accelerated radiotherapy with carbogen and nicotinamide; ATG: autophagy related; BrdUrd: bromodeoxyuridine; CA9/CAIX: carbonic anhydrase 9; HIF1A/HIF1α: hypoxia inducible factor 1 subunit alpha; HNSCC: head and neck squamous cell carcinoma; HPV: human papilloma virus; HR: hazard ratio; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; mRNA: messenger ribonucleic acid; PCR: polymerase chain reaction; SLC2A1/GLUT1: solute carrier family 2 member 1; TCGA: the Cancer Genome Atlas; TME: tumor microenvironment; UTR: untranslated region; VEGF: vascular endothelial growth factor

Radiotherapy is an important treatment modality of head and neck squamous cell carcinomas (HNSCC). Multiple links have been described between the metabolic activity of tumors and their clinical outcome. Here we test the hypothesis that metabolic features determine radiosensitivity, explaining the relationship between metabolism and clinical outcome. Radiosensitivity of 14 human HNSCC cell lines was determined using colony forming assays and the expression profile of approximately 200 metabolic and cancer-related genes was generated using targeted RNA sequencing by single molecule molecular inversion probes. Results: Correlation between radiosensitivity data and expression profiles yielded 18 genes associated with radiosensitivity or radioresistance, of which adenosine triphosphate (ATP) citrate lyase (ACLY) was of particular interest. Pharmacological inhibition of ACLY caused an impairment of DNA damage repair, specifically homologous recombination, and lead to radiosensitization in HNSCC cell lines. Examination of a The Cancer Genome Atlas (TCGA) cohort of HNSCC patients revealed that high expression of ACLY was predictive for radiotherapy failure, as it was only associated with poor overall survival in patients who received radiotherapy (hazard ratio of 2.00, 95% CI: 1.12–3.55; p = 0.0184). These data were further validated in an independent cohort of HNSCC patients treated with chemoradiation. Furthermore, patients with poor locoregional control after radiotherapy have significantly higher nuclear ACLY protein levels. Together, we here show that ACLY affects DNA damage repair, and is a predictive factor for radiotherapy outcome in HNSCC.

Hypoxic head and neck tumors respond poorly to radiotherapy and can be identified using gene expression profiles. However, it is unknown whether treatment outcome is driven by acute or chronic hypoxia. Gene expression data of 398 head and neck cancers was collected. Four clinical hypoxia profiles were compared to in vitro acute and chronic hypoxia profiles. Chronic and acute hypoxia profiles were tested for their association to outcome using Cox proportional hazard analyses. In an initial set of 224 patients, scores of the four clinical hypoxia profiles correlated with each other and with chronic hypoxia. However, the acute hypoxia profile showed a stronger association with local recurrence after chemoradiotherapy (p = 0.02; HR = 3.1) than the four clinical (chronic hypoxia) profiles (p = 0.2; HR = 0.9). An independent set of 174 patients confirmed that acute hypoxia is a stronger prognostic factor than chronic hypoxia for overall survival, progression-free survival, local and locoregional control. Multivariable analyses accounting for known prognostic factors substantiate this finding (p = 0.045; p = 0.042; p = 0.018 and p = 0.003, respectively). In conclusion, the four clinical hypoxia profiles are related to chronic hypoxia and not acute hypoxia. The acute hypoxia profile shows a stronger association with patient outcome and should be incorporated into existing prediction models.

The combination of radiotherapy (RT) with targeted agents or immunotherapy may result in improved outcomes, but it can also increase toxicity. However, there is a paucity of high-quality toxicity data, leading to an absence of evidence-based guidelines. To address this, European Society for Medical Oncology (ESMO) and European SocieTy for Radiotherapy and Oncology (ESTRO) initiated a series of systematic reviews followed by a Delphi consensus process to develop multidisciplinary, evidence-based consensus statements regarding the safety of combining RT with such agents. The current publication describes the combination of RT with immune checkpoint inhibitors (ICIs), vascular endothelial growth factor (receptor) [VEGF(R)] inhibitors, or multitargeted tyrosine kinase inhibitors (TKIs). By systematically covering different drug classes and irradiated areas, 76 clinical scenarios were evaluated during two Delphi rounds with 20 international experts. Safety statements were developed for each scenario, based on the systematic literature reviews. A total of 5921 records were screened during the systematic literature review process for ICIs, VEGF(R) inhibitors, and multitargeted TKIs, and 159 reports were selected for inclusion in the final literature reviews and the database. During the two Delphi rounds, agreement was reached regarding the safety statements for 74 clinical scenarios. Generally, the expected toxicity of combining RT with ICIs is low, particularly for programmed death(-ligand) 1 inhibitors. For most combinations with VEGF(R) inhibitors and multitargeted TKIs, exercising caution is recommended. The evidence-based safety statements developed during this comprehensive project provide practical guidance on combining RT with targeted cancer therapies and immunotherapy.

While combining radiotherapy (RT) with targeted agents or immunotherapy may improve outcomes, it may also increase toxicity. High-quality toxicity data and multidisciplinary, evidence-based guidelines on the combination of these treatment modalities are scarce. The European Society for Medical Oncology (ESMO) and the European SocieTy for Radiotherapy and Oncology (ESTRO) developed a series of systematic reviews with multidisciplinary, tumor-agnostic, evidence-based Delphi consensus statements regarding the safety of combining RT with targeted agents or immunotherapy. The current study addresses the safety of combining RT with CDK4/6 inhibitors, anti-HER2 monoclonal antibodies, PARP inhibitors, or mTOR-inhibitors. During the modified Delphi process, two digital voting rounds were organized with 18 international experts. By systematically evaluating the different drug classes and irradiated areas, 74 clinical scenarios were assessed. Safety statements were formulated for all scenarios, based on the evidence from the systematic literature reviews. A total of 1,341 records were screened during the systematic literature reviews, of which 107 studies were ultimately included in the final reviews and the literature database. After two Delphi voting rounds, agreement was reached on all 74 scenario-specific safety statements. The expected combined toxicity is often low for anti-HER2 monoclonal antibodies. For most scenarios with CDK4/6, PARP, or mTOR inhibitors, exercising caution is recommended.