Immunological impact of cell death signaling driven by radiation on the tumor microenvironment.

Therapeutic irradiation of the tumor microenvironment causes differential activation of pro-survival and pro-death pathways in malignant, stromal, endothelial and immune cells, hence causing a profound cellular and biological reconfiguration via multiple, non-redundant mechanisms. Such mechanisms include the selective elimination of particularly radiosensitive cell types and consequent loss of specific cellular functions, the local release of cytokines and danger signals by dying radiosensitive cells, and altered cytokine secretion by surviving radioresistant cells. Altogether, these processes create chemotactic and immunomodulatory cues for incoming and resident immune cells. Here we discuss how cytoprotective and cytotoxic signaling modules activated by radiation in specific cell populations reshape the immunological tumor microenvironment.

Cell-type-specific tumour sensitivity identified with a bromodomain targeting PROTAC in adenoid cystic carcinoma.

Salivary gland adenoid cystic carcinoma (ACC) is a rare malignancy with limited treatment options. The development of novel therapies is hindered by a lack of preclinical models. We have generated ACC patient-derived xenograft (PDX) lines that retain the physical and genetic properties of the original tumours, including the presence of the common MYB::NFIB or MYBL1::NFIB translocations. We have developed the conditions for the generation of both 2D and 3D tumour organoid patient-derived ACC models that retain MYB expression and can be used for drug studies. Using these models, we show in vitro and in vivo sensitivity of ACC cells to the bromodomain degrader, dBET6. Molecular studies show a decrease in BRD4 and MYB protein levels and target gene expression with treatment. The most prominent effect of dBET6 on tumours in vivo was a change in the relative composition of ACC cell types expressing either myoepithelial or ductal markers. We show that dBET6 inhibits the progenitor function of ACC cells, particularly in the myoepithelial marker-expressing population, revealing a cell-type-specific sensitivity. These studies uncover a novel mechanistic effect of bromodomain inhibitors on tumours and highlight the need to impact both cell-type populations for more effective treatments in ACC patients. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Pembrolizumab plus concurrent chemoradiotherapy versus placebo plus concurrent chemoradiotherapy in patients with locally advanced squamous cell carcinoma of the head and neck (KEYNOTE-412): a randomised, double-blind, phase 3 trial.

BACKGROUND: Despite multimodal therapy, 5-year overall survival for locally advanced head and neck squamous cell carcinoma (HNSCC) is about 50%. We assessed the addition of pembrolizumab to concurrent chemoradiotherapy for locally advanced HNSCC. METHODS: In the randomised, double-blind, phase 3 KEYNOTE-412 trial, participants with newly diagnosed, high-risk, unresected locally advanced HNSCC from 130 medical centres globally were randomly assigned (1:1) to pembrolizumab (200 mg) plus chemoradiotherapy or placebo plus chemoradiotherapy. Randomisation was done using an interactive response technology system and was stratified by investigator's choice of radiotherapy regimen, tumour site and p16 status, and disease stage, with participants randomly assigned in blocks of four per stratum. Participants, investigators, and sponsor personnel were masked to treatment assignments. Local pharmacists were aware of assignments to support treatment preparation. Pembrolizumab and placebo were administered intravenously once every 3 weeks for up to 17 doses (one before chemoradiotherapy, two during chemoradiotherapy, 14 as maintenance therapy). Chemoradiotherapy included cisplatin (100 mg/m2) administered intravenously once every 3 weeks for two or three doses and accelerated or standard fractionation radiotherapy (70 Gy delivered in 35 fractions). The primary endpoint was event-free survival analysed in all randomly assigned participants. Safety was analysed in all participants who received at least one dose of study treatment. This study is registered with ClinicalTrials.gov, NCT03040999, and is active but not recruiting. FINDINGS: Between April 19, 2017, and May 2, 2019, 804 participants were randomly assigned to the pembrolizumab group (n=402) or the placebo group (n=402). 660 (82%) of 804 participants were male, 144 (18%) were female, and 622 (77%) were White. Median study follow-up was 47·7 months (IQR 42·1-52·3). Median event-free survival was not reached (95% CI 44·7 months-not reached) in the pembrolizumab group and 46·6 months (27·5-not reached) in the placebo group (hazard ratio 0·83 [95% CI 0·68-1·03]; log-rank p=0·043 [significance threshold, p≤0·024]). 367 (92%) of 398 participants treated in the pembrolizumab group and 352 (88%) of 398 participants treated in the placebo group had grade 3 or worse adverse events. The most common grade 3 or worse adverse events were decreased neutrophil count (108 [27%] of 398 participants in the pembrolizumab group vs 100 [25%] of 398 participants in the placebo group), stomatitis (80 [20%] vs 69 [17%]), anaemia (80 [20%] vs 61 [15%]), dysphagia (76 [19%] vs 62 [16%]), and decreased lymphocyte count (76 [19%] vs 81 [20%]). Serious adverse events occurred in 245 (62%) participants in the pembrolizumab group versus 197 (49%) participants in the placebo group, most commonly pneumonia (43 [11%] vs 25 [6%]), acute kidney injury (33 [8%] vs 30 [8%]), and febrile neutropenia (24 [6%] vs seven [2%]). Treatment-related adverse events led to death in four (1%) participants in the pembrolizumab group (one participant each from aspiration pneumonia, end-stage renal disease, pneumonia, and sclerosing cholangitis) and six (2%) participants in the placebo group (three participants from pharyngeal haemorrhage and one participant each from mouth haemorrhage, post-procedural haemorrhage, and sepsis). INTERPRETATION: Pembrolizumab plus chemoradiotherapy did not significantly improve event-free survival compared with chemoradiotherapy alone in a molecularly unselected, locally advanced HNSCC population. No new safety signals were seen. Locally advanced HNSCC remains a challenging disease that requires better treatment approaches. FUNDING: Merck Sharp & Dohme, a subsidiary of Merck & Co, Rahway, NJ, USA.

Long-term Outcomes with Nivolumab as First-line Treatment in Recurrent or Metastatic Head and Neck Cancer: Subgroup Analysis of CheckMate 141.

In the randomized, phase 3 CheckMate 141 trial, nivolumab significantly improved overall survival (OS) versus investigator's choice (IC) of chemotherapy at primary analysis among 361 patients with recurrent or metastatic squamous cell carcinoma of the head and neck (R/M SCCHN) post-platinum therapy. Nivolumab versus IC as first-line treatment also improved OS among patients with R/M SCCHN who progressed on platinum therapy for locally advanced disease in the adjuvant or primary setting at 1-year follow-up. In the present long-term follow-up analysis of patients receiving first-line treatment, OS benefit with nivolumab (n = 50) versus IC (n = 26) was maintained (median: 7.7 months versus 3.3 months; hazard ratio: 0.56; 95% confidence interval, 0.34-0.94) at 2 years. No new safety signals were identified. In summary, this long-term 2-year analysis of CheckMate 141 supports the use of nivolumab as a first-line treatment for patients with platinum-refractory R/M SCCHN.

Triggering anti-GBM immune response with EGFR-mediated photoimmunotherapy.

BACKGROUND: Surgical resection followed by chemo-radiation postpones glioblastoma (GBM) progression and extends patient survival, but these tumours eventually recur. Multimodal treatment plans combining intraoperative techniques that maximise tumour excision with therapies aiming to remodel the immunologically cold GBM microenvironment could improve patients' outcomes. Herein, we report that targeted photoimmunotherapy (PIT) not only helps to define tumour location and margins but additionally promotes activation of anti-GBM T cell response. METHODS: EGFR-specific affibody molecule (ZEGFR:03115) was conjugated to IR700. The response to ZEGFR:03115-IR700-PIT was investigated in vitro and in vivo in GBM cell lines and xenograft model. To determine the tumour-specific immune response post-PIT, a syngeneic GBM model was used. RESULTS: In vitro findings confirmed the ability of ZEGFR:03115-IR700 to produce reactive oxygen species upon light irradiation. ZEGFR:03115-IR700-PIT promoted immunogenic cell death that triggered the release of damage-associated molecular patterns (DAMPs) (calreticulin, ATP, HSP70/90, and HMGB1) into the medium, leading to dendritic cell maturation. In vivo, therapeutic response to light-activated conjugate was observed in brain tumours as early as 1 h post-irradiation. Staining of the brain sections showed reduced cell proliferation, tumour necrosis, and microhaemorrhage within PIT-treated tumours that corroborated MRI T2*w acquisitions. Additionally, enhanced immunological response post-PIT resulted in the attraction and activation of T cells in mice bearing murine GBM brain tumours. CONCLUSIONS: Our data underline the potential of ZEGFR:03115-IR700 to accurately visualise EGFR-positive brain tumours and to destroy tumour cells post-conjugate irradiation turning an immunosuppressive tumour environment into an immune-vulnerable one.

Pembrolizumab versus methotrexate, docetaxel, or cetuximab for recurrent or metastatic head-and-neck squamous cell carcinoma (KEYNOTE-040): a randomised, open-label, phase 3 study.

BACKGROUND: There are few effective treatment options for patients with recurrent or metastatic head-and-neck squamous cell carcinoma. Pembrolizumab showed antitumour activity and manageable toxicity in early-phase trials. We aimed to compare the efficacy and safety of pembrolizumab versus standard-of-care therapy for the treatment of head-and-neck squamous cell carcinoma. METHODS: We did a randomised, open-label, phase 3 study at 97 medical centres in 20 countries. Patients with head-and-neck squamous cell carcinoma that progressed during or after platinum-containing treatment for recurrent or metastatic disease (or both), or whose disease recurred or progressed within 3-6 months of previous multimodal therapy containing platinum for locally advanced disease, were randomly assigned (1:1) in blocks of four per stratum with an interactive voice-response and integrated web-response system to receive pembrolizumab 200 mg every 3 weeks intravenously or investigator's choice of standard doses of methotrexate, docetaxel, or cetuximab intravenously (standard-of-care group). The primary endpoint was overall survival in the intention-to-treat population. Safety was analysed in the as-treated population. This trial is registered with ClinicalTrials.gov, number NCT02252042, and is no longer enrolling patients. FINDINGS: Between Dec 24, 2014, and May 13, 2016, 247 patients were randomly allocated to pembrolizumab and 248 were randomly allocated to standard of care. As of May 15, 2017, 181 (73%) of 247 patients in the pembrolizumab group and 207 (83%) of 248 patients in the standard-of-care group had died. Median overall survival in the intention-to-treat population was 8·4 months (95% CI 6·4-9·4) with pembrolizumab and 6·9 months (5·9-8·0) with standard of care (hazard ratio 0·80, 0·65-0·98; nominal p=0·0161). Fewer patients treated with pembrolizumab than with standard of care had grade 3 or worse treatment-related adverse events (33 [13%] of 246 vs 85 [36%] of 234). The most common treatment-related adverse event was hypothyroidism with pembrolizumab (in 33 [13%] patients) and fatigue with standard of care (in 43 [18%]). Treatment-related death occurred in four patients treated with pembrolizumab (unspecified cause, large intestine perforation, malignant neoplasm progression, and Stevens-Johnson syndrome) and two patients treated with standard of care (malignant neoplasm progression and pneumonia). INTERPRETATION: The clinically meaningful prolongation of overall survival and favourable safety profile of pembrolizumab in patients with recurrent or metastatic head and neck squamous cell carcinoma support the further evaluation of pembrolizumab as a monotherapy and as part of combination therapy in earlier stages of disease. FUNDING: Merck Sharp & Dohme, a subsidiary of Merck & Co.

Pembrolizumab alone or with chemotherapy versus cetuximab with chemotherapy for recurrent or metastatic squamous cell carcinoma of the head and neck (KEYNOTE-048): a randomised, open-label, phase 3 study.

BACKGROUND: Pembrolizumab is active in head and neck squamous cell carcinoma (HNSCC), with programmed cell death ligand 1 (PD-L1) expression associated with improved response. METHODS: KEYNOTE-048 was a randomised, phase 3 study of participants with untreated locally incurable recurrent or metastatic HNSCC done at 200 sites in 37 countries. Participants were stratified by PD-L1 expression, p16 status, and performance status and randomly allocated (1:1:1) to pembrolizumab alone, pembrolizumab plus a platinum and 5-fluorouracil (pembrolizumab with chemotherapy), or cetuximab plus a platinum and 5-fluorouracil (cetuximab with chemotherapy). Investigators and participants were aware of treatment assignment. Investigators, participants, and representatives of the sponsor were masked to the PD-L1 combined positive score (CPS) results; PD-L1 positivity was not required for study entry. The primary endpoints were overall survival (time from randomisation to death from any cause) and progression-free survival (time from randomisation to radiographically confirmed disease progression or death from any cause, whichever came first) in the intention-to-treat population (all participants randomly allocated to a treatment group). There were 14 primary hypotheses: superiority of pembrolizumab alone and of pembrolizumab with chemotherapy versus cetuximab with chemotherapy for overall survival and progression-free survival in the PD-L1 CPS of 20 or more, CPS of 1 or more, and total populations and non-inferiority (non-inferiority margin: 1·2) of pembrolizumab alone and pembrolizumab with chemotherapy versus cetuximab with chemotherapy for overall survival in the total population. The definitive findings for each hypothesis were obtained when statistical testing was completed for that hypothesis; this occurred at the second interim analysis for 11 hypotheses and at final analysis for three hypotheses. Safety was assessed in the as-treated population (all participants who received at least one dose of allocated treatment). This study is registered at ClinicalTrials.gov, number NCT02358031. FINDINGS: Between April 20, 2015, and Jan 17, 2017, 882 participants were allocated to receive pembrolizumab alone (n=301), pembrolizumab with chemotherapy (n=281), or cetuximab with chemotherapy (n=300); of these, 754 (85%) had CPS of 1 or more and 381 (43%) had CPS of 20 or more. At the second interim analysis, pembrolizumab alone improved overall survival versus cetuximab with chemotherapy in the CPS of 20 or more population (median 14·9 months vs 10·7 months, hazard ratio [HR] 0·61 [95% CI 0·45-0·83], p=0·0007) and CPS of 1 or more population (12·3 vs 10·3, 0·78 [0·64-0·96], p=0·0086) and was non-inferior in the total population (11·6 vs 10·7, 0·85 [0·71-1·03]). Pembrolizumab with chemotherapy improved overall survival versus cetuximab with chemotherapy in the total population (13·0 months vs 10·7 months, HR 0·77 [95% CI 0·63-0·93], p=0·0034) at the second interim analysis and in the CPS of 20 or more population (14·7 vs 11·0, 0·60 [0·45-0·82], p=0·0004) and CPS of 1 or more population (13·6 vs 10·4, 0·65 [0·53-0·80], p<0·0001) at final analysis. Neither pembrolizumab alone nor pembrolizumab with chemotherapy improved progression-free survival at the second interim analysis. At final analysis, grade 3 or worse all-cause adverse events occurred in 164 (55%) of 300 treated participants in the pembrolizumab alone group, 235 (85%) of 276 in the pembrolizumab with chemotherapy group, and 239 (83%) of 287 in the cetuximab with chemotherapy group. Adverse events led to death in 25 (8%) participants in the pembrolizumab alone group, 32 (12%) in the pembrolizumab with chemotherapy group, and 28 (10%) in the cetuximab with chemotherapy group. INTERPRETATION: Based on the observed efficacy and safety, pembrolizumab plus platinum and 5-fluorouracil is an appropriate first-line treatment for recurrent or metastatic HNSCC and pembrolizumab monotherapy is an appropriate first-line treatment for PD-L1-positive recurrent or metastatic HNSCC. FUNDING: Merck Sharp & Dohme.

The immunological consequences of radiation-induced DNA damage.

Historically, our understanding of the cytotoxicity of radiation has centred on tumour cell-autonomous mechanisms of cell death. Here, tumour cell death occurs when a threshold number of radiation-induced non-reparable double-stranded DNA breaks is exceeded. However, in recent years, the importance of immune mechanisms of cell death has been increasingly recognised, as well as the impact of radiotherapy on non-malignant cellular components of the tumour microenvironment. Conserved antiviral pathways that detect foreign nucleic acid in the cytosol and drive downstream interferon (IFN) responses via the cyclic guanosine monophosphate-adenosine monophosphate synthase/stimulator of IFN genes (cGAS/STING) pathway are key components of the immune response to radiation-induced DNA damage. In preclinical models, acute induction of a type 1 IFN response is important for both direct and abscopal tumour responses to radiation. Inhibitors of the DNA damage response show promise in augmenting this inflammatory IFN response. However, a substantial proportion of tumours show chronic IFN signalling prior to radiotherapy, which paradoxically drives immunosuppression. This chronic IFN signalling leads to treatment resistance, and heterotypic interactions between stromal fibroblasts and tumour cells contribute to an aggressive tumour phenotype. The effect of radiotherapy on myeloid cell populations, particularly tumour-associated macrophages, has an additional impact on the immune tumour microenvironment. It is not yet clear how the above preclinical findings translate into a human context. Human tumours show greater intratumoural genomic heterogeneity and more variable levels of chromosomal instability than experimental murine models. High-quality translational studies of immunological changes occurring during radiotherapy that incorporate intrinsic tumour biology will enable a better understanding of the immunological consequences of radiation-induced DNA damage in patients. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Potentiating Oncolytic Virus-Induced Immune-Mediated Tumor Cell Killing Using Histone Deacetylase Inhibition.

A clinical oncolytic herpes simplex virus (HSV) encoding granulocyte-macrophage colony-stimulating factor (GM-CSF), talimogene laherparepvec, causes regression of injected and non-injected melanoma lesions in patients and is now licensed for clinical use in advanced melanoma. To date, limited data are available regarding the mechanisms of human anti-tumor immune priming, an improved understanding of which could inform the development of future combination strategies with improved efficacy. This study addressed direct oncolysis and innate and adaptive human immune-mediated effects of a closely related HSV encoding GM-CSF (HSVGM-CSF) alone and in combination with histone deacetylase inhibition. We found that HSVGM-CSF supported activation of anti-melanoma immunity via monocyte-mediated type I interferon production, which activates NK cells, and viral maturation of immature dendritic cells (iDCs) into potent antigen-presenting cells for cytotoxic T lymphocyte (CTL) priming. Addition of the histone deacetylase inhibitor valproic acid (VPA) to HSVGM-CSF treatment of tumor cells increased viral replication, viral GM-CSF production, and oncolysis and augmented the development of anti-tumor immunity. Mechanistically, VPA increased expression of activating ligands for NK cell recognition and induced expression of tumor-associated antigens, supporting innate NK cell killing and CTL priming. These data support the clinical combination of talimogene laherparepvec with histone deacetylase inhibition to enhance oncolysis and anti-tumor immunity.

Nivolumab treatment beyond RECIST-defined progression in recurrent or metastatic squamous cell carcinoma of the head and neck in CheckMate 141: A subgroup analysis of a randomized phase 3 clinical trial.

BACKGROUND: Response patterns with immune checkpoint inhibitors may be different from those with chemotherapy. Therefore, assessment of response to immunotherapy with the Response Evaluation Criteria in Solid Tumors (RECIST), version 1.1, could result in premature treatment termination. The randomized, open-label, phase 3 CheckMate 141 trial (NCT02105636), which evaluated nivolumab in recurrent/metastatic squamous cell carcinoma of the head and neck after platinum therapy, allowed treatment beyond first RECIST-defined progression (TBP) according to protocol-specified criteria. METHODS: In CheckMate 141, patients with RECIST-defined progression who had a stable performance status and demonstrated clinical benefit without rapid disease progression were permitted to receive TBP with nivolumab at 3 mg/kg every 2 weeks until further progression, which was defined as an additional ≥10% increase in tumor volume. This post hoc analysis evaluated outcomes for patients who received TBP with nivolumab. RESULTS: Of 240 patients randomized to nivolumab, 146 experienced RECIST-defined progression. Sixty-two of these patients received TBP, and 84 discontinued treatment (no TBP). Among the 60 TBP patients evaluable for response, 15 (25%) had no change in their tumor burden, and 15 (25%) had reductions in target lesion size; 3 patients (5%) had reductions >30%. The median overall survival among TBP patients was 12.7 months (95% confidence interval, 9.7-14.6 months). No new safety signals were observed with TBP. Exploratory analyses of immune cell biomarkers suggested a potential relationship with initial and TBP responses. CONCLUSIONS: Tumor burden reduction was noted in a proportion of patients who received TBP with nivolumab in CheckMate 141. Additional research is warranted to identify factors predictive of a TBP benefit in this population.

Near-infrared photoimmunotherapy targeting EGFR-Shedding new light on glioblastoma treatment.

Glioblastomas (GBMs) are high-grade brain tumors, differentially driven by alterations (amplification, deletion or missense mutations) in the epidermal growth factor receptor (EGFR), that carry a poor prognosis of just 12-15 months following standard therapy. A combination of interventions targeting tumor-specific cell surface regulators along with convergent downstream signaling pathways may enhance treatment efficacy. Against this background, we investigated a novel photoimmunotherapy approach combining the cytotoxicity of photodynamic therapy with the specificity of immunotherapy. An EGFR-specific affibody (ZEGFR:03115 ) was conjugated to the phthalocyanine dye, IR700DX, which when excited with near-infrared light produces a cytotoxic response. ZEGFR:03115 -IR700DX EGFR-specific binding was confirmed by flow cytometry and confocal microscopy. The conjugate showed effective targeting of EGFR positive GBM cells in the brain. The therapeutic potential of the conjugate was assessed both in vitro, in GBM cell lines and spheroids by the CellTiter-Glo® assay, and in vivo using subcutaneous U87-MGvIII xenografts. In addition, mice were imaged pre- and post-PIT using the IVIS/Spectrum/CT to monitor treatment response. Binding of the conjugate correlated to the level of EGFR expression in GBM cell lines. The cell proliferation assay revealed a receptor-dependent response between the tested cell lines. Inhibition of EGFRvIII+ve tumor growth was observed following administration of the immunoconjugate and irradiation. Importantly, this response was not seen in control tumors. In conclusion, the ZEGFR:03115 -IR700DX showed specific uptake in vitro and enabled imaging of EGFR expression in the orthotopic brain tumor model. Moreover, the proof-of-concept in vivo PIT study demonstrated therapeutic efficacy of the conjugate in subcutaneous glioma xenografts.

CheckMate 141: 1-Year Update and Subgroup Analysis of Nivolumab as First-Line Therapy in Patients with Recurrent/Metastatic Head and Neck Cancer.

Nivolumab significantly improved overall survival (OS) vs investigator's choice (IC) of chemotherapy at the primary analysis of randomized, open-label, phase 3 CheckMate 141 in patients with recurrent or metastatic (R/M) squamous cell carcinoma of the head and neck (SCCHN). Here, we report that OS benefit with nivolumab was maintained at a minimum follow-up of 11.4 months. Further, OS benefit with nivolumab vs IC was also noted among patients who received first-line treatment for R/M SCCHN after progressing on platinum therapy for locally advanced disease in the adjuvant or primary (i.e., with radiation) setting.

Changes in multimodality functional imaging parameters early during chemoradiation predict treatment response in patients with locally advanced head and neck cancer.

OBJECTIVE: To assess the optimal timing and predictive value of early intra-treatment changes in multimodality functional and molecular imaging (FMI) parameters as biomarkers for clinical remission in patients receiving chemoradiation for head and neck squamous cell carcinoma (HNSCC). METHODS: Thirty-five patients with stage III-IVb (AJCC 7th edition) HNSCC prospectively underwent 18F-FDG-PET/CT, and diffusion-weighted (DW), dynamic contrast-enhanced (DCE) and susceptibility-weighted MRI at baseline, week 1 and week 2 of chemoradiation. Patients with evidence of persistent or recurrent disease during follow-up were classed as non-responders. Changes in FMI parameters at week 1 and week 2 were compared between responders and non-responders with the Mann-Whitney U test. The significance threshold was set at a p value of <0.05. RESULTS: There were 27 responders and 8 non-responders. Responders showed a greater reduction in PET-derived tumor total lesion glycolysis (TLG40%; p = 0.007) and maximum standardized uptake value (SUVmax; p = 0.034) after week 1 than non-responders but these differences were absent by week 2. In contrast, it was not until week 2 that MRI-derived parameters were able to discriminate between the two groups: larger fractional increases in primary tumor apparent diffusion coefficient (ADC; p < 0.001), volume transfer constant (Ktrans; p = 0.012) and interstitial space volume fraction (Ve; p = 0.047) were observed in responders versus non-responders. ADC was the most powerful predictor (∆ >17%, AUC 0.937). CONCLUSION: Early intra-treatment changes in FDG-PET, DW and DCE MRI-derived parameters are predictive of ultimate response to chemoradiation in HNSCC. However, the optimal timing for assessment with FDG-PET parameters (week 1) differed from MRI parameters (week 2). This highlighted the importance of scanning time points for the design of FMI risk-stratified interventional studies.

Nivolumab versus standard, single-agent therapy of investigator's choice in recurrent or metastatic squamous cell carcinoma of the head and neck (CheckMate 141): health-related quality-of-life results from a randomised, phase 3 trial.

BACKGROUND: Patients with platinum-refractory recurrent or metastatic squamous cell carcinoma of the head and neck have few treatment options and poor prognosis. Nivolumab significantly improved survival of this patient population when compared with standard single-agent therapy of investigator's choice in Checkmate 141; here we report the effect of nivolumab on patient-reported outcomes (PROs). METHODS: CheckMate 141 was a randomised, open-label, phase 3 trial in patients with recurrent or metastatic squamous cell carcinoma of the head and neck who progressed within 6 months after platinum-based chemotherapy. Patients were randomly assigned (2:1) to nivolumab 3 mg/kg every 2 weeks (n=240) or investigator's choice (n=121) of methotrexate (40-60 mg/m2 of body surface area), docetaxel (30-40 mg/m2), or cetuximab (250 mg/m2 after a loading dose of 400 mg/m2) until disease progression, intolerable toxicity, or withdrawal of consent. On Jan 26, 2016, the independent data monitoring committee reviewed the data at the planned interim analysis and declared overall survival superiority for nivolumab over investigator's choice therapy (primary endpoint; described previously). The protocol was amended to allow patients in the investigator's choice group to cross over to nivolumab. All patients not on active therapy are being followed for survival. As an exploratory endpoint, PROs were assessed at baseline, week 9, and every 6 weeks thereafter using the European Organisation for Research and Treatment of Cancer (EORTC) Quality of Life Questionnaire-Core 30 (QLQ-C30), the EORTC head and neck cancer-specific module (EORTC QLQ-H&N35), and the three-level European Quality of Life-5 Dimensions (EQ-5D) questionnaire. Differences within and between treatment groups in PROs were analysed by ANCOVA among patients with baseline and at least one other assessment. All randomised patients were included in the time to clinically meaningful deterioration analyses. Median time to clinically meaningful deterioration was analysed by Kaplan-Meier methods. CheckMate 141 was registered with ClinicalTrials.org, number NCT02105636. FINDINGS: Patients were enrolled between May 29, 2014, and July 31, 2015, and subsequently 361 patients were randomly assigned to receive nivolumab (n=240) or investigator's choice (n=121). Among them, 129 patients (93 in the nivolumab group and 36 in the investigator's choice group) completed any of the PRO questionnaires at baseline and at least one other assessment. Treatment with nivolumab resulted in adjusted mean changes from baseline to week 15 ranging from -2·1 to 5·4 across functional and symptom domains measured by the EORTC QLQ-C30, with no domains indicating clinically meaningful deterioration. By contrast, eight (53%) of the 15 domains in the investigator's choice group showed clinically meaningful deterioration (10 points or more) at week 15 (change from baseline range, -24·5 to 2·4). Similarly, on the EORTC QLQ-H&N35, clinically meaningful worsening at week 15 was seen in no domains in the nivolumab group and eight (44%) of 18 domains in the investigator's choice group. Patients in the nivolumab group had a clinically meaningful improvement (according to a difference of 7 points or greater) in adjusted mean change from baseline to week 15 on the EQ-5D visual analogue scale, in contrast to a clinically meaningful deterioration in the investigator's choice group (7·3 vs -7·8). Differences between groups were significant and clinically meaningful at weeks 9 and 15 in favour of nivolumab for role functioning, social functioning, fatigue, dyspnoea, and appetite loss on the EORTC QLQ-C30 and pain and sensory problems on the EORTC QLQ-H&N35. Median time to deterioration was significantly longer with nivolumab versus investigator's choice for 13 (37%) of 35 domains assessed across the three questionnaires. INTERPRETATION: In this exploratory analysis of CheckMate 141, nivolumab stabilised symptoms and functioning from baseline to weeks 9 and 15, whereas investigator's choice led to clinically meaningful deterioration. Nivolumab delayed time to deterioration of patient-reported quality-of-life outcomes compared with single-agent therapy of investigator's choice in patients with platinum-refractory recurrent or metastatic squamous cell carcinoma of the head and neck. In view of the major unmet need in this population and the importance of maintaining or improving quality of life for patients with recurrent or metastatic squamous cell carcinoma of the head and neck, these data support nivolumab as a new standard-of-care option in this setting. FUNDING: Bristol-Myers Squibb.

HSP90 inhibition sensitizes head and neck cancer to platin-based chemoradiotherapy by modulation of the DNA damage response resulting in chromosomal fragmentation.

BACKGROUND: Concurrent cisplatin radiotherapy (CCRT) is a current standard-of-care for locally advanced head and neck squamous cell carcinoma (HNSCC). However, CCRT is frequently ineffective in patients with advanced disease. It has previously been shown that HSP90 inhibitors act as radiosensitizers, but these studies have not focused on CCRT in HNSCC. Here, we evaluated the HSP90 inhibitor, AUY922, combined with CCRT. METHODS: The ability of AUY922 to sensitize to CCRT was assessed in p53 mutant head and neck cell lines by clonogenic assay. Modulation of the CCRT induced DNA damage response (DDR) by AUY922 was characterized by confocal image analysis of RAD51, BRCA1, 53BP1, ATM and mutant p53 signaling. The role of FANCA depletion by AUY922 was examined using shRNA. Cell cycle checkpoint abrogation and chromosomal fragmentation was assessed by western blot, FACS and confocal. The role of ATM was also assessed by shRNA. AUY922 in combination with CCRT was assessed in vivo. RESULTS: The combination of AUY922 with cisplatin, radiation and CCRT was found to be synergistic in p53 mutant HNSCC. AUY922 leads to significant alterations to the DDR induced by CCRT. This comprises inhibition of homologous recombination through decreased RAD51 and pS1524 BRCA1 with a corresponding increase in 53BP1 foci, activation of ATM and signaling into mutant p53. A shift to more error prone repair combined with a loss of checkpoint function leads to fragmentation of chromosomal material. The degree of disruption to DDR signalling correlated to chromosomal fragmentation and loss of clonogenicity. ATM shRNA indicated a possible rationale for the combination of AUY922 and CCRT in cells lacking ATM function. CONCLUSIONS: This study supports future clinical studies combining AUY922 and CCRT in p53 mutant HNSCC. Modulation of the DDR and chromosomal fragmentation are likely to be analytical points of interest in such trials.

Isolated limb perfusion with biochemotherapy and oncolytic virotherapy combines with radiotherapy and surgery to overcome treatment resistance in an animal model of extremity soft tissue sarcoma.

The management of locally advanced or recurrent extremity sarcoma often necessitates multimodal therapy to preserve a limb, of which isolated limb perfusion (ILP) is a key component. However, with standard chemotherapeutic agents used in ILP, the duration of response is limited. Novel agents or treatment combinations are urgently needed to improve outcomes. Previous work in an animal model has demonstrated the efficacy of oncolytic virotherapy when delivered by ILP and, in this study, we report further improvements from combining ILP-delivered oncolytic virotherapy with radiation and surgical resection. In vitro, the combination of radiation with an oncolytic vaccinia virus (GLV-1h68) and melphalan demonstrated increased cytotoxicity in a panel of sarcoma cell lines. The effects were mediated through activation of the intrinsic apoptotic pathway. In vivo, combinations of radiation, oncolytic virotherapy and standard ILP resulted in delayed tumour growth and prolonged survival when compared with standard ILP alone. However, local disease control could only be secured when such treatment was combined with surgical resection, the timing of which was crucial in determining outcome. Combinations of oncolytic virotherapy with surgical resection and radiation have direct clinical relevance in extremity sarcoma and represent an exciting prospect for improving outcomes in this pathology.

HOX transcription factors are potential targets and markers in malignant mesothelioma.

BACKGROUND: The HOX genes are a family of homeodomain-containing transcription factors that determine cellular identity during development and which are dys-regulated in some cancers. In this study we examined the expression and oncogenic function of HOX genes in mesothelioma, a cancer arising from the pleura or peritoneum which is associated with exposure to asbestos. METHODS: We tested the sensitivity of the mesothelioma-derived lines MSTO-211H, NCI-H28, NCI-H2052, and NCI-H226 to HXR9, a peptide antagonist of HOX protein binding to its PBX co-factor. Apoptosis was measured using a FACS-based assay with Annexin, and HOX gene expression profiles were established using RT-QPCR on RNA extracted from cell lines and primary mesotheliomas. The in vivo efficacy of HXR9 was tested in a mouse MSTO-211H flank tumor xenograft model. RESULTS: We show that HOX genes are significantly dysregulated in malignant mesothelioma. Targeting HOX genes with HXR9 caused apoptotic cell death in all of the mesothelioma-derived cell lines, and prevented the growth of mesothelioma tumors in a mouse xenograft model. Furthermore, the sensitivity of these lines to HXR9 correlated with the relative expression of HOX genes that have either an oncogenic or tumor suppressive function in cancer. The analysis of HOX expression in primary mesothelioma tumors indicated that these cells could also be sensitive to the disruption of HOX activity by HXR9, and that the expression of HOXB4 is strongly associated with overall survival. CONCLUSION: HOX genes are a potential therapeutic target in mesothelioma, and HOXB4 expression correlates with overall survival.