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Despite recent advances, the treatment of head and neck squamous cell carcinoma (HNSCC) remains an area of high unmet medical need. HNSCC is frequently associated with either amplification or mutational changes in the PI3K pathway, making PI3K an attractive target particularly in cetuximab-resistant tumors. Here, we explored the antitumor activity of the selective, pan-class I PI3K inhibitor copanlisib with predominant activity towards PI3Kα and δ in monotherapy and in combination with cetuximab using a mouse clinical trial set-up with 33 patient-derived xenograft (PDX) models with known HPV and PI3K mutational status and available data on cetuximab sensitivity. Treatment with copanlisib alone resulted in moderate antitumor activity with 12/33 PDX models showing either tumor stabilization or regression. Combination treatment with copanlisib and cetuximab was superior to either of the monotherapies alone in the majority of the models (21/33), and the effect was particularly pronounced in cetuximab-resistant tumors (14/16). While no correlation was observed between PI3K mutation status and response to either cetuximab or copanlisib, increased PI3K signaling activity evaluated through gene expression profiling showed a positive correlation with response to copanlisib. Together, these data support further investigation of PI3K inhibition in HNSCC and suggests gene expression patterns associated with PI3K signaling as a potential biomarker for predicting treatment responses.

Comprehensive molecular characterization of head and neck squamous cell carcinoma (HNSCC) has led to the identification of distinct molecular subgroups with fundamental differences in biological properties and clinical behavior. Despite improvements in tumor classification and increased understanding about the signaling pathways involved in neoplastic transformation and disease progression, current standard-of-care treatment for HNSCC mostly remains to be based on a stage-dependent strategy whereby all patients at the same stage receive the same treatment. Preclinical models that closely resemble molecular HNSCC subgroups that can be exploited for dissecting the biological function of genetic variants and/or altered gene expression will be highly valuable for translating molecular findings into improved clinical care. In the present review, we merge and discuss existing and new information on established cell lines, primary two- and three-dimensional ex vivo tumor cultures from HNSCC patients, and animal models. We review their value in elucidating the basic biology of HNSCC, molecular mechanisms of treatment resistance and their potential for the development of novel molecularly stratified treatment.

Immune checkpoint inhibition leads to response in some patients with head and neck squamous cell carcinoma (HNSCC). Robust biomarkers are lacking to date. We analyzed viral status, gene expression signatures, mutational load and mutational signatures in whole exome and RNA-sequencing data of the HNSCC TCGA dataset (n = 496) and a validation set (DKTK MASTER cohort, n = 10). Public single-cell gene expression data from 17 HPV-negative HNSCC were separately reanalyzed. APOBEC3-associated TCW motif mutations but not total single nucleotide variant burden were significantly associated with inflammation. This association was restricted to HPV-negative HNSCC samples. An APOBEC-enriched, HPV-negative subgroup was identified, that showed higher T-cell inflammation and immune checkpoint expression, as well as expression of APOBEC3 genes. Mutations in immune-evasion pathways were also enriched in these tumors. Analysis of single-cell sequencing data identified expression of APOBEC3B and 3C genes in malignant cells. We identified an APOBEC-enriched subgroup of HPV-negative HNSCC with a distinct immunogenic phenotype, potentially mediating response to immunotherapy.

Biomarkers with relevance for loco-regional therapy are needed in human papillomavirus negative aka HPV(-) head and neck squamous cell carcinoma (HNSCC). Based on the premise that DNA methylation pattern is highly conserved, we sought to develop a reliable and robust methylome-based classifier identifying HPV(-) HNSCC patients at risk for loco-regional recurrence (LR) and all-event progression after postoperative radiochemotherapy (PORT-C). The training cohort consisted of HPV-DNA negative HNSCC patients (n = 128) homogeneously treated with PORT-C in frame of the German Cancer Consortium-Radiation Oncology Group (DKTK-ROG) multicenter biomarker trial. DNA Methylation analysis was performed using Illumina 450 K and 850 K-EPIC microarray technology. The performance of the classifier was integrated with a series of biomarkers studied in the training set namely hypoxia-, 5-microRNA (5-miR), stem-cell gene-expression signatures and immunohistochemistry (IHC)-based immunological characterization of tumors (CD3/CD8/PD-L1/PD1). Validation occurred in an independent cohort of HPV(-) HNSCC patients, pooled from two German centers (n = 125). We identified a 38-methylation probe-based HPV(-) Independent Classifier of disease Recurrence (HICR) with high prognostic value for LR, distant metastasis and overall survival (P < 10-9 ). HICR remained significant after multivariate analysis adjusting for anatomical site, lymph node extracapsular extension (ECE) and size (T-stage). HICR high-risk tumors were enriched for younger patients with hypoxic tumors (15-gene signature) and elevated 5-miR score. After adjustment for hypoxia and 5-miR covariates, HICR maintained predicting all endpoints. HICR provides a novel mean for assessing the risk of LR in HPV(-) HNSCC patients treated with PORT-C and opens a new opportunity for biomarker-assisted stratification and therapy adaptation in these patients. Keywords: DNA methylation; disease recurrence; head and neck cancers; radiotherapy; stratification

Current algorithms for the clinical management of patients with squamous cell carcinoma of the head and neck (HNSCC) are based on a stage-dependent strategy where all patients at the same TNM stage receive the same treatment. Patient outcomes might be substantially improved by biomarker-guided treatment selection based on individual differences in the genetic and biological characteristics of tumours. Rapid technical advances enabling fast and affordable comprehensive molecular characterisation of tumours have led to increased knowledge of the molecular pathways involved in neoplastic transformation and disease progression in HNSCC. Despite notable successes in other tumour entities, the exploitation of molecular data for the improvement of tumour staging, prognosis, and individual treatment selection for patients with HNSCC has not yet become clinical routine. In this Review, we discuss and merge existing and new information on prognostic biomarkers for HNSCC, with the potential to improve clinical management of patients in the near future.

The prevalence of oropharyngeal squamous cell carcinoma (OPSCC) is increasing globally while the prevalence of other head and neck cancers is decreasing. The most likely reasons for this are a decreasing influence of smoking and the growing relevance of infections with the human papilloma virus (HPV) as a risk factor. A rise in the HPV‐attributable fraction (HPV‐AF) of OPSCC has been observed in many countries, yet a comprehensive review of prevalence rates and trends in Germany is lacking. To determine the current HPV‐AF of OPSCC in Germany and to assess whether it has changed during the last decades, we performed a systematic literature review. We screened Medline and EMBASE for studies that reported the tumor HPV status of newly diagnosed OPSCC patients treated at medical centers in Germany by testing for both HPV DNA and p16INK4a overexpression to confirm involvement of HPV in tumorigenesis. Out of 287 screened studies, 14 studies with data from a total of 1819 OPSCC patients treated between 1988 and 2015 were included in the data synthesis. The reported average HPV‐AF varied considerably between the studies, ranging from 11.5% (1988‐2008) to 55.0% (2004‐2009). Two of the included studies did not only provide the HPV‐AF for the entire observed calendar period but also for separate years, allowing to more accurately assess changes over time. These studies reported increases in the HPV‐AF from 21% in 2000 to 53% in 2015 and from 38% in 2004 to 71% in 2013, respectively.

Genetic investigation of tumor heterogeneity and clonal evolution in solid cancers could be assisted by the analysis of liquid biopsies. However, tumors of various entities might release different quantities of circulating tumor cells (CTCs) and cell-free DNA (cfDNA) into the bloodstream, potentially limiting the diagnostic potential of liquid biopsy in distinct tumor histologies. Patients with advanced colorectal cancer (CRC), head and neck squamous cell carcinoma (HNSCC), and melanoma (MEL) were enrolled in the study, representing tumors with different metastatic patterns. Mutation profiles of cfDNA, CTCs, and tumor tissue were assessed by panel sequencing, targeting 327 cancer-related genes. In total, 30 tissue, 18 cfDNA, and 7 CTC samples from 18 patients were sequenced. Best concordance between the mutation profile of tissue and cfDNA was achieved in CRC and MEL, possibly due to the remarkable heterogeneity of HNSCC (63%, 55% and 11%, respectively). Concordance especially depended on the amount of cfDNA used for library preparation. While 21 of 27 (78%) tissue mutations were retrieved in high-input cfDNA samples (30–100 ng, N = 8), only 4 of 65 (6%) could be detected in low-input samples (N = 10). CTCs were detected in 13 of 18 patients (72%). However, downstream analysis was limited by poor DNA quality, allowing targeted sequencing of only seven CTC samples isolated from four patients. Only one CTC sample reflected the mutation profile of the respective tumor. Private mutations, which were detected in CTCs but not in tissue, suggested the presence of rare subclones. Our pilot study demonstrated superiority of cfDNA- compared to CTC-based mutation profiling. It was further shown that CTCs may serve as additional means to detect rare subclones possibly involved in treatment resistance. Both findings require validation in a larger patient cohort.

Body fluids of cancer patients have attracted increasing attention in biomedical research within the last 15 years since—as so-called liquid biopsies—they represent a non-invasive source of clinically exploitable biomarkers, including circulating tumor cells (CTCs) and cell-free tumor DNA (ctDNA). Assessment of CTCs in peripheral blood from solid cancer patients has proven useful for detection of subclinical disease which otherwise remains invisible for current staging techniques. Based on results from large cohort studies in breast and colon cancer, diagnostic tests for enumeration of CTCs have been developed which can be used for tumor staging, prognosis, and post-treatment surveillance. Circulating plasma DNA derived from Epstein–Barr or human papilloma viruses has been established as a sensitive and highly specific biomarker for early cancer detection and disease monitoring. More recently, first studies have been initiated for studying the diagnostic value of mutant variants in plasma-derived ctDNA for treatment selection, response assessment and early detection of treatment failure.Advanced Head and Neck Squamous Cell Carcinoma (HNSCC) represents a malignancy associated with locoregionally advanced stage at presentation, dismal prognosis and little improvement in treatment outcome over the past decade, especially for patients with metastatic disease. HNSCC patients might therefore benefit from incorporation of liquid biopsy-based assays in clinical management. In the following chapters, I will summarize current evidence of the diagnostic value of liquid biopsies in HNSCC and give examples of potential clinical applications.

3D tumor models clearly outperform 2D cell cultures in recapitulating tissue architecture and drug response. However, their potential in understanding treatment efficacy and resistance development should be better exploited if also long-term effects of treatment could be assessed in vitro. The main disadvantages of the matrices commonly used for in vitro culture are their limited cultivation time and the low comparability with patient-specific matrix properties. Extended cultivation periods are feasible when primary human cells produce the extracellular matrix in situ. Herein, we adapted the hyalograft-3D approach from reconstructed human skin to normal and tumor oral mucosa models and compared the results to bovine collagen-based models. The hyalograft models showed similar morphology and cell proliferation after 7 weeks compared to collagen-based models after 2 weeks of cultivation. Tumor thickness and VEGF expression increased in hyalograft-based tumor models, whereas expression of laminin-332, tenascin C, and hypoxia-inducible factor 1α was lower than in collagen-based models. Taken together, the in situ produced extracellular matrix better confined tumor invasion in the first part of the cultivation period, with continuous tumor proliferation and increasing invasion later on. This proof-of-concept study showed the successful transfer of the hyalograft approach to tumor oral mucosa models and lays the foundation for the assessment of long-term drug treatment effects. Moreover, the use of an animal-derived extracellular matrix is avoided.

Objectives Fever-range whole body hyperthermia (FRWBH) has been shown to improve tumor oxygenation in vivo. A prospective pilot study addressed the question if addition of FRWBH to re-irradiation is feasible in recurrent head and neck squamous cell carcinomas (HNSCC) with unfavorable prognostic features. Materials and methods The study completed accrual with the recruitment of ten patients between April 2018 and March 2020. Re-irradiation was administered using volumetric arc hyperfractionated radiotherapy with bi-daily 1.2 Gray (Gy) single fractions and a total dose of 66 Gy to all macroscopic tumor lesions. Concomitant chemotherapy consisted mostly of cisplatin (7 patients). FRWBH was scheduled weekly during re-irradiation. The study was registered in the clinicaltrials.gov database (NCT03547388). Results Only five patients received all cycles of FRWBH. Poor patient compliance, active infections during treatment and study restrictions due to the Covid-19 pandemic were the main reasons for omitting FRWBH. No increase of acute toxicity was observed by FRWBH. Exploratory evaluation of outcome data suggests that FRWBH treatment according to protocol does not seem to have a detrimental effect on tumor control or survival and might even increase treatment efficacy. Conclusion FRWBH is difficult to apply concomitant to re-irradiation in HNSCC. No excess toxicity was observed in patients receiving FRWBH and exploratory analyses suggest potential anti-tumor activity and decreased patient-reported depression scores after FRWBH.

Pharmacotherapy of head and neck squamous cell carcinoma (HNSCC) often fails due to the development of chemoresistance and severe systemic side effects of current regimens limiting dose escalation. Preclinical models comprising all major elements of treatment resistance are urgently needed for the development of new strategies to overcome these limitations. For model establishment, we used tumor cells from patient-derived HNSCC xenografts or cell lines (SCC-25, UM-SCC-22B) and characterized the model phenotype. Docetaxel and cetuximab were selected for comparative analysis of drug-related effects at topical and systemic administration. Cetuximab cell binding was mapped by cluster-based fluorescence lifetime imaging microscopy.The tumor oral mucosa (TOM) models displayed unstructured, hyper-proliferative, and pleomorphic cell layers, reflecting well the original tumor morphology and grading. Dose- and time-dependent effects of docetaxel on tumor size, apoptosis, hypoxia, and interleukin-6 release were observed. Although the spectrum of effects was comparable, significantly lower doses were required to achieve similar docetaxel-induced changes at topical compared to systemic application. Despite displaying anti-proliferative effects in monolayer cultures, cetuximab treatment showed only minor effects in TOM models. This was not due to inefficient cetuximab uptake or target cell binding but likely mediated by microenvironmental components.We developed multi-layered HNSCC models, closely reflecting tumor morphology and displaying complex interactions between the tumor and its microenvironment. Topical application of docetaxel emerged as promising option for HNSCC treatment. Aside from the development of novel strategies for topical drug delivery, our tumor model might help to better understand key regulators of drug-tumor-interactions.

Identifying patients with locally advanced head and neck carcinoma on high risk of recurrence after definitive concurrent radiochemotherapy is of key importance for the selection for consolidation therapy and for individualized treatment intensification. In this multicenter study we analyzed recurrence-associated single-nucleotide polymorphisms (SNPs) in DNA repair genes in tumor DNA from 132 patients with locally advanced head and neck carcinoma (LadHnSCC). Patients were treated with definitive radiotherapy and simultaneous cisplatin-based chemotherapy at six partner sites of the German Cancer Consortium (DKTK) Radiation Oncology Group from 2005 to 2011. For validation, a group of 20 patients was available. Score selection method using proportional hazard analysis and leave-one-out cross-validation were performed to identify markers associated with outcome. The SNPs rs1799793 and rs13181 were associated with survival and the same SNPs and in addition rs17655 with freedom from loco-regional relapse (ffLRR) in the trainings datasets from all patients. The homozygote major rs1799793 genotype at the ERCC2 gene was associated with better (Hazard ratio (HR): 0.418 (0.234–0.744), p = 0.003) and the homozygote minor rs13181 genotype at ERCC2 with worse survival (HR: 2.074, 95% CI (1.177–3.658), p = 0.017) in comparison to the other genotypes. At the ffLRR endpoint, rs1799793 and rs13181 had comparable prognostic value. The rs1799793 and rs13181 genotypes passed the leave-one-out cross-validation procedure and associated with survival and ffLRR in patients with LadHnSCC treated with definitive radiochemotherapy. While findings were confirmed in a small validation dataset, further validation is underway within a prospective biomarker study of the DKTK.

This article compares the expression and applicability of biomarkers, from single genes and gene signatures, identified in patients with locally advanced head and neck squamous cell carcinoma using the GeneChip Human Transcriptome Array 2.0, nCounter, and real-time PCR analyses. Two multicenter, retrospective cohorts of patients with head and neck squamous cell carcinoma from the German Cancer Consortium Radiation Oncology Group who received postoperative radiochemotherapy or primary radiochemotherapy were considered. Real-time PCR was performed for a limited number of 38 genes of the cohort who received postoperative radiochemotherapy only. Correlations between the methods were evaluated by the Spearman rank correlation coefficient. Patients were stratified based on the expression of putative cancer stem cell markers, hypoxia-associated gene signatures, and a previously developed seven-gene signature. Locoregional tumor control was compared between these patient subgroups using log-rank tests. Gene expressions obtained from nCounter analyses were moderately correlated to GeneChip analyses (median ρ = approximately 0.68). A higher correlation was obtained between nCounter analyses and real-time PCR (median ρ = 0.84). Significant associations with locoregional tumor control were observed for most of the considered biomarkers evaluated by GeneChip and nCounter analyses. In general, all applied biomarkers (single genes and gene signatures) classified approximately 70% to 85% of the patients similarly. Overall, gene signatures seem to be more robust and had a better transferability among different measurement methods.

Purpose: Platinum-based drugs, in particular cisplatin (cis-diamminedichloridoplatinum(II), CDDP), are used for treatment of squamous cell carcinoma of the head and neck (SCCHN). Despite initial responses, CDDP treatment often results in chemoresistance, leading to therapeutic failure. The role of primary resistance at subclonal level and treatment-induced clonal selection in the development of CDDP resistance remains unknown. Experimental Design: By applying targeted next-generation sequencing, fluorescence in situ hybridization, microarray-based transcriptome, and mass spectrometry-based phosphoproteome analysis to the CDDP-sensitive SCCHN cell line FaDu, a CDDP-resistant subline, and single-cell derived subclones, the molecular basis of CDDP resistance was elucidated. The causal relationship between molecular features and resistant phenotypes was determined by siRNA-based gene silencing. The clinical relevance of molecular findings was validated in patients with SCCHN with recurrence after CDDP-based chemoradiation and the TCGA SCCHN dataset. Results: Evidence of primary resistance at clonal level and clonal selection by long-term CDDP treatment was established in the FaDu model. Resistance was associated with aneuploidy of chromosome 17, increased TP53 copy-numbers and overexpression of the gain-of-function (GOF) mutant variant p53R248L. siRNA-mediated knockdown established a causal relationship between mutant p53R248L and CDDP resistance. Resistant clones were also characterized by increased activity of the PI3K–AKT–mTOR pathway. The poor prognostic value of GOF TP53 variants and mTOR pathway upregulation was confirmed in the TCGA SCCHN cohort. Conclusions: Our study demonstrates a link of intratumoral heterogeneity and clonal evolution as important mechanisms of drug resistance in SCCHN and establishes mutant GOF TP53 variants and the PI3K/mTOR pathway as molecular targets for treatment optimization. Clin Cancer Res; 24(1); 158–68. ©2017 AACR.

Cetuximab is the single targeted therapy approved for the treatment of head and neck cancer (HNSCC). Predictive biomarkers have not been established and patient stratification based on molecular tumor profiles has not been possible. Since EGFR pathway activation is pronounced in basal subtype, we hypothesized this activation could be a predictive signature for an EGFR directed treatment. From our patient-derived xenograft platform of HNSCC, 28 models were subjected to Affymetrix gene expression studies on HG U133+ 2.0. Based on the expression of 821 genes, the subtype of each of the 28 models was determined by integrating gene expression profiles through centroid-clustering with previously published gene expression data by Keck et al. The models were treated in groups of 5-6 animals with docetaxel, cetuximab, everolimus, cis- or carboplatin and 5-fluorouracil. Response was evaluated by comparing tumor volume at treatment initiation and after 3 weeks of treatment (RTV). Tumors distributed over the 3 signature-defined subtypes: 5 mesenchymal/inflamed phenotype (MS), 15 basal type (BA), 8 classical type (CL). Cluster analysis revealed a strong correlation between response to cetuximab and the basal subtype. RTV MS 3.32 vs. BA 0.78 (MS vs. BA, unpaired t-test, p 0.0002). Cetuximab responders were distributed as following: 1/5 in MS, 5/8 in CL and 13/15 in the BA group. Activity of classical chemotherapies did not differ between the subtypes. In conclusion basal subtype was associated with response to EGFR directed therapy in head and neck squamous cell cancer patient-derived xenografts.

Kopf-Hals-Tumoren sind weltweit auf Platz 6 der haufigsten Krebsarten. Neben den klassischen Noxen Alkohol und Tabak sind fur Oropharynxkarzinome mittlerweile Infektionen mit humanen Papillomviren (HPV) als pathogenetisch auslosende Faktoren identifiziert worden. HPV-assoziierte Oropharynxkarzinome steigen in Europa, Japan und den USA immer weiter an. Die Onkoproteine E6 und E7 stellen dabei die pathogenetisch entscheidenden Faktoren dar. Die Prognose der Patienten mit HPV-assoziierten Oropharynxkarzinomen ist unter allen primar oder sekundar eingesetzten Therapiemodalitaten (Operation, adjuvante oder palliative Chemo- oder Radiotherapie, molekular zielgerichtete Therapie oder Immuncheckpointblockade) gunstiger. Das 2017 aktualisierte Cancer Staging System des American Joint Committee on Cancer fuhrt HPV-assoziierte Oropharynxkarzinome nun als gesonderte Entitat auf. Trotz der gunstigeren Prognose wird in aktuellen Leitlinien eine Deeskalation der Therapie bei Patienten mit HPV- oder p16-positivem Oropharynxkarzinom auserhalb kontrollierter klinischer Studien abgelehnt. Eine HPV-Impfung junger Manner sollte empfohlen werden.

Highlights • Next-Generation Sequencing cost has significantly decreased recently. • NGS is used to comprehensively assess tumoral radiosensitivity. • Personalized and dose-adapted radiotherapy could be achieved through the use of these technologies., Technological advances have led to more precise radiation delivery, which has resulted in significant clinical gains. A better understanding of tumoral radiosensitivity is still needed to develop strategies and further personalize radiation treatments. Next-Generation Sequencing (NGS) and system biology have significantly transformed the field of oncology in the last two decades, but have only a few clinical applications in radiation oncology. This review describes the technical aspects and evolutions of NGS and discusses the latest clinical applications of genomics to predict tumoral radiosensitivity.

Background Reliable and reproducible interpretation of molecular aberrations constitutes a bottleneck of precision medicine. Evidence-based decision management systems may improve rational therapy recommendations. To cope with an increasing amount of complex molecular data in the clinical care of patients with cancer, we established a workflow for the interpretation of molecular analyses. Methods A specialized physician screened results from molecular analyses for potential biomarkers, irrespective of the diagnostic modality. Best available evidence was retrieved and categorized through establishment of an in-house database and interrogation of publicly available databases. Annotated biomarkers were ranked using predefined evidence levels and subsequently discussed at a molecular tumour board (MTB), which generated treatment recommendations. Subsequent translation into patient treatment and clinical outcomes were followed up. Results One hundred patients were discussed in the MTB between January 2016 and May 2017. Molecular data were obtained for 70 of 100 patients (50 whole exome/RNA sequencing, 18 panel sequencing, 2 immunohistochemistry (IHC)/microsatellite instability analysis). The MTB generated a median of two treatment recommendations each for 63 patients. Thirty-nine patients were treated: 6 partial responses and 12 stable diseases were achieved as best responses. Genetic counselling for germline events was recommended for seven patients. Conclusion The development of an evidence-based workflow allowed for the clinical interpretation of complex molecular data and facilitated the translation of personalized treatment strategies into routine clinical care. The high number of treatment recommendations in patients with comprehensive genomic data and promising responses in patients treated with combination therapy warrant larger clinical studies.

Dysphagia represents one of the most serious adverse events after curative-intent treatments with a tremendous impact on quality of life in patients with head and neck cancers. Novel surgical and radiation therapy techniques have been developed to better preserve swallowing function, while not negatively influencing local control and/or overall survival. This review focuses on the current literature of swallowing outcomes after curative treatment strategies. Available results from recent studies relevant to this topic are presented, demonstrating the potential role of new treatment modalities for early- and intermediate-stage oropharyngeal cancers. Based on this, we present the rationale and design of the currently active EORTC 1420 "Best of" trial, and highlight the potential of this study to help prioritizing either surgery- or radiation-based treatment modalities for the treatment of oropharyngeal cancer in the future. ispartof: FRONTIERS IN ONCOLOGY vol:9 ispartof: location:Switzerland status: published

Background: Intra-tumoral CD8 + T-cell infiltration in squamous cell carcinoma of the head and neck (HNSCC) has previously been linked to the efficacy of cisplatin-based chemoradiation (CDDP-CRTX) and immune checkpoint inhibitor (ICI) monotherapy. Further detailed characterization of the tumor immune-micromilieu and its influence on outcome may guide the development of CRTX-ICI combinations. Methods: Comprehensive immune transcriptome analysis was applied to a training set of tumor specimens from oropharyngeal squamous cell carcinoma (OPSCC) patients treated with CDDP-CRTX in the ARO-0401 phase III study (n = 33). A composite immune signature risk score (ISRS) for survival prediction was developed, and subsequently validated in two independent OPSCC cohorts treated with either CDDP-CRTX (n = 36) or mitomycin-based CRTX (MMC-CRTX, n = 31). Further validation of the ISRS was performed in the OPSCC subset (n = 79) of the TCGA HNSCC cohort. Potential interference between immune signatures and HPV status was evaluated in multivariate Cox regression models. Results: Significant differences according to the 3-y OS status in the abundance of tumor-infiltrating T- and B-cells, and the expression levels of 51 immune-related genes were observed. A risk score based on 13 differentially expressed genes involved in cytokine signaling, T-cell effector functions and the TNFR pathway was established as robust predictive factor of OS. Its predictive power was superior to the 6-gene interferon-gamma signature of ICI efficacy and independent of the HPV status. Conclusions: This study further elucidates the complex interaction of the tumor immune microenvironment with the efficacy of CDDP-CRTX in OPSCC. The results suggest immune markers for selection of patients treated with CRTX-ICI combinations.

Immunotherapy is effective in head and neck squamous cell carcinoma (HNSCC), but only a minority of patients responds to immune checkpoint blockade (ICB). To contribute to a better understanding of the underlying immune biology, we combined histomorphological evaluation and molecular analysis of the HNSCC immune microenvironment in the TCGA cohort. Analyzing digital HE-stained slides, a method for classification of tumor infiltrating lymphocytes (TILs) in the intra-epithelial compartment (ieTILs, present vs. absent) and the stromal compartment (strTILs, high vs. low) was established. We also analyzed the abundance of eight immune cell populations (estimated from RNAseq data) and PD-L1 mRNA expression. Status of ieTILs and status of strTILs were concordant for 61%, but discordant for 39% of tumors. In univariate survival analysis, ieTILs were a positive prognostic marker for DFS in the study cohort (HR = 0.66, p = 0.015) and in the HPV− subcohort (HR = 0.68, p = 0.04), but not in the HPV + subcohort. T cells were a positive prognostic marker for DFS in the study cohort (HR = 0.80, p = 0.03) and in the HPV + subcohort (HR = 0.20, p = 0.001), but not in the HPV− subcohort. In univariate survival analysis, PD-L1 mRNA expression was neither associated with DFS nor with OS. However, in bivariate and multivariate analyses including both PD-L1 mRNA levels and T cells, PD-L1 was a negative prognostic marker of DFS and OS, while T cells remained a positive prognostic marker. In conclusion, ieTILs and strTILs were non-redundant biomarkers in HNSCC and should be evaluated separately. The identified prognostic markers should be evaluated for predictivity in ICB-treated patients.

Immune checkpoint inhibition leads to response in some patients with head and neck squamous cell carcinoma (HNSCC). Robust biomarkers are lacking to date. We analyzed viral status, gene expression signatures, mutational load and mutational signatures in whole exome and RNA-sequencing data of the HNSCC TCGA dataset (N = 496) and a validation set (DKTK MASTER cohort, N = 10). Public single-cell gene expression data from 17 HPV-negative HNSCC were separately re-analyzed. APOBEC3-associated TCW motif mutations but not total single nucleotide variant burden were significantly associated with inflammation. This association was restricted to HPV-negative HNSCC samples. An APOBEC-enriched, HPV-negative subgroup was identified, that showed higher T-cell inflammation and immune checkpoint expression, as well as expression of APOBEC3 genes. Mutations in immune-evasion pathways were also enriched in these tumors. Analysis of single-cell sequencing data identified expression of APOBEC3B and 3C genes in malignant cells. We identified an APOBEC-enriched subgroup of HPV-negative HNSCC with a distinct immunogenic phenotype, potentially mediating response to immunotherapy.

// Evelyn Kidess-Sigal 1, 2, * , Haiyan E. Liu 3, * , Melanie M. Triboulet 2 , James Che 3 , Vishnu C. Ramani 2 , Brendan C. Visser 2 , George A. Poultsides 2 , Teri A. Longacre 4 , Andre Marziali 5 , Valentina Vysotskaia 6 , Matthew Wiggin 5 , Kyra Heirich 2 , Violet Hanft 2 , Ulrich Keilholz 7 , Ingeborg Tinhofer 8 , Jeffrey A. Norton 2 , Mark Lee 9 , Elodie Sollier-Christen 3 , Stefanie S. Jeffrey 2 1 Department of Medicine, Division of Hepatology and Gastroenterology, Charite University Hospital, Berlin, Germany 2 Department of Surgery, Stanford University School of Medicine, Stanford, CA, USA 3 Vortex BioSciences, Inc., Menlo Park, CA, USA 4 Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA 5 Boreal Genomics, Vancouver, BC, Canada 6 Counsyl Inc., San Francisco, CA, USA 7 Comprehensive Cancer Center Charite, Berlin, Germany 8 Department of Radiooncology and Radiotherapy, Charite University Hospital, Berlin, Germany 9 GRAIL, Redwood City, CA, USA * These authors have contributed equally to this work Correspondence to: Stefanie S. Jeffrey, email: ssj@stanford.edu Keywords: colorectal cancer, circulating tumor cells, circulating tumor DNA, liquid biopsy, Vortex Received: June 30, 2016 Accepted: October 26, 2016 Published: November 15, 2016 ABSTRACT Treatment of advanced colorectal cancer (CRC) requires multimodal therapeutic approaches and need for monitoring tumor plasticity. Liquid biopsy biomarkers, including CTCs and ctDNA, hold promise for evaluating treatment response in real-time and guiding therapeutic modifications. From 15 patients with advanced CRC undergoing liver metastasectomy with curative intent, we collected 41 blood samples at different time points before and after surgery for CTC isolation and quantification using label-free Vortex technology. For mutational profiling, KRAS, BRAF, and PIK3CA hotspot mutations were analyzed in CTCs and ctDNA from 23 samples, nine matched liver metastases and three primary tumor samples. Mutational patterns were compared. 80% of patient blood samples were positive for CTCs, using a healthy baseline value as threshold (0.4 CTCs/mL), and 81.4% of captured cells were EpCAM+ CTCs. At least one mutation was detected in 78% of our blood samples. Among 23 matched CTC and ctDNA samples, we found a concordance of 78.2% for KRAS, 73.9% for BRAF and 91.3% for PIK3CA mutations. In several cases, CTCs exhibited a mutation that was not detected in ctDNA, and vice versa. Complementary assessment of both CTCs and ctDNA appears advantageous to assess dynamic tumor profiles.

// Stefano Meucci 1 , Ulrich Keilholz 1 , Ingeborg Tinhofer 2 and Oliva A. Ebner 1 1 Charite Comprehensive Cancer Center, Charite University Hospital, Chariteplatz, Berlin, Germany 2 Department of Radiation Oncology and Radiotherapy, Charite University Hospital Berlin, Translational Radiation Oncology Research Laboratory, Chariteplatz, Berlin, Germany Correspondence to: Ulrich Keilholz, email: // Stefano Meucci, email: // Keywords : head and neck squamous cell carcinoma; aging; somatic mutations; sequencing; genomics; Gerotarget Received : December 21, 2015 Accepted : July 23, 2016 Published : August 16, 2016 Abstract Head and neck squamous cell carcinoma (HNSCC) is a cancer with well-defined tumor causes such as HPV infection, smoking and drinking. Using The Cancer Genome Atlas (TCGA) HNSCC cohort we systematically studied the mutational load as well as patterns related to patient age in HNSCC. To obtain a homogenous set we excluded all patients with HPV infection as well as wild type TP53. We found that the overall mutational load is higher in patients of old age. Through unsupervised hierarchical clustering, we detected distinct mutational clusters in very young as well as very old patients. In the group of old patients, we identified four enriched pathways (“Axon Guidance”, “ECM-Receptor Interaction”, “Focal Adhesion” and “Notch Signaling”) that are only sporadically mutated in the other age groups. Our findings indicate that the four pathways regulate cell motility, tumor invasion and angiogenesis supposedly leading to less aggressive tumors in older age patients. Importantly, we did not see a strict pattern of genes always mutated in older age but rather an accumulation of mutations in the same pathways. Our study provides indications of age-dependent differences in mutational backgrounds of tumors that might be relevant for treatment approaches of HNSCCs patients.

Despite recent advances in radiochemotherapy, treatment of locally advanced head and neck squamous cell carcinoma is still challenging, and survival rates have improved only slightly. This is due to the high frequency of metastases and local and/or regional tumor recurrences that have acquired radio- or chemoresistance. MiRNAs regulate diverse processes in tumorigenesis, metastasis, and therapy resistance in head and neck squamous cell carcinoma. Hence, miRNAs are highly valued in biomarker studies. Establishment of the miRNA profiles of oropharyngeal tumors enables personalized treatment selection, since expression of distinct miRNAs can predict the response to two different radiochemotherapy regimens. Development of novel miRNA therapeutics has a high clinical potential for further improving treatment of cancerous disease. The use of nanoparticles with distinct surface modifications as miRNA vectors permits prolonged bioavailability, high efficacy in tumor targeting, and low toxicity. Nevertheless, the efficacy of miRNA therapy has only been shown in animal models to date.

Detection of circulating tumor cells (CTCs) has been established as a blood-based biomarker of poor prognosis in most types of epithelial cancer. The observation of increased CTC detection rates in patients with metastatic disease supported the hypothesis that CTCs might represent the precursor cells of distant metastases. Experimental proof for this hypothesis was provided by studies in experimental mouse models in which single metastasis-initiating cells within the total pool of CTCs could be causally linked to the formation of distant metastases. In addition, a potential of CTCs of reseeding at the primary site has been reported, suggesting that CTCs could also play a role in local tumor recurrence. Enumeration and molecular characterization of CTCs might thus be clinically exploited for improving the clinical management of epithelial cancers both in the locally advanced and recurrent/metastatic disease setting. Numerous studies reported on the presence of CTCs in peripheral blood samples of head and neck squamous cell carcinoma (HNSCC) patients. Significant associations with clinical high-risk parameters such as advanced stage, high tumor burden, nodal disease, and distant metastasis were reported. Accordingly, CTC detection was correlated with increased risk of local and/or distant recurrence, progression-free and overall survival. Current data in HNSCC are not yet robust enough for the implementation of CTC detection in clinical routine. In addition, the mere enumeration of CTCs has proven insufficiently informative to prompt widespread clinical adoption. There is accumulating evidence that more extended phenotyping of CTCs might be necessary for improving their diagnostic value. Indeed, despite the estimated daily shedding of millions of tumor cells from the bulk tumor tissue, only a small subpopulation of CTCs is able to reach and survive in the blood circulation and to contribute to the formation of distant metastasis. Identification of the true metastasis-inducing subclones within the bulk CTC population remains a challenge but is imperative in order to improve the diagnostic potential of CTCs. In addition, improved knowledge of the relevant biologic mechanisms endowing CTCs with the potential to emigrate from the primary site to blood circulation, to survive their journey, and to reseed at distant organs will support the development of novel antimetastatic therapies. Several technologies are currently available that allow multiparametric CTC phenotyping at the single-cell level. These platforms include imaging flow cytometry (IFC), by which multispectral images from a large number of cells acquired in suspension can be analyzed. By combining the features of flow cytometry and fluorescent microscopy, information on the cell morphology together with the extent of target protein expression and its subcellular localization can be collected. This provides a unique opportunity for statistical analysis of large populations, but also helps in avoiding artifacts. In the talk, the evidence of a prognostic role of CTCs in HNSCc will be summarized. Results from recent studies reporting on multiparametric CTC phenotyping at the single-cell level will be presented. Examples of CTC-based biomarkers that could be exploited for the optimization of current molecular treatment strategies in HNSCC will be discussed. Citation Format: Ingeborg Tinhofer. Beyond counting: Imaging flow cytometry-based detection of therapeutic molecular targets in circulating tumor cells [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr IA09.

Background Tumour mutational burden (TMB) estimated from whole exome sequencing or comprehensive gene panels has previously been established as predictive factor of response to immune checkpoint inhibitors (ICIs). Its predictive value for the efficacy of concurrent chemoradiation (cCRTX), a potential combination partner of ICI, remains unknown. Methods The accuracy of TMB estimation by an in-house 327-gene panel was established in the Cancer Genome Atlas (TCGA) head and neck squamous cell carcinoma (HNSCC) data set. Interference of TMB with outcome after cCRTX was determined in a multicentre cohort of patients with locally advanced HNSCC uniformly treated with cCRTX. Targeted next-generation sequencing was successfully applied in 101 formalin-fixed, paraffin-embedded pretreatment tumour samples. In a subset of cases (n = 40), tumour RNA was used for immune-related gene expression profiling by the nanoString platform. TMB was correlated with TP53 genotype, human papilloma virus (HPV) status, immune expression signatures and survival parameters. Results were validated in the TCGA HNSCC cohort. Results A high accuracy of TMB estimation by the 327-gene panel was established. High TMB was significantly associated with an increased prevalence of TP53 mutations and immune gene expression patterns unrelated to T cell–inflamed gene expression profiles. Kaplan-Meier analysis revealed significantly reduced overall survival in the patient group with high TMB (hazard ratio for death: 1.79, 95% confidence interval: 1.02–3.14; P = 0.042) which remained significant after correcting for confounding factors in the multivariate model. The prognostic value of TMB was confirmed in the TCGA HNSCC cohort. Conclusion High TMB identifies HNSCC patients with poor outcome after cCRTX who might preferentially benefit from CRTX-ICI combinations.

Assessment of circulating tumor cells (CTCs) in peripheral blood from solid cancer patients including head and neck squamous cell carcinoma (HNSCC) has proven useful for detection of subclinical disease that otherwise remains invisible for current staging techniques. Based on large cohort studies, diagnostic tests for enumeration of CTCs have been developed, which can be used for tumor staging, prognosis, treatment monitoring, and post-treatment surveillance. Areas covered: Here, we briefly summarize the history of CTC discovery. We review the current evidence of a diagnostic potential of CTCs in HNSCC. We present recent technical advancements in the tools for enrichment, detection, and molecular characterization of CTCs. We also discuss potential clinical applications of CTC detection for personalized treatment strategies. Expert commentary: Recent technical advances in the platforms for enrichment, detection, and comprehensive molecular characterization of CTC represent a great opportunity to improve our understanding in the biology of CTCs and will allow to expand the clinical utility of CTCs in HNSCC. Especially, HNSCC patients with recurrent/metastatic disease associated dismal prognosis and little improvement in treatment outcome over the past decade might benefit in the future from incorporation of CTC assays in clinical management.

Rapid technical advances in the molecular characterization of tumors, enabling complete gene sequencing of multiple cancers in the Cancer Genome Project, have led to a largely increased knowledge of the molecular pathways that underlie cancer. The integration of high-throughput sequencing, known more commonly as “next-generation” sequencing (NGS), into standard clinical practice has enabled more targeted treatment of various malignancies based on the presence of specific alterations. Despite notable successes in other tumor entities, individual treatment selection for patients with squamous cell carcinoma of the head and neck (HNSCC) based on molecular tumor profiles has not yet become clinical practice. In the following chapters, we describe the genomic alterations detected in NGS studies of HNSCC which define specific molecular subgroups and whose contribution to oncogenesis provide a biological rationale for the use of a specific targeted therapy. We highlight different tumor sequencing strategies currently used for precision oncology and describe their individual strengths and weaknesses. We also discuss unresolved hurdles in the translation of molecular findings into concepts for personalized HNSCC treatment.

Purpose: The aim of this study was to identify and independently validate a novel gene signature predicting locoregional tumor control (LRC) for treatment individualization of patients with locally advanced HPV-negative head and neck squamous cell carcinomas (HNSCC) who are treated with postoperative radio(chemo)therapy (PORT-C). Experimental Design: Gene expression analyses were performed using NanoString technology on a multicenter training cohort of 130 patients and an independent validation cohort of 121 patients. The analyzed gene set was composed of genes with a previously reported association with radio(chemo)sensitivity or resistance to radio(chemo)therapy. Gene selection and model building were performed comparing several machine-learning algorithms. Results: We identified a 7-gene signature consisting of the three individual genes HILPDA, CD24, TCF3, and one metagene combining the highly correlated genes SERPINE1, INHBA, P4HA2, and ACTN1. The 7-gene signature was used, in combination with clinical parameters, to fit a multivariable Cox model to the training data (concordance index, ci = 0.82), which was successfully validated (ci = 0.71). The signature showed improved performance compared with clinical parameters alone (ci = 0.66) and with a previously published model including hypoxia-associated genes and cancer stem cell markers (ci = 0.65). It was used to stratify patients into groups with low and high risk of recurrence, leading to significant differences in LRC in training and validation (P < 0.001). Conclusions: We have identified and validated the first hypothesis-based gene signature for HPV-negative HNSCC treated by PORT-C including genes related to several radiobiological aspects. A prospective validation is planned in an ongoing prospective clinical trial before potential application in clinical trials for patient stratification. Clin Cancer Res; 24(6); 1364–74. ©2018 AACR.

The molecular landscape of squamous cell carcinoma of the head and the neck (SCCHN) has been characterized and actionable or targetable genomic alterations have been identified. However, targeted therapies have very limited activity in unselected SCCHN, and the current treatment strategy is still based on tumor location and disease stage and not on tumor biology. Trying to select upfront the patients who will benefit from a specific treatment might be a way to improve patients' outcome. With the objective of optimizing the activity of targeted therapies and immunotherapy, we have designed an umbrella biomarker-driven study dedicated to recurrent and/or metastatic SCCHN patients (EORTC-1559-HNCG, NCT03088059). In this article, we review not only the different trial designs for biomarker-driven studies with their respective advantages and opportunities but also the potential pitfalls that led to the design of the EORTC-1559-HNCG protocol. We also discuss the scientific and logistic challenges of biomarker-driven trials.

Background The prognostic role of persistence of circulating tumor cells (CTC) after upfront tumor surgery for outcome of adjuvant (chemo)radiation in locally advanced squamous cell carcinoma of the head and neck (LASCCHN) was evaluated. Patients and methods In this prospective study, peripheral blood samples from 144 patients with LASCCHN presenting after tumor resection for adjuvant treatment were analyzed for CTC. Their detection was correlated with tumor site, clinical risk factors, disease-free (DFS) and overall survival (OS). Results CTC were detected in 42 of 144 patients (29%). CTC detection was higher in cases with nodal involvement and in carcinomas located at the tonsil or base of tongue but was not influenced by age, smoking history, T stage, extracapsular lymph node extension, surgical margins or the human papillomavirus status. Overall, the presence of CTC was not predictive for OS or DFS. However, while in oropharyngeal carcinomas (OPC, n = 63), the detection of CTC was associated per trend with improved DFS [CTC+ versus CTC- (% of patients without evidence of disease at 2 years): 100% versus 79%; log rank: P = 0.059]; the reverse was observed for carcinomas from other sites (non-OPC, n = 81; CTC+ versus CTC-: 29% versus 75%; P = 0.001). In multivariate analysis, CTC remained an independent prognostic marker for DFS [hazard ratio (HR) 4.3, 95% confidence interval (CI) 1.7–10.9, P = 0.002] and OS (HR 2.7, 95% CI 1.2–6.3, P = 0.016) in non-OPC. Conclusions Assessment of CTC in non-OPC should prove useful for identification of patients who benefit from treatment intensification. The basis for the good prognostic value of CTC in OPC has to be elucidated in future studies.

Background Despite clear differences in clinical presentation and outcome, squamous cell carcinomas of the head and neck (SCCHN) arising from human papilloma virus (HPV) infection or heavy tobacco/alcohol consumption are treated equally. Next-generation sequencing is expected to reveal novel targets for more individualised treatment. Patients and methods Tumour specimens from 208 patients with locally advanced squamous cell carcinoma of the hypopharynx, oropharynx or oral cavity, all uniformly treated with adjuvant cisplatin-based chemoradiation, were included. A customised panel covering 211 exons from 45 genes frequently altered in SCCHN was used for detection of non-synonymous point and frameshift mutations. Mutations were correlated with HPV status and treatment outcome. Results Mutational profiles and HPV status were successfully established for 179 cases. HPV– tumours showed an increased frequency of alterations in tumour suppressor genes compared to HPV+ cases ( TP53 67% versus 4%, CDKN2A 18% versus 0%). Conversely, HPV+ carcinomas were enriched for activating mutations in driver genes compared to HPV– cases ( PIK3CA 30% versus 12%, KRAS 6% versus 1%, and NRAS 4% versus 0%). Hotspot TP53 missense mutations in HPV– carcinomas correlated with an increased risk of locoregional recurrence (hazard ratio [HR] 4.3, 95% confidence interval [CI] 1.5–12.1, P =0.006) and death (HR 2.2, 95% CI 1.1–4.4, P =0.021). In HPV+ SCCHN, driver gene mutations were associated per trend with a higher risk of death (HR 3.9, 95% CI 0.7–21.1, P =0.11). Conclusions Distinct mutation profiles in HPV– and HPV+ SCCHN identify subgroups with poor outcome after adjuvant chemoradiation. Mutant p53 and the phosphoinositide 3-kinase pathway were identified as potential druggable targets for subgroup-specific treatment optimisation.

In solid tumours millions of cells are shed into the blood circulation each day. Only a subset of these circulating tumour cells (CTCs) survive, many of them presumable because of their potential to form multi-cellular clusters also named spheroids. Tumour cells within these spheroids are protected from anoikis, which allows them to metastasize to distant organs or re-seed at the primary site. We used spheroid cultures of head and neck squamous cell carcinoma (HNSCC) cell lines as a model for such CTC clusters for determining the role of the epidermal growth factor receptor (EGFR) in cluster formation ability and cell survival after detachment from the extra-cellular matrix. The HNSCC cell lines FaDu, SCC-9 and UT-SCC-9 (UT-SCC-9P) as well as its cetuximab (CTX)-resistant sub-clone (UT-SCC-9R) were forced to grow in an anchorage- independent manner by coating culture dishes with the anti-adhesive polymer poly-2-hydroxyethylmethacrylate (poly-HEMA). The extent of apoptosis, clonogenic survival and EGFR signalling under such culture conditions was evaluated. The potential of spheroid formation in suspension culture was found to be positively correlated with the proliferation rate of HNSCC cell lines as well as their basal EGFR expression levels. CTX and gefitinib blocked, whereas the addition of EGFR ligands promoted anchorage-independent cell survival and spheroid formation. Increased spheroid formation and growth were associated with persistent activation of EGFR and its downstream signalling component (MAPK/ERK). Importantly, HNSCC cells derived from spheroid cultures retained their clonogenic potential in the absence of cell-matrix contact. Addition of CTX under these conditions strongly inhibited colony formation in CTX- sensitive cell lines but not their resistant subclones. Altogether, EGFR activation was identified as crucial factor for anchorage-independent survival of HNSCC cells. Targeting EGFR in CTC cluster formation might represent an attractive anti-metastatic treatment approach in HNSCC.

Purpose: To investigate the impact of hypoxia-induced gene expression and cancer stem cell (CSC) marker expression on outcome of postoperative cisplatin-based radiochemotherapy (PORT-C) in patients with locally advanced head and neck squamous cell carcinoma (HNSCC). Experimental Design: Expression of the CSC markers CD44, MET, and SLC3A2, and hypoxia gene signatures were analyzed in the resected primary tumors using RT-PCR and nanoString technology in a multicenter retrospective cohort of 195 patients. CD44 protein expression was further analyzed in tissue microarrays. Primary endpoint was locoregional tumor control. Results: Univariate analysis showed that hypoxia-induced gene expression was significantly associated with a high risk of locoregional recurrence using the 15-gene signature (P = 0.010) or the 26-gene signature (P = 0.002). In multivariate analyses, in patients with HPV16 DNA–negative but not with HPV16 DNA–positive tumors the effect of hypoxia-induced genes on locoregional control was apparent (15-gene signature: HR 4.54, P = 0.006; 26-gene signature: HR 10.27, P = 0.024). Furthermore, MET, SLC3A2, CD44, and CD44 protein showed an association with locoregional tumor control in multivariate analyses (MET: HR 3.71, P = 0.016; SLC3A2: HR 8.54, P = 0.037; CD44: HR 3.36, P = 0.054; CD44 protein n/a because of no event in the CD44-negative group) in the HPV16 DNA–negative subgroup. Conclusions: We have shown for the first time that high hypoxia-induced gene expression and high CSC marker expression levels correlate with tumor recurrence after PORT-C in patients with HPV16 DNA–negative HNSCC. After validation in a currently ongoing prospective trial, these parameters may help to further stratify patients for individualized treatment de-escalation or intensification strategies. Clin Cancer Res; 22(11); 2639–49. ©2016 AACR.

Background and purpose The role of epithelial–mesenchymal transition (EMT) in the poor outcome of EGFR-overexpressing SCCHN was evaluated. Material and methods SCCHN cell lines were characterized for their cell morphology and expression of EGFR and the EMT-associated factors E-cadherin, vimentin and Snail1. The migratory potential of cells was assessed in motility assays. Response to irradiation and cetuximab was determined using clonogenic survival assays. Results High basal expression of E-cadherin but low to absent vimentin expression could be observed in all SCCHN cell lines. Although E-cadherin expression levels did not change after treatment with EGF we observed a significant change in cell morphology resembling EMT. SCCHN cells with high basal levels of Snail1 resulting from constitutive EGFR activation were characterized by mesenchymal-like morphology, elevated migratory potential, reduced sensitivity to irradiation and cetuximab but increased sensitivity to the combined treatment. Conclusions Autocrine activation of EGFR leading to EMT is associated with a metastatic phenotype and reduced sensitivity of SCCHN cells to single-modality treatment with cetuximab or irradiation. The potential of Snail1 as biomarker for selection of patients who will mostly benefit from a combination of cetuximab and radiotherapy has to be evaluated in future clinical studies.

For breast cancer patients, treatment decisions based on the molecular profile of the primary tumor have been used for many years. In Her2-overexpressing tumors, trastuzumab is a key component of therapy. However, despite persistent expression of Her2, most tumors eventually become resistant to trastuzumab. When this happens, the patients benefit from a regime containing lapatinib, a dual EGFR and Her2 tyrosine kinase inhibitor. We report on a patient affected by chemo-refractory metastatic Her2-positive breast cancer enrolled in a translational research program for the detection and characterization of circulating tumor cells (CTCs). Depletion of the EGFR-positive CTC pool in the blood was associated with tumor response, whereas disease progression was related to a recurrence in CTCs, which were both EGFR and Her2 negative. Although a proof for the clinical significance of EGFR-positive circulating tumor cells is currently lacking, expression of EGFR may predict response to lapatinib-based treatments as in the case presented.

Recently, nivolumab and pembrolizumab have been approved as second-line treatment for recurrent and/or metastatic squamous cell carcinomas of the head and neck (SCCHN). However, while nivolumab improved response rate and overall survival in the CheckMate-141 trail and led to long-lasting responses, the overall response rate of 13% was limited. Therefore, biomarkers to select patients for immune therapy and to analyze and overcome mechanisms of resistance are urgently needed. To gain inside into the immunological status of SCCHN, we evaluated digital slides (HE stain) and molecular data of 528 primary tumors from TCGA. Tumor infiltrating lymphocytes (TILs) were evaluated separately intra-epithelial and in tumor stroma using a semi-quantitative method. The content of T cells, CD8+ T cells, cytotoxic lymphocytes, B lineage cells, NK cells, monocytic lineage cells, myeloid dendritic cells, neutrophils, fibroblasts and endothelial cells was estimated from RNA-seq data using the MCP-counter method. The T cell level was classified as high and low using a median cutoff point. As both intra-epithelial TILs and stromal TILs followed bimodal distributions, cutoff points were introduced leading to the flowing classification: Intra-epithelial TILs were present in 61% and absent in the remaining 39% of tumors. Levels of stromal TILs were high in 52% and low in the remaining 48% of tumors. Intra-epithelial and stromal TILs correlated highly significant (p=1.1E-06), but there were considerable proportions of tumors with absent intra-epithelial and high stromal TILs (13%) as well was with present intra-epithelial and low stromal TILs (21%). Intra-epithelial TILs correlated significantly with HPV status, but not with PD-L1 mRNA. Stromal TILs correlated significantly with PD-L1 mRNA, but not with HPV status. In univariate Kaplan-Meier analysis, intra-epithelial TILs correlated significantly with better DFS (p=0.009), but not with OS, while high stromal TILs correlated borderline significantly with better OS (p=0.071), but not with DFS. Tumors with many T cells had both significantly better DFS (p=0.018) and better OS (p Citation Format: Jan Budczies, Mohamed Badr, Ingeborg Tinhofer, Carsten Denkert, Korinna Jöhrens. Histomorphological and molecular immune classification of head and neck cancer and its relation to prognosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5682.

Background The mammalian target of rapamycin (mTOR) signaling pathway plays a pivotal role in numerous cellular processes involving growth, proliferation and survival. The purpose of this study was to investigate the anti-tumoral effect of the mTOR inhibitor (mTORi) CCI-779 in HNSCC cell lines and its potency in cisplatin- and cetuximab-resistant cells. Methods A panel of 10 HNSCC cell lines with differences in TP53 mutational status and basal cisplatin sensitivity and two isogenic models of acquired resistance to cisplatin and cetuximab, respectively were studied. Cell survival after treatment with CCI-779, cisplatin and cetuximab alone or in combination was determined by MTT assays. Potential predictive biomarkers for tumor cell sensitivity to CCI-779 were evaluated. Results We observed considerable heterogeneity in sensitivity of HNSCC cell lines to CCI-779 monotherapy. Sensitivity was observed in TP53 mutated as well as wild-type cell lines. Total and p-EGFR expression levels but not the basal activity of the mTOR and MAPK signaling pathways were correlated with sensitivity to CCI-779. Resistant cells with increased EGFR activation could be sensitized by the combination of CCI-779 with cetuximab. Interestingly, cell lines with acquired resistance to cisplatin displayed a higher sensitivity to CCI-779 whereas cetuximab-resistant cells were less sensitive to the drug, but could be sensitized to CCI-779 by EGFR blockade. Conclusions Activity of CCI-779 in HNSCC cells harboring TP53 mutations and displaying a phenotype of cisplatin resistance suggests its clinical potential even in patients with dismal outcome after current standard treatment. Cetuximab/mTORi combinations might be useful for treatment of tumors with high expression of EGFR/p-EGFR and/or acquired cetuximab resistance. This combinatorial treatment modality needs further evaluation in future translational and clinical studies. Electronic supplementary material The online version of this article (doi:10.1186/s12967-015-0456-6) contains supplementary material, which is available to authorized users.

Exosomes are membrane vesicles of endocytic origin that participate in inter-cellular communication. Environmental and physiological conditions affect composition of secreted exosomes, their abundance and potential influence on recipient cells. Here, we analyzed protein component of exosomes released in vitro from cells exposed to ionizing radiation (2Gy dose) and compared their content with composition of exosomes released from control not irradiated cells. Exosomes secreted from FaDu cells originating from human squamous head and neck cell carcinoma were analyzed using LC-MS/MS approach. We have found that exposure to ionizing radiation resulted in gross changes in exosomal cargo. There were 217 proteins identified in exosomes from control cells and 384 proteins identified in exosomes from irradiated cells, including 148 "common" proteins, 236 proteins detected specifically after irradiation and 69 proteins not detected after irradiation. Among proteins specifically overrepresented in exosomes from irradiated cells were those involved in transcription, translation, protein turnover, cell division and cell signaling. This indicated that exosomal cargo reflected radiation-induced changes in cellular processes like transient suppression of transcription and translation or stress-induced signaling.

Increases in incidence of oropharyngeal squamous cell carcinoma (OPSCC) in countries with falling tobacco use have been attributed to a growing role of human papilloma virus (HPV) in the carcinogenesis. Trends of HPV prevalence in populations with persistently high portions of smokers are poorly characterised.Registry data from East Germany were used to determine incidence trends between 1998 and 2011. Data from patients treated at the Charité University Medicine Berlin between 2004 and 2013 (cohort 1, N=436) were used for estimation of trends in HPV prevalence, smoking and survival. HPV prevalence was prospectively confirmed in cohort 2 (N=213) comprising all primary HNSCC cases at the Charité in 2013.Between 1998 and 2011 incidence of both OPSCC and non-OPSCC increased. An increase in HPV prevalence (% of HPV+ cases in 2004-2006 versus 2012-2013: 27% versus 59%, P=0.0004) accompanied by a moderate decrease in the portion of current smokers was observed in OPSCC but not in non-OPSCC. The change in disease epidemiology in OPSCC was associated with significant improvement in overall survival. Increased HPV prevalence in OPSCC (48%) compared to non-OPSCC (11%) was confirmed in cohort 2.Despite clear differences to the United States in terms of tobacco use, the increase in OPSCC incidence in a European population was also mainly attributed to HPV, and the HPV status significantly affected prognosis. For clinical trial design it is important to consider the large group of smokers within HPV-induced OPSCC.

Summary Objectives To evaluate loco-regional control, survival, toxicities and patterns of failure of adjuvant intensity-modulated radiotherapy (IMRT) with a simultaneous integrated boost (SIB) for head and neck cancer (HNC) patients according to risk features. Materials and methods 129 HNC patients who were treated between January 2001 and June 2010 at our institute with adjuvant SIB-IMRT with or without concurrent chemotherapy (CTX-SIB-IMRT) were included. High-risk (HR) patients with extracapsular tumor extension (ECE) and/or close resection margins had CTX-SIB-IMRT to 54/63.9 Gy and intermediate-risk (IR) patients had SIB-IMRT to 50/56 Gy. The primary endpoints were local (LC) and regional control (RC). Secondary endpoints included distant control rate (DC), overall survival (OS), acute and late toxicities and patterns of failure. Results 79/129 Patients were HR. 50/129 patients IR. 5-year LC was 87% and 89%, RC was 97% and 86%, DC was 95% and 77% and the OS 73% and 67% for IR and HR respectively. 43 deaths occurred. Acute toxicity CTCAE ⩾ grade 3 was observed in 55% and 56% and late toxicities in 10% and 15% of the IR and HR-group respectively. Fifteen patients developed loco-regional failure. Conclusion We observed significantly more patients with distant metastases in the HR group and no difference in LC, RC or OS between the two groups. The majority of the analyzed recurrences were in-field, in the high dose volume. Acute and late toxicity was moderate.