Advertisement

Biomarkers for immunotherapy response in head and neck cancer

  • Niki Gavrielatou
    Affiliations
    Section of Medical Oncology, 2nd Department of Internal Medicine, School of Medicine, National and Kapodistrian University of Athens, Attikon University Hospital, 1st Rimini St, 12462 Haidari, Athens, Greece
    Search for articles by this author
  • Stergios Doumas
    Affiliations
    Maxillofacial Unit, Brighton and Sussex University Hospitals NHS, UK
    Search for articles by this author
  • Panagiota Economopoulou
    Affiliations
    Section of Medical Oncology, 2nd Department of Internal Medicine, School of Medicine, National and Kapodistrian University of Athens, Attikon University Hospital, 1st Rimini St, 12462 Haidari, Athens, Greece
    Search for articles by this author
  • Periklis G. Foukas
    Affiliations
    2nd Department of Pathology, School of Medicine, National and Kapodistrian University of Athens, Attikon University Hospital, 1st Rimini St, 12462 Haidari, Athens, Greece
    Search for articles by this author
  • Amanda Psyrri
    Correspondence
    Corresponding author at: Section of Medical Oncology, 2nd Department of Internal Medicine, School of Medicine, National and Kapodistrian University of Athens, Attikon University Hospital, 1st Rimini St, 12462 Haidari, Athens, Greece.
    Affiliations
    Section of Medical Oncology, 2nd Department of Internal Medicine, School of Medicine, National and Kapodistrian University of Athens, Attikon University Hospital, 1st Rimini St, 12462 Haidari, Athens, Greece
    Search for articles by this author
Published:January 24, 2020DOI:https://doi.org/10.1016/j.ctrv.2020.101977

      Highlights

      • Nivolumab and Pembrolizumab have been recently approved for advanced HNSCC.
      • PD-L1 correlates with improved efficacy to anti-PD-1 monoclonal antibodies.
      • Tumor mutational burden has been associated with immunotherapy (IO) response.
      • Interferon-γ signature has prognostic and predictive significance for response to IO.
      • Tumor microenvironment is a potential source for the identification of biomarkers.

      Abstract

      Preclinical data suggest that head and neck squamous cell carcinoma (HNSCC) is a profoundly immunosuppressive disease, characterized by abnormal secretion of proinflammatory cytokines and dysfunction of immune effector cells. Based on landmark phase III trials, two anti-Programmed Cell Death-1 (PD-1) antibodies, pembrolizumab and nivolumab have been approved for HNSCC by FDA and EMEA in the recurrent/metastatic setting; in addition, pembrolizumab has recently received FDA and EMEA approval as first line treatment. In clinical practice, only a minority of patients with HNSCC derive benefit from immunotherapy and the need for the discovery of novel biomarkers to optimize treatment strategies is becoming increasingly more relevant. Although currently only PD-L1 is widely used as a predictive biomarker for response to immune checkpoint inhibitors in HNSCC, there are many ongoing trials focusing on the identification of new biomarkers. This review will summarize current data on emerging biomarkers for response to immunotherapy in HNSCC.

      Keywords

      Introduction

      Harnessing immune system responses against cancer cells ushered a new era for oncology. Deciphering the mechanisms of cancer cell deregulation and resistance to conventional treatments resulted in preclinical evidence-based findings and translated into groundbreaking clinical results with substantial impact on survival and quality of life of some cancer patients even of terminal stage.
      Immunotherapy outcomes are closely linked to the seven steps of Cancer-Immunity Cycle as proposed by Chen, Coukos and Mellman. According to this cycle, cancer cell eradication by the immune cells is a stepwise process beginning with cancer immune recognition and mounting of an adaptive immune response, to cancer cell elimination; each of them represents an eligible target for treatment, as well as a potential strategy for cancer immune escape [
      • Chen D.S.
      • Mellman I.
      Oncology meets immunology: the cancer-immunity cycle.
      ]. In this context inhibition of PD-1/L1 checkpoint axis using monoclonal antibodies (mAbs) has shown a well-established efficacy in the treatment of numerous cancer types including head and neck squamous cell carcinoma (HNSCC) and has found wide clinical application in recent years, while novel approaches using combinations of checkpoint inhibitors with radiotherapy and/or chemotherapy, cytokine-based and/or adoptive T cell therapies are investigated in ongoing clinical trials [
      • Kareemaghay S.
      • Tavassoli M.
      Clinical immunotherapeutic approaches for the treatment of head and neck cancer.
      ].
      Moreover, in 2016, the US Food and Drug Administration (FDA) approved two immune checkpoint inhibitors (ICI), the anti-PD-1 mabs, nivolumab (CheckMate141) (Opdivo, Bristol-Myers Squibb) and pembrolizumab (KEYNOTE-012) (Keytruda, Merck), as second line treatment of patients with recurrent/metastatic (R/M) HNSCC refractory to platinum-based therapy. A year later, Nivolumab was approved by the European Medicines Agency (EMEA) in the platinum refractory R/M setting while Pembrolizumab approval by EMEA in the same setting was restricted to those patients with PD-L1 Tumor Positive Score (TPS) ≥ 50% . In 2019, the FDA and EMEA approved pembrolizumab (KEYNOTE-048) for the first-line treatment of patients with R/M HNSCC who express PD-L1 Combined Positive Score (CPS) ≥ 1% . FDA approved Pembrolizumab with chemotherapy in patients with R/M HNSCC as a first line treatment regardless of PD-L1 score while EMEA restricted approval in patients with CPS PD-L1 expression ≥ 1% [
      • Burtness B.
      • Harrington K.J.
      • Greil R.
      • Soulières D.
      • Tahara M.
      • de Castro G.
      • et al.
      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.
      ].
      Despite the initial enthusiasm, the clinical benefit to ICI varies among R/M HNSCC patients with only 18% of them experiencing positive outcomes as shown in phase 1/2 KEYNOTE-012 [
      • Seiwert T.Y.
      • Burtness B.
      • Mehra R.
      • Weiss J.
      • Berger R.
      • Eder J.P.
      • et al.
      Safety and clinical activity of pembrolizumab for treatment of recurrent or metastatic squamous cell carcinoma of the head and neck (KEYNOTE-012): an open-label, multicentre, phase 1b trial.
      ]. ICI mechanism of action is not by direct cytotoxic effect as in traditional chemotherapy rendering more challenging their therapeutic response evaluation with the current radiographic response criteria like RECISTv1.1. Although ICI have better toxicity profile compared to EXTREME regimen, their immune-related adverse effects are entirely different to the traditional cytotoxic agents. Altogether, cost-benefit analysis of these new therapies emphasizes the necessity of effective patient selection. The need for predictive biomarkers in this setting has become imperative not only for providing the drug of choice with maximal patient benefit using a more individualized approach, but also for shedding light to newer combination therapeutic regimens and possible targets against this anatomically and biologically heterogeneous malignancy.
      In July 2019, the Society for Immunotherapy of Cancer issued the first guidelines on immunotherapy for the treatment of HNSCC. The subcommittee among others has attested to the need for identification and understanding of factors with predictive value so we can maximise immunotherapy efficacy and spare toxicities in selected patients [
      • Cohen E.E.W.
      • Bell R.B.
      • Bifulco C.B.
      • Burtness B.
      • Gillison M.L.
      • Harrington K.J.
      • et al.
      The society for immunotherapy of cancer consensus statement on immunotherapy for the treatment of squamous cell carcinoma of the head and neck (HNSCC).
      ]. Aim of this review is to provide current knowledge for biomarkers in HNSCC.

      Tumour related factors

      PDL-1 expression

      PD-1 checkpoint receptor expressed on activated T cells has been shown to promote immunosuppression upon interaction with its ligands, PD-L1 and PD-L2, located on cancer cells and immune infiltrating cells [
      • Freeman G.J.
      • Long A.J.
      • Iwai Y.
      • Bourque K.
      • Chernova T.
      • Nishimura H.
      • et al.
      Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation.
      ,
      • Dong H.
      • Strome S.E.
      • Salomao D.R.
      • Tamura H.
      • Hirano F.
      • Flies D.B.
      • et al.
      Tumor-associated B7–H1 promotes T-cell apoptosis: a potential mechanism of immune evasion.
      ,
      • Topalian S.L.
      • Drake C.G.
      • Pardoll D.M.
      Targeting the PD-1/B7-H1(PD-L1) pathway to activate anti-tumor immunity.
      ]. PD-L1 expression on immune cells in pre-treatment tumour biopsies is indicative of formerly elicited anti-tumour adaptive immune response [
      • Tumeh P.C.
      • Harview C.L.
      • Yearley J.H.
      • Shintaku I.P.
      • Taylor E.J.
      • Robert L.
      • et al.
      PD-1 blockade induces responses by inhibiting adaptive immune resistance.
      ] and has been associated with improved treatment outcomes [
      • Herbst R.S.
      • Soria J.C.
      • Kowanetz M.
      • Fine G.D.
      • Hamid O.
      • Gordon M.S.
      • et al.
      Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients.
      ]. Consequently, blocking PD-1/PD-L1 interaction with anti PD-1 or anti PD-L1 mAbs promotes the reactivation of immunity, resulting in durable anti-tumour effects in a fraction of patients with various solid tumours [
      • Topalian S.L.
      • Hodi F.S.
      • Brahmer J.R.
      • Gettinger S.N.
      • Smith D.C.
      • McDermott D.F.
      • et al.
      Safety, activity, and immune correlates of anti-PD-1 antibody in cancer.
      ,
      • Brahmer J.R.
      • Tykodi S.S.
      • Chow L.Q.
      • Hwu W.J.
      • Topalian S.L.
      • Hwu P.
      • et al.
      Safety and activity of anti-PD-L1 antibody in patients with advanced cancer.
      ] Current evidence in HNSCC suggests that combined positive score (CPS), the number of PD-L1 positive cells including tumour, lymphocytes and macrophages, in relation to total tumour cells, offers a more effective evaluation than tumour proportion score (TPS), which measures PD-L1 expression on tumour cells alone [
      • Cohen E.E.W.
      • Bell R.B.
      • Bifulco C.B.
      • Burtness B.
      • Gillison M.L.
      • Harrington K.J.
      • et al.
      The society for immunotherapy of cancer consensus statement on immunotherapy for the treatment of squamous cell carcinoma of the head and neck (HNSCC).
      ].
      The superiority of CPS versus TPS in correlation to clinical response to immunotherapy in HNSCC was first described in phase 3 KEYNOTE-048 trial supporting the use of CPS as the optimal biomarker for patient selection in this type of cancer [
      • Burtness B.
      • Harrington K.J.
      • Greil R.
      • Soulières D.
      • Tahara M.
      • de Castro G.
      • et al.
      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.
      ,
      • Cohen E.E.W.
      • Bell R.B.
      • Bifulco C.B.
      • Burtness B.
      • Gillison M.L.
      • Harrington K.J.
      • et al.
      The society for immunotherapy of cancer consensus statement on immunotherapy for the treatment of squamous cell carcinoma of the head and neck (HNSCC).
      ]. This was a 1:1:1 prospective randomised trial in 882 patients (updated format presented in ASCO 2019) with incurable R/M HSNCC with pembrolizumab (P) (IgG4 humanised anti- PD-1 mab) vs. (P) + chemotherapy (C) vs. the EXTREME. At the second interim analysis (IA2), pembrolizumab significantly improved OS vs. cetuximab-chemotherapy in the CPS ≥ 20 (median 14.9 vs. 10.7 months, HR 0.61 [95% CI, 0.45–0.83]; p = 0·0007) and CPS ≥ 1 (12.3 vs. 10.3 months, 0.78 [0·64–0·96], p = 0·0086) populations and was non inferior in the total population (11.6 vs. 10.7 months, 0.85 [0.71–1.03]). P + C significantly improved overall survival (OS) vs. EXTREME in the total population (13.0 vs. 10.7 months, HR 0.77 [95% CI, 0.63–0.93], p = 0.0034) at IA2 and in the CPS ≥ 20 (14.7 vs. 11.0 months, 0.60 [0.45–0.82], p = 0.0004) and CPS ≥ 1 (13.6 vs. 10.4 months, 0.65 [0.53–0·0.80], p less than 0.0001) populations at final analysis. Neither pembrolizumab nor pembrolizumab-chemotherapy improved PFS at IA2. Overall, KEYNOTE-048 showed that P in CPS ≥ 20 and CPS ≥ 1 and P + C in the total population had superior efficacy to the standard of care, hence backed the FDA approval for pembrolizumab as first line treatment in the incurable R/M HNSCC in June 2019.
      Similarly, phase 3 KEYNOTE-040, a multicentre, open-label prospective randomised trial that evaluated the clinical efficacy (OS) and adverse effects of 2nd line (P) vs investigator’s choice of standard of care (SOC) treatment (weekly methotrexate, weekly cetuximab or Q3W docetaxel). In the intention-to-treat population, irrespective of PD-L1 status, median OS was 8.4 months (95% CI 6.4–9.4) with (P) vs. 6.9 months (5.9–8.0) with SOC (HR 0.80, 0.65–0.98; p = 0.0161). Among patients with a CPS ≥ 1, median OS was 8.7 months (95% CI 6.9–11.4) with (P) compared to 7.1 months (5·7–8·3) with SOC (HR 0.74; 95% CI: 0.58–0.93, p = 0.0070). Among patients with a CPS score of less than 1, median OS was 6.3 months (3.9–8.9) with (P) vs. 7.0 months (5.1–9.0) with SOC (HR 1.28; 95% CI: 0.8–2.07, p = 0.8476) [
      • Cohen E.E.W.
      • Soulières D.
      • Le Tourneau C.
      • Dinis J.
      • Licitra L.
      • Ahn M.J.
      • et al.
      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.
      ].
      PD-L1 expression, along with p16 status for HPV testing in oropharyngeal cancer, has been the most widely used biomarker for patient selection in head and neck cancer clinical trials to date. However, diverse results coming from multiple clinical studies have failed to establish PD-L1 infallibility in patient selection [
      • Seiwert T.Y.
      • Burtness B.
      • Mehra R.
      • Weiss J.
      • Berger R.
      • Eder J.P.
      • et al.
      Safety and clinical activity of pembrolizumab for treatment of recurrent or metastatic squamous cell carcinoma of the head and neck (KEYNOTE-012): an open-label, multicentre, phase 1b trial.
      ,
      • Ferris R.L.
      • Blumenschein G.
      • Fayette J.
      • Guigay J.
      • Colevas A.D.
      • Licitra L.
      • et al.
      Nivolumab for recurrent squamous-cell carcinoma of the head and neck.
      ,
      • Bauml J.
      • Seiwert T.Y.
      • Pfister D.G.
      • Worden F.
      • Liu S.V.
      • Gilbert J.
      • et al.
      Pembrolizumab for platinum- and cetuximab-refractory head and neck cancer: results from a single-arm phase II study.
      ]. Longer overall survival (OS) was observed in phase 1b KEYNOTE 012 trial in 192 patients (60 patients in initial cohort and 132 in the expansion cohort) with PD-L1≥1. The median (95% CI) OS rates were also significantly different when CPS was used (PD-L1+, 10 m [9–13 months] vs. PD-L1-, 5 [3–8 months]; one-sided P = 0.008), a finding confirmed also by long-term follow-up analysis, and additionally PD-L2 was associated with better overall response to treatment both on its own and co-expressed with PD-L1 (both ORR 23% vs. 10%). However, the authors noticed responses in PD-L1 – tumours in a significant portion of patients (9%) questioning the validity of PD-L1 as biomarkers for the use of (P) [
      • Seiwert T.Y.
      • Burtness B.
      • Mehra R.
      • Weiss J.
      • Berger R.
      • Eder J.P.
      • et al.
      Safety and clinical activity of pembrolizumab for treatment of recurrent or metastatic squamous cell carcinoma of the head and neck (KEYNOTE-012): an open-label, multicentre, phase 1b trial.
      ,
      • Mehra R.
      • Seiwert T.Y.
      • Gupta S.
      • Weiss J.
      • Gluck I.
      • Eder J.P.
      • et al.
      Efficacy and safety of pembrolizumab in recurrent/metastatic head and neck squamous cell carcinoma: pooled analyses after long-term follow-up in KEYNOTE-012.
      ].
      Nonetheless, in KEYNOTE 055, a phase II study on the use of (P) as 2nd line treatment for R/M HNSCC, although PD-L1 positive patients demonstrated, as expected, higher response rates, PD-L1 negative patients also had significant therapeutic benefit, suggesting that PD-L1 alone should not be used as a determinant for patient exclusion from immunotherapy and alternative biomarkers need to be explored [
      • Bauml J.
      • Seiwert T.Y.
      • Pfister D.G.
      • Worden F.
      • Liu S.V.
      • Gilbert J.
      • et al.
      Pembrolizumab for platinum- and cetuximab-refractory head and neck cancer: results from a single-arm phase II study.
      ]. Similar findings were seen in phase III CHECKMATE 141 trial investigating nivolumab treatment for R/M HNSCC, where although patients with >1% TPS showed better PFS, there was no significant difference in overall survival (OS) between patients expressing and those not expressing PD-L1 [
      • Ferris R.L.
      • Blumenschein G.
      • Fayette J.
      • Guigay J.
      • Colevas A.D.
      • Licitra L.
      • et al.
      Nivolumab for recurrent squamous-cell carcinoma of the head and neck.
      ,
      • Ferris R.L.
      • Blumenschein G.
      • Fayette J.
      • Guigay J.
      • Colevas A.D.
      • Licitra L.
      • et al.
      Nivolumab vs investigator's choice in recurrent or metastatic squamous cell carcinoma of the head and neck: 2-year long-term survival update of CheckMate 141 with analyses by tumor PD-L1 expression.
      ].
      Two more interesting issues pertained to PD-L1 evaluation are the intra-, inter-tumour heterogeneity and the differences in “cut-offs” defining positive or negative, as well as the reagents used for staining [
      • Cohen E.E.W.
      • Bell R.B.
      • Bifulco C.B.
      • Burtness B.
      • Gillison M.L.
      • Harrington K.J.
      • et al.
      The society for immunotherapy of cancer consensus statement on immunotherapy for the treatment of squamous cell carcinoma of the head and neck (HNSCC).
      ]. Recently, Rasmussen et al raised the intratumoural heterogeneity in PD-L1 after prospectively studying 33 whole surgical specimens of 16 patients with HNSCC [
      • Rasmussen J.H.
      • Lelkaitis G.
      • Hakansson K.
      • Vogelius I.R.
      • Johannesen H.H.
      • Fischer B.M.
      • et al.
      Intratumor heterogeneity of PD-L1 expression in head and neck squamous cell carcinoma.
      ]. They reported that with 1% cut off, 36% of the specimens were concordant with TPS and 52% with CPS, whereas a 50% cut-off value would yield a concordance of 70% with TPS and 55% with CPS. Defining a tumour as positive if just a single-one of the biopsies was positive, the negative predictive value (NPV) of a single negative core biopsy was 38.9 and 0% (1% cut off), and 79.9% and 62.8% (50% cut off) for TPS and CPS, respectively. This fact could account for the PD-L1- responders in ICI treatment. Also, Ratcliff et al. presented in ESMO 2016 their comparative study between 3 different PD-L1 staining assays used in HNSCC clinical trials to date (the Ventana SP263 assay in durvalumab (anti-PD-L1) clinical trials, the Dako 28–8 and Dako 22C3 assays, commonly used in nivolumab (Opdivo®) and pembrolizumab (Keytruda®) trials, respectively). After staining 108 tumour biopsies they found that overall percent agreement was >90% [

      M.J. Ratcliffe AS, M. Rebelatto, M. Scott, C. Barker, P. Scorer, J. Walker. A comparative study of PD-L1 diagnostic assays in squamous cell carcinoma of the head and neck (SCCHN). Annals Oncol. 2016 27 (6): 328–350 101093/annonc/mdw376. 2016.

      ].
      Taken together, the SITC subcommittee comments that Tumour PD-L1 expression generally correlates with improved efficacy with anti-PD-1/PD-L1 ICIs in R/M HNSCC, with increased predictive value when including PD-L1 expression on tumour infiltrating immune cells i.e. CPS. Nevertheless, some patients who are PD-L1 negative still benefit from treatment with these agents [
      • Cohen E.E.W.
      • Bell R.B.
      • Bifulco C.B.
      • Burtness B.
      • Gillison M.L.
      • Harrington K.J.
      • et al.
      The society for immunotherapy of cancer consensus statement on immunotherapy for the treatment of squamous cell carcinoma of the head and neck (HNSCC).
      ].

      Tumour mutational burden/ neo-antigens

      Recently a significant number of studies have established the major role of neo-epitopes, resulting from non-synonymous mutations on tumour cells, in cancer immune recognition and specific T-cell activation [
      • Robbins P.F.
      • Lu Y.C.
      • El-Gamil M.
      • Li Y.F.
      • Gross C.
      • Gartner J.
      • et al.
      Mining exomic sequencing data to identify mutated antigens recognized by adoptively transferred tumor-reactive T cells.
      ,
      • Linnemann C.
      • van Buuren M.M.
      • Bies L.
      • Verdegaal E.M.
      • Schotte R.
      • Calis J.J.
      • et al.
      High-throughput epitope discovery reveals frequent recognition of neo-antigens by CD4+ T cells in human melanoma.
      ]. Although tumours with high frequency of missense mutations demonstrate increased density of infiltrating CD8 + T cells and are related to better outcomes [
      • Brown S.D.
      • Warren R.L.
      • Gibb E.A.
      • Martin S.D.
      • Spinelli J.J.
      • Nelson B.H.
      • et al.
      Neo-antigens predicted by tumor genome meta-analysis correlate with increased patient survival.
      ], only a small proportion of these mutations results in the production of neo-antigens and only a part of these neo-antigens leads to T-cell recognition and reactivity [
      • Schumacher T.N.
      • Schreiber R.D.
      Neoantigens in cancer immunotherapy.
      ]. Analysis of various types of tumours using RNA sequencing revealed that it is specifically immunogenic mutations, rather than whole mutational load, that are associated with better survival prognosis and lead to increased expression of CD8A and immune exhaustion markers (PDCD1, CTLA4) creating thus a favourable setting for immunotherapy response [
      • Brown S.D.
      • Warren R.L.
      • Gibb E.A.
      • Martin S.D.
      • Spinelli J.J.
      • Nelson B.H.
      • et al.
      Neo-antigens predicted by tumor genome meta-analysis correlate with increased patient survival.
      ]. Consequently, as is challenging to predict the ultimate effect of mutations in anti-tumour immune activation, the prognostic value of TMB in respect to immune modulating treatments remains limited.
      Nevertheless, TMB has been shown to be a promising biomarker for immunotherapy response by multiple studies. Increased mutational burden has been related to improved response to PD-1 inhibition and prolonged PFS in Non Small Cell Lung Cancer (NSCLC) [
      • Rizvi N.A.
      • Hellmann M.D.
      • Snyder A.
      • Kvistborg P.
      • Makarov V.
      • Havel J.J.
      • et al.
      Cancer immunology. Mutational landscape determines sensitivity to PD-1 blockade in non-small cell lung cancer.
      ], while research on anti-CTLA4 treatment in melanoma has depicted the association of TMB with clinical response and OS [
      • Van Allen E.M.
      • Miao D.
      • Schilling B.
      • Shukla S.A.
      • Blank C.
      • Zimmer L.
      • et al.
      Genomic correlates of response to CTLA-4 blockade in metastatic melanoma.
      ,
      • Snyder A.
      • Makarov V.
      • Merghoub T.
      • Yuan J.
      • Zaretsky J.M.
      • Desrichard A.
      • et al.
      Genetic basis for clinical response to CTLA-4 blockade in melanoma.
      ]. Another study focused on the effect of neo-antigen heterogeneity within both NSCLC and melanoma tumours revealed that high clonality of neo-antigen load predisposes to an inflamed TME and enhances the benefit of immunotherapy suggesting that multiple tumour subclones increase the risk of one of them escaping host immunity [
      • McGranahan N.
      • Furness A.J.
      • Rosenthal R.
      • Ramskov S.
      • Lyngaa R.
      • Saini S.K.
      • et al.
      Clonal neoantigens elicit T cell immunoreactivity and sensitivity to immune checkpoint blockade.
      ].
      Regarding HNSCC, total mutational load, using a cutoff of ≥102 mutations per exome, was evaluated in KEYNOTE 012 trial and demonstrated a positive correlation with immunotherapy response [
      • Seiwert T.Y.
      • Burtness B.
      • Mehra R.
      • Weiss J.
      • Berger R.
      • Eder J.P.
      • et al.
      Safety and clinical activity of pembrolizumab for treatment of recurrent or metastatic squamous cell carcinoma of the head and neck (KEYNOTE-012): an open-label, multicentre, phase 1b trial.
      ,
      • Cristescu R.
      • Mogg R.
      • Ayers M.
      • Albright A.
      • Murphy E.
      • Yearley J.
      • et al.
      Pan-tumor genomic biomarkers for PD-1 checkpoint blockade-based immunotherapy.
      ]. Additional data from a cohort of 126 patients receiving anti-PD-1/L1 therapy have revealed that higher TMB was observed among responders and it was found to be a positive predictor among HPV-/EBV- patients of the same group. NOTCH1 and SMARCA4 mutations showed noticeably greater occurrence in responders in comparison to non-responders and when HPV/EBV viral status was taken into account the above results were only noticed among HPV-/EBV- responders. Moreover, microsatellite instability was higher among responders. In contrast, copy-number alterations indicated no association with response regardless of viral status [
      • Hanna G.J.
      • Lizotte P.
      • Cavanaugh M.
      • Kuo F.C.
      • Shivdasani P.
      • Frieden A.
      • et al.
      Frameshift events predict anti-PD-1/L1 response in head and neck cancer.
      ].
      Recently, two more studies shed light to the TMB prognostic role in HNSCC [
      • Cohen E.E.W.
      • Bell R.B.
      • Bifulco C.B.
      • Burtness B.
      • Gillison M.L.
      • Harrington K.J.
      • et al.
      The society for immunotherapy of cancer consensus statement on immunotherapy for the treatment of squamous cell carcinoma of the head and neck (HNSCC).
      ,
      • Topalian S.L.
      • Drake C.G.
      • Pardoll D.M.
      Targeting the PD-1/B7-H1(PD-L1) pathway to activate anti-tumor immunity.
      ,
      • Allen C.T.
      • Judd N.P.
      • Bui J.D.
      • Uppaluri R.
      The clinical implications of antitumor immunity in head and neck cancer.
      ] analysing the RNA sequencing data from HNSCC by The Cancer Tumour Atlas (TCGA) and the Chicago Head Neck Genomics. They both concluded that TMB has no correlation with immune cell infiltrates. FDA has yet to recommend about TMB testing [
      • Cohen E.E.W.
      • Bell R.B.
      • Bifulco C.B.
      • Burtness B.
      • Gillison M.L.
      • Harrington K.J.
      • et al.
      The society for immunotherapy of cancer consensus statement on immunotherapy for the treatment of squamous cell carcinoma of the head and neck (HNSCC).
      ]. Also, given that microsatellite instability (MSI) is only reported in 1–3%, SITC subcommittee recommends against MSI testing [
      • Cohen E.E.W.
      • Bell R.B.
      • Bifulco C.B.
      • Burtness B.
      • Gillison M.L.
      • Harrington K.J.
      • et al.
      The society for immunotherapy of cancer consensus statement on immunotherapy for the treatment of squamous cell carcinoma of the head and neck (HNSCC).
      ].

      Interferon-γ gene signature

      Type I interferons (IFNs) are implicated in the mechanism by which tumour innate immune sensing results in spontaneous cytotoxic T cell recruitment, a key step for establishing an inflamed type of TME [
      • Gajewski T.F.
      • Fuertes M.B.
      • Woo S.R.
      Innate immune sensing of cancer: clues from an identified role for type I IFNs.
      ,
      • Corrales L.
      • Gajewski T.F.
      Molecular Pathways: targeting the stimulator of interferon genes (STING) in the immunotherapy of cancer.
      ,
      • Woo S.R.
      • Corrales L.
      • Gajewski T.F.
      Innate immune recognition of cancer.
      ]. Specifically for HNSCC, the relation of interferons to ICI therapy was investigated in KEYNOTE-O12 trial where a six-gene IFN-γ signature (including IDO1, CXCL10, CXCL9, HLA-DRA, STAT1, IFN-γ gene expression) was examined in pretreatment biopsies. Results showed that IFN-γ gene signature exhibits statistically significant association with Best Overall Response (BOR) and PFS and could become a potential biomarker for patient exclusion from immunotherapy due to its high negative predictive value [
      • Seiwert T.Y.
      • Burtness B.
      • Mehra R.
      • Weiss J.
      • Berger R.
      • Eder J.P.
      • et al.
      Safety and clinical activity of pembrolizumab for treatment of recurrent or metastatic squamous cell carcinoma of the head and neck (KEYNOTE-012): an open-label, multicentre, phase 1b trial.
      ]. Research performed on mice using melanoma cell lines has shown that tumour- derived DNA located within the cytosol of host APCs induces IFN-β secretion through STING and IRF3 activation via the cytosolic DNA sensor cGAS suggesting that a functional STING pathway is essential for an endogenous T cell anti-tumour response and consequently for the effectiveness of immunotherapy [
      • Woo S.R.
      • Fuertes M.B.
      • Corrales L.
      • Spranger S.
      • Furdyna M.J.
      • Leung M.Y.
      • et al.
      STING-dependent cytosolic DNA sensing mediates innate immune recognition of immunogenic tumors.
      ]. The spontaneous formation of micronuclei within tumour cells is also involved in this process as micronuclear membrane has been shown to be prone to collapse resulting in cGAS relocalisation to the micronuclei and anti-tumour immune activation [
      • Mackenzie K.J.
      • Carroll P.
      • Martin C.A.
      • Murina O.
      • Fluteau A.
      • Simpson D.J.
      • et al.
      cGAS surveillance of micronuclei links genome instability to innate immunity.
      ]. In an analysis performed by Ayers et al [
      • Ayers M.
      • Lunceford J.
      • Nebozhyn M.
      • Murphy E.
      • Loboda A.
      • Kaufman D.R.
      • et al.
      IFN-γ-related mRNA profile predicts clinical response to PD-1 blockade.
      ], IFN-γ gene expression profile of various types of cancers was examined and positively correlated to clinical response in patients treated with the anti-PD-L1 inhibitor pembrolizumab. STING ‘agonists’ have been developed in order to overcome immunosuppression caused by defective STING pathway and are currently investigated in early phase clinical trials. (NCT03010176, NCT02675439, NCT03172936).

      Tumour microenvironment

      Inflamed - Non-Inflamed

      Tumour microenvironment (TME) and its implication in cancer development and progression is a promising source for developing predictive immunotherapy biomarkers. TME is classified by its immune cell components into three distinct phenotypes: inflamed tumours, immune-excluded and immune-desert. Inflamed tumours are characterized by abundant intratumoural and stromal infiltration with immune cells, whereas in immune-excluded phenotype immune cell presence is restricted to stroma and immune-desert phenotype is void of T-cells both in tumour bed and stroma [
      • Chen D.S.
      • Mellman I.
      Elements of cancer immunity and the cancer-immune set point.
      ]. Tolerogenic pathways such as PD-L1 and IDO overexpression stimulated by interferon-γ, in addition to FoxP3 + regulatory cell infiltration, driven by increased CD8 + presence within the TME, are identified as promoters of tumour immune escape for the T-cell inflamed subtype [
      • Spranger S.
      • Spaapen R.M.
      • Zha Y.
      • Williams J.
      • Meng Y.
      • Ha T.T.
      • et al.
      Up-regulation of PD-L1, IDO, and T(regs) in the melanoma tumor microenvironment is driven by CD8(+) T cells.
      ]. CTLA4 methylation was proposed as an eligible IO biomarker in a melanoma study. While CTLA4 expression was positively correlated with IFN-γ/JAK/STAT1 pathway genes, CTLA4 methylation was inversely correlated with IFN-γ expression and higher levels of methylated CTLA4 were associated with poor response to ICB therapy [
      • Goltz D.
      • Gevensleben H.
      • Vogt T.J.
      • Dietrich J.
      • Golletz C.
      • Bootz F.
      • et al.
      CTLA4 methylation predicts response to anti-PD-1 and anti-CTLA-4 immunotherapy in melanoma patients.
      ]. Elimination of immune inhibition with immunotherapeutic agents results in the reactivation of pre-existing effector T-cells within the TME rather than in the recruitment of new ones [
      • Tumeh P.C.
      • Harview C.L.
      • Yearley J.H.
      • Shintaku I.P.
      • Taylor E.J.
      • Robert L.
      • et al.
      PD-1 blockade induces responses by inhibiting adaptive immune resistance.
      ,
      • Spranger S.
      • Koblish H.K.
      • Horton B.
      • Scherle P.A.
      • Newton R.
      • Gajewski T.F.
      Mechanism of tumor rejection with doublets of CTLA-4, PD-1/PD-L1, or IDO blockade involves restored IL-2 production and proliferation of CD8(+) T cells directly within the tumor microenvironment.
      ] which indicates why immunotherapy has been shown to exert positive clinical effects exclusively on T-cell inflamed tumours [
      • Gajewski T.F.
      The next hurdle in cancer immunotherapy: overcoming the non-T-cell-inflamed tumor microenvironment.
      ]. Conversely, in non-inflamed tumours immune evasion is achieved through complete T-cell exclusion from the tumour site due to the involvement of various oncogenic pathways. WNT-β-catenin pathway upregulation, impaired Basic leucine zipper transcriptional factor ATF-like 3 (BATF3) DCs, Myc overexpression, LKB1 and PTEN gene mutations or deletion, p53 inactivation, activating mutations of IDH1/IDH2 and FGFR3 genes and STAT3 and Peroxisome proliferator-activated receptor gamma (PPARγ) signalling amplification, all favour an immunosuppressive TME as shown in several preclinical studies on a variety of tumours [
      • Spranger S.
      • Gajewski T.F.
      Impact of oncogenic pathways on evasion of antitumour immune responses.
      ].
      Tertiary lymphoid structures (TLS) constitute yet another important promoter of anti-tumour immune response. By mimicking the normal function and architecture of secondary lymphoid organs, TLS have been shown to positively affect prognosis in the majority of solid cancers including HNSCC. Chronic inflammatory state, an invariable component of cancer pathophysiology, stimulates chemokine and cytokine expression within the TME, resulting in the recruitment of lymphocytes from a rich network of high endothelial venules [
      • Ager A.
      • May M.J.
      Understanding high endothelial venules: Lessons for cancer immunology.
      ] and their arrangement into TLS, with a follicular zone of B-cells organized in germinal centres, surrounded by a T-cell zone. TLS presence in stroma or tumour bed is associated with a direct and prolonged immune attack on the malignant cells. Consequently, in immune exhausted tumours, checkpoint inhibitors could reactivate TLS function and reattain their benefits while in non inflamed tumours, induced TLS generation might enhance anti-neoplastic effects [
      • Sautès-Fridman C.
      • Petitprez F.
      • Calderaro J.
      • Fridman W.H.
      Tertiary lymphoid structures in the era of cancer immunotherapy.
      ].

      The effect of tumour microenvironment in HNSCC

      As TME structure, components and their effects vary among different malignancies, it is important to outline its specific characteristics present in HNC. Immune infiltration is notable in the majority of HNSCCs indicating an ongoing natural (spontaneous) immune response. However, the fact that even immune-inflamed tumours ultimately manage to evade host immunity and continue to progress, insinuates that interactions between immune cells, tumour cells and their products within TME restrict adaptive immunity and eventually promote immunosuppression [
      • Allen C.T.
      • Judd N.P.
      • Bui J.D.
      • Uppaluri R.
      The clinical implications of antitumor immunity in head and neck cancer.
      ,
      • Whiteside T.L.
      • Demaria S.
      • Rodriguez-Ruiz M.E.
      • Zarour H.M.
      • Melero I.
      Emerging opportunities and challenges in cancer immunotherapy.
      ,
      • Davis R.J.
      • Van Waes C.
      • Allen C.T.
      Overcoming barriers to effective immunotherapy: MDSCs, TAMs, and Tregs as mediators of the immunosuppressive microenvironment in head and neck cancer.
      ]. Chen et al. proposed a novel stratification of HNSC tumours regarding their immune cell composition and inflammatory marker expression. Using non-negative matrix factorization they were able to identify two distinct subtypes, named Active and Exhausted Immune Class, with the former incorporating characteristics related to favourable immunotherapy outcomes such as high levels of B-cell immune signatures, increased cytolytic activity and M1 macrophage infiltration in contrast with the latter which demonstrates activated stroma, M2 macrophage infiltration and activation of WNT/TGF-β, factors known to favour tumourigenesis [
      • Chen Y.P.
      • Wang Y.Q.
      • Lv J.W.
      • Li Y.Q.
      • Chua M.L.K.
      • Le Q.T.
      • et al.
      Identification and validation of novel microenvironment-based immune molecular subgroups of head and neck squamous cell carcinoma: implications for immunotherapy.
      ].
      Specific immune cell types such as CD3+,CD8 + and Foxp3 + tumor-infiltrating lymphocytes (TILs) are associated with better prognosis in HNSCC [
      • de Ruiter E.J.
      • Ooft M.L.
      • Devriese L.A.
      • Willems S.M.
      The prognostic role of tumor infiltrating T-lymphocytes in squamous cell carcinoma of the head and neck: a systematic review and meta-analysis.
      ]. The positive effect of CD4 + Foxp3 + infiltrating T cells in survival and locoregional disease control might appear inconsistent to their immunosuppressive nature; however, it has been hypothesized that it derives from inhibition of tumour-promoting inflammatory factors, which are abundant in HNC and possibly a direct cytotoxic effect [
      • Badoual C.
      • Hans S.
      • Rodriguez J.
      • Peyrard S.
      • Klein C.
      • NlH Agueznay
      • et al.
      Prognostic value of tumor-infiltrating CD4+ T-cell subpopulations in head and neck cancers.
      ,
      • Badoual Cecile
      • Hans Stephane
      • Fridman Wolf H.
      • Brasnu Daniel
      • Erdman Susan
      • Tartour Eric
      Revisiting the prognostic value of regulatory T cells in patients with cancer.
      ]. In addition, higher CD8 + T-cell infiltration has been observed among anti PD1/L1 therapy responders and has been proven to be an independent predictive factor for improved prognosis [
      • Hanna G.J.
      • Lizotte P.
      • Cavanaugh M.
      • Kuo F.C.
      • Shivdasani P.
      • Frieden A.
      • et al.
      Frameshift events predict anti-PD-1/L1 response in head and neck cancer.
      ]. Supporting these findings, results from a study of PD-1 inhibition in melanoma suggest that tumour infiltration by CD8 + T cells under PD-1/PD-L1 inhibition as well as T-cell clonality are predictive of better therapeutic outcome [
      • Tumeh P.C.
      • Harview C.L.
      • Yearley J.H.
      • Shintaku I.P.
      • Taylor E.J.
      • Robert L.
      • et al.
      PD-1 blockade induces responses by inhibiting adaptive immune resistance.
      ]. TME infiltration by NK cells is also related to better prognosis and their anti-tumour effect may be even more crucial for non-inflamed tumours as they act independently to tumour antigen recognition and presentation [
      • Mandal R.
      • Şenbabaoğlu Y.
      • Desrichard A.
      • Havel J.J.
      • Dalin M.G.
      • Riaz N.
      • et al.
      The head and neck cancer immune landscape and its immunotherapeutic implications.
      ]. Alternatively, the presence of myeloid-derived suppressor cells (MDSCs), which are attracted to tumour site by GM-CSF, IL-1 and IL-6 expression from cancer cells, M2 polarized tumour-associated macrophages (TAMs) and N2 tumour associated neutrophils (TANs) characterizes a tolerogenic tumour immune landscape [
      • Allen C.T.
      • Judd N.P.
      • Bui J.D.
      • Uppaluri R.
      The clinical implications of antitumor immunity in head and neck cancer.
      ] but also indicates potential targets for immunosuppression reversal. Data from preclinical research on mice revealed that inhibition of MDSC function against T cell proliferation with the addition of a PI3Kγ/δ inhibitor resulted in improved response to PD-L1 inhibition in T cell-inflamed tumours in contrast to non-inflamed [
      • Davis R.J.
      • Moore E.C.
      • Clavijo P.E.
      • Friedman J.
      • Cash H.
      • Chen Z.
      • et al.
      Anti-PD-L1 efficacy can be enhanced by inhibition of myeloid-derived suppressor cells with a selective inhibitor of PI3Kδ/γ.
      ]. Indoleamine 2,3-dioxygenase (IDO), a tryptophan metabolizing enzyme induced by inflammation, normally promotes immunosuppression in order to control harmful inflammatory responses. This otherwise beneficial effect, however, is also responsible for cancer immune evasion and IO resistance as IDO shows increased expression in tumour cells, tumour-related immune cells, dendritic cells and macrophages [
      • Munn D.H.
      • Mellor A.L.
      IDO in the tumor microenvironment: inflammation, counter-regulation, and tolerance.
      ]. Tryptophan depletion and immune inhibitory tryptophan metabolites have been described as potential mechanisms by which IDO achieves tumour-specific T cell suppression and anergy as well as Tregs activation [
      • Munn D.H.
      • Mellor A.L.
      Indoleamine 2,3-dioxygenase and tumor-induced tolerance.
      ] and its expression has been associated with increased MDSC tumour infiltration, induced by Tregs [
      • Holmgaard R.B.
      • Zamarin D.
      • Li Y.
      • Gasmi B.
      • Munn D.H.
      • Allison J.P.
      • et al.
      Tumor-expressed IDO recruits and activates MDSCs in a treg-dependent manner.
      ]. Although results from clinical trial investigating IDO inhibitors in various cancers are far from promising [
      • Garber K.
      A new cancer immunotherapy suffers a setback.
      ], preclinical research on melanoma has shown that IDO promotes resistance to anti-CTLA4 therapy, while IDO inhibition reverses it [
      • Holmgaard R.B.
      • Zamarin D.
      • Munn D.H.
      • Wolchok J.D.
      • Allison J.P.
      Indoleamine 2,3-dioxygenase is a critical resistance mechanism in antitumor T cell immunotherapy targeting CTLA-4.
      ] and IDO activity has also been associated with resistance to anti-PD-1 therapy in NSCLC [
      • Botticelli A.
      • Cerbelli B.
      • Lionetto L.
      • Zizzari I.
      • Salati M.
      • Pisano A.
      • et al.
      Can IDO activity predict primary resistance to anti-PD-1 treatment in NSCLC?.
      ]. Consequently, the value of IDO as an immunotherapy response biomarker remains to be determined.
      Additionally, a specific subpopulation of tumour cells characterized by CD44 expression on their surface, known to display cancer stem cell properties, is implicated in therapy resistance and disease aggressiveness [
      • Prince M.E.
      • Sivanandan R.
      • Kaczorowski A.
      • Wolf G.T.
      • Kaplan M.J.
      • Dalerba P.
      • et al.
      Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma.
      ]. Research on their function and possible interactions within the TME revealed that CD44 + HNSCC cells have a negative interference with anti-tumour immunity by downregulating effector T-cell and Th1-cell activity and inducing regulatory T-cell and MDSC function [
      • Chikamatsu K.
      • Takahashi G.
      • Sakakura K.
      • Ferrone S.
      • Masuyama K.
      Immunoregulatory properties of CD44+ cancer stem-like cells in squamous cell carcinoma of the head and neck.
      ]. Another proposed mechanism of CD44 + cell mediated immunosuppression is attributed to their increased PD-L1 expression which shields them from host immune responses and links them to disease recurrence, making CD44- rich tumours ideal targets for anti-PD-1 therapy, even after surgical intervention [
      • Lee Y.
      • Shin J.H.
      • Longmire M.
      • Wang H.
      • Kohrt H.E.
      • Chang H.Y.
      • et al.
      CD44+ cells in head and neck squamous cell carcinoma suppress T-cell-mediated immunity by selective constitutive and inducible expression of PD-L1.
      ].
      Exploring the possibility that immune cell distribution within TME might affect response to immunotherapy, Wood et al. assessed biopsies of 16 HNSCC patients at diagnosis and resection using RNA sequencing and immunohistochemistry assays. The results depicted a stable immune cell signature within various regions of the same tumours at different timepoints prior to treatment, contradicting the hypothesis that HNSCC immunotherapy failure could be attributed to immunological diversity within primary tumours [
      • Wood O.
      • Clarke J.
      • Woo J.
      • Mirza A.H.
      • Woelk C.H.
      • Thomas G.J.
      • et al.
      Head and neck squamous cell carcinomas are characterized by a stable immune signature within the primary tumor over time and space.
      ]. Studies examining the impact of stromal TILs on patient survival have shown conflicting results. Vassilakopoulou et al demonstrated that stromal TIL (strTIL) abundance exerts positive impact on both disease free survival (DFS) and OS in HNSCC and associates with higher PD-L1 protein expression levels, suggesting a possible concordance with IO response [
      • Vassilakopoulou M.
      • Avgeris M.
      • Velcheti V.
      • Kotoula V.
      • Rampias T.
      • Chatzopoulos K.
      • et al.
      Evaluation of PD-L1 expression and associated tumor-infiltrating lymphocytes in laryngeal squamous cell carcinoma.
      ]. Conversely, Badr et al., combining H/E stained slide evaluation with molecular analysis of immune cell signatures in treatment-naïve tumors, they showeda positive effect of intraepithelial TILs (ieTILs) on DFS, which was retained in HPV negative tumors after HPV status stratification, while strTILs failed to show association with survival [
      • Badr M.
      • Jöhrens K.
      • Allgäuer M.
      • Boxberg M.
      • Weichert W.
      • Tinhofer I.
      • et al.
      Morphomolecular analysis of the immune tumor microenvironment in human head and neck cancer.
      ]. An analysis by multiparametric flow cytometry in 34 patients with R/M HNSCC found that high co-expression of CD8 and PD-1/TIM3 checkpoints on tumour infiltrating T cells (inflamed tumours) was associated with longer survival, in comparison with non-inflamed tumours, from the time of starting anti PD-1 therapy. However, this result comes from a small subgroup of only 9 patients so further research towards this direction is mandatory before definite conclusions can be drawn [
      • Hanna G.J.
      • Liu H.
      • Jones R.E.
      • Bacay A.F.
      • Lizotte P.H.
      • Ivanova E.V.
      • et al.
      Defining an inflamed tumor immunophenotype in recurrent, metastatic squamous cell carcinoma of the head and neck.
      ]. Incorporation of the evaluation of all these heterogeneous factors into clinical practice would be yet another challenge. Analysing results from previous studies Bates et al. proposed a predictive computational model which utilizes expression of various factors, including PD-L1 and chemokines responsible for dendritic-cell migration as well as immunosuppressive biomarkers, in order to stratify HNSCC patients regarding their potential to respond to immunotherapy [
      • Bates A.M.
      • Lanzel E.A.
      • Qian F.
      • Abbasi T.
      • Vali S.
      • Brogden K.A.
      Cell genomics and immunosuppressive biomarker expression influence PD-L1 immunotherapy treatment responses in HNSCC-a computational study.
      ].
      Molecular analysis of HNSCC tumours has identified significant correlations of specific immune cell subpopulations’ infiltration with distinct gene expression patterns such as HPV, genetic alterations, neoantigens and smoking mutational signature as well as with survival. Notably, the HPV-related (atypical) molecular subgroup demonstrates the highest immune infiltration and cytolytic activity in addition to increased Treg/CD8 + Tcells ratio while the smoking related (classical) subgroup has the lowest levels of immune infiltration and IFN-γ signalling among HNSCCs. Regarding survival, adaptive immune response cell infiltrates and mutation dominated tumours exhibit better outcomes in comparison with tumours rich in components of innate immune response and copy number alterations [
      • Mandal R.
      • Şenbabaoğlu Y.
      • Desrichard A.
      • Havel J.J.
      • Dalin M.G.
      • Riaz N.
      • et al.
      The head and neck cancer immune landscape and its immunotherapeutic implications.
      ]. Additionally, in a study focused on SCCs by Li et al., gene expression analysis using the Cancer Genome Atlas (TCGA) identified distinct immune entities with common characteristics and effect on prognosis. Tumours categorized as immune-cold had the lowest lymphocyte infiltration, TCR diversity and leukocyte/stroma ratio and highest aneyploidy and were associated with the worst prognosis while immune-hot tumours were characterized by the highest levels of lymphocyte infiltration, IFN-γ response, TCR diversity, M1/M2 ratio and cytolytic activity and the lowest levels of genomic aberrations and TGF-β expression and were related to the best prognosis. A third subtype demonstrated a molecular pattern consistent with immune-hot but tolerogenic tumours with high infiltration with M2 polarized macrophages, high TGF-β expression, TCR diversity and reactive stroma and relatively worse prognosis, while other subtypes with mixed characteristics showed association with intermediate outcomes [
      • Li B.
      • Cui Y.
      • Nambiar D.K.
      • Sunwoo J.B.
      • Li R.
      The immune subtypes and landscape of squamous cell carcinoma.
      ]. These findings are promising and could point towards patients with the highest probability to benefit from IO and combination treatments.
      Recently, a 12-gene chemokine gene expression (the Messina signature) was defined as T-cell inflamed phenotype (TCIP) in HNSCC in analogy to melanoma studies [
      • Cohen E.E.W.
      • Bell R.B.
      • Bifulco C.B.
      • Burtness B.
      • Gillison M.L.
      • Harrington K.J.
      • et al.
      The society for immunotherapy of cancer consensus statement on immunotherapy for the treatment of squamous cell carcinoma of the head and neck (HNSCC).
      ,
      • Topalian S.L.
      • Drake C.G.
      • Pardoll D.M.
      Targeting the PD-1/B7-H1(PD-L1) pathway to activate anti-tumor immunity.
      ]. TCIP-H tumours abounded PD-L1, PD-1, CTLA4, TIM3, CEACAM1, LAG3, CD206, FoxP3 along with M2 macrophages and FoxP3 + Treg conferring strong immunosuppressive milieu to CD8 + T-cells, albeit granzyme B and IFNγ levels were increased too. Interestingly, PD-L1 was not necessarily associated with CD8 + T cell infiltration, thus providing a plausible explanation why ICI in PD-L1 + tumours does not always elicit T-cell immune responses [
      • Cohen E.E.W.
      • Bell R.B.
      • Bifulco C.B.
      • Burtness B.
      • Gillison M.L.
      • Harrington K.J.
      • et al.
      The society for immunotherapy of cancer consensus statement on immunotherapy for the treatment of squamous cell carcinoma of the head and neck (HNSCC).
      ,
      • Topalian S.L.
      • Drake C.G.
      • Pardoll D.M.
      Targeting the PD-1/B7-H1(PD-L1) pathway to activate anti-tumor immunity.
      ]. However no correlation with OS was found in multivariate analysis.

      Host factors

      HPV profile

      HNSCCs are divided in two major subgroups in respect to their causative agents: HPV infection and tobacco use/alcohol consumption. HPV positivity has been identified as a favourable prognostic factor for survival in HNSCC patients treated with standard chemotherapy and radiotherapy by multiple clinical trials [
      • Posner M.R.
      • Lorch J.H.
      • Goloubeva O.
      • Tan M.
      • Schumaker L.M.
      • Sarlis N.J.
      • et al.
      Survival and human papillomavirus in oropharynx cancer in TAX 324: a subset analysis from an international phase III trial.
      ,
      • Lassen P.
      • Eriksen J.G.
      • Krogdahl A.
      • Therkildsen M.H.
      • Ulhøi B.P.
      • Overgaard M.
      • et al.
      The influence of HPV-associated p16-expression on accelerated fractionated radiotherapy in head and neck cancer: evaluation of the randomised DAHANCA 6&7 trial.
      ,
      • Vermorken J.B.
      • Psyrri A.
      • Mesía R.
      • Peyrade F.
      • Beier F.
      • de Blas B.
      • et al.
      Impact of tumor HPV status on outcome in patients with recurrent and/or metastatic squamous cell carcinoma of the head and neck receiving chemotherapy with or without cetuximab: retrospective analysis of the phase III EXTREME trial.
      ,
      • Argiris A.
      • Li S.
      • Ghebremichael M.
      • Egloff A.M.
      • Wang L.
      • Forastiere A.A.
      • et al.
      Prognostic significance of human papillomavirus in recurrent or metastatic head and neck cancer: an analysis of eastern cooperative oncology group trials.
      ,
      • Fakhry C.
      • Zhang Q.
      • Nguyen-Tan P.F.
      • Rosenthal D.
      • El-Naggar A.
      • Garden A.S.
      • et al.
      Human papillomavirus and overall survival after progression of oropharyngeal squamous cell carcinoma.
      ]. However, the value of HPV as a predictor of immunotherapy response remains to be determined. Although clinical trials so far have failed to show a clear association of HPV status with response to PD-1 inhibition therapy [
      • Seiwert T.Y.
      • Burtness B.
      • Mehra R.
      • Weiss J.
      • Berger R.
      • Eder J.P.
      • et al.
      Safety and clinical activity of pembrolizumab for treatment of recurrent or metastatic squamous cell carcinoma of the head and neck (KEYNOTE-012): an open-label, multicentre, phase 1b trial.
      ,
      • Ferris R.L.
      • Blumenschein G.
      • Fayette J.
      • Guigay J.
      • Colevas A.D.
      • Licitra L.
      • et al.
      Nivolumab for recurrent squamous-cell carcinoma of the head and neck.
      ,
      • Bauml J.
      • Seiwert T.Y.
      • Pfister D.G.
      • Worden F.
      • Liu S.V.
      • Gilbert J.
      • et al.
      Pembrolizumab for platinum- and cetuximab-refractory head and neck cancer: results from a single-arm phase II study.
      ], viral protein expression within HPV + tumours is known to serve as a trigger for immune activation and implications of viral status in the effect of immunotherapy have been presented in a large number of studies. Well- defined differences have been observed regarding the immune landscape of HPV related and unrelated head and neck tumours making the former better candidates for IO. Specifically, HPV + cancers are characterized by marked increase of intratumoral CD8 + INFγ producing T cells, CD4 + Th1 cells, DCs and macrophages as well as higher chemokine (CXCL9, CXCL10, CXCL12, CCL17,CCL21) and proinflammatory cytokine (IL-2, IL-17, IL-23, IFN-γ) production and PD-L1 expression [
      • Partlová S.
      • Bouček J.
      • Kloudová K.
      • Lukešová E.
      • Zábrodský M.
      • Grega M.
      • et al.
      Distinct patterns of intratumoral immune cell infiltrates in patients with HPV-associated compared to non-virally induced head and neck squamous cell carcinoma.
      ]. Additional data from transcriptomic analysis of 280 HNSCCs using TCGA showed that HPV-positive tumours demonstrate higher immunogenicity with larger infiltration of activated CD8 T cells in comparison with HPV-negative tumours. Moreover, although PD-1 and PDL-1 expression was not altered by HPV status, HPV-positive tumours had higher expression of CTLA-4 as well as Tregs infiltration and Tregs/CD8 ratio, leading to the conclusion that HPV positivity might enhance responsiveness to ICIs [
      • Mandal R.
      • Şenbabaoğlu Y.
      • Desrichard A.
      • Havel J.J.
      • Dalin M.G.
      • Riaz N.
      • et al.
      The head and neck cancer immune landscape and its immunotherapeutic implications.
      ]. In accordance to this, further research has revealed that HPV + status in head and neck cancer prior to treatment translates into an inflamed tumour phenotype [
      • Gameiro S.F.
      • Ghasemi F.
      • Barrett J.W.
      • Koropatnick J.
      • Nichols A.C.
      • Mymryk J.S.
      • et al.
      Treatment-naïve HPV+ head and neck cancers display a T-cell-inflamed phenotype distinct from their HPV- counterparts that has implications for immunotherapy.
      ], while in another study, gene analysis performed on 544 head and neck tumours, revealed that higher expression of CTLA-4, PD-1 and TIM3 encoding genes was observed among HPV + compared to HPV- tumours, indicating that HPV positivity is linked to greater T-cell activation and immune exhaustion. In addition, TILs specifically in HPV + tumours were characterized by an increased B-cell signature [
      • Wood O.
      • Woo J.
      • Seumois G.
      • Savelyeva N.
      • McCann K.J.
      • Singh D.
      • et al.
      Gene expression analysis of TIL rich HPV-driven head and neck tumors reveals a distinct B-cell signature when compared to HPV independent tumors.
      ]. PD-1 expression in correlation with HPV status was also examined in a study of 64 previously untreated HNSCCs. The results demonstrated PD-1 positivity in a considerably larger number of CD4 + and CD8 + infiltrating lymphocytes in HPV + compared to HPV- tumours. Remarkably, HPV+/high PD-1 lymphocytic infiltration subgroup presented with better overall survival suggesting that PD-1 constitutes a marker of T-cell activation and priory elicited anti-tumour immune response rather than immune exhaustion. Possible implications with immunotherapy were explored as anti- PD-1 mAb in vitro treatment of HPV+/high PD-1 TILs tumours resulted in increased IFN-γ secretion [
      • Badoual C.
      • Hans S.
      • Merillon N.
      • Van Ryswick C.
      • Ravel P.
      • Benhamouda N.
      • et al.
      PD-1-expressing tumor-infiltrating T cells are a favorable prognostic biomarker in HPV-associated head and neck cancer.
      ]. Another study showed overexpression of T-cell exhaustion genes in HPV + compared to HPV- tumours. Particularly, IDO-1 expression was associated with specific HPV antigenicity and combined PD-1/IDO-1 inhibition elicited HPV specific cytotoxicity, defining IDO-1 as an ideal target for reversal of immune exhaustion in HPV + HNSCC [
      • Krishna S.
      • Ulrich P.
      • Wilson E.
      • Parikh F.
      • Narang P.
      • Yang S.
      • et al.
      Human papilloma virus specific immunogenicity and dysfunction of CD8.
      ].
      Both HPV positive and HPV negative HNSCC carries a high level of nonsynonymous mutations which have been implicated in the activation of immune response through MHC class I molecules [
      • Mandal R.
      • Şenbabaoğlu Y.
      • Desrichard A.
      • Havel J.J.
      • Dalin M.G.
      • Riaz N.
      • et al.
      The head and neck cancer immune landscape and its immunotherapeutic implications.
      ]. However, each of these two tumour subgroups defined by HPV status has been associated with a distinct genetic profile and is characterized by different subsets of mutations. HPV + tumours are characterized by PIK3CA activating mutations, inactivating mutations of CYLD and amplification of FGFR2 and FGFR3 cell cycle genes while HPV- tumours show a prevalence of inactivating mutations in tumour suppressor genes TP53, CDKN2A [
      • Seiwert T.Y.
      • Zuo Z.
      • Keck M.K.
      • Khattri A.
      • Pedamallu C.S.
      • Stricker T.
      • et al.
      Integrative and comparative genomic analysis of HPV-positive and HPV-negative head and neck squamous cell carcinomas.
      ]. Apolipoprotein-B mRNA editing catalytic polypeptide like (APOBEC), a family cytidine deaminase enzyme, has been associated with mutagenesis in HPV + HNSCC. It has been shown that specifically APOBEC3 enzymes demonstrate increased activity in HPV16 infected tumours [
      • Mori S.
      • Takeuchi T.
      • Ishii Y.
      • Kukimoto I.
      Identification of APOBEC3B promoter elements responsible for activation by human papillomavirus type 16 E6.
      ] and in addition to participating in antiviral immunity, they promote tumorigenesis by driving a distinct set of mutations [
      • Henderson S.
      • Fenton T.
      APOBEC3 genes: retroviral restriction factors to cancer drivers.
      ] including PIK3CA activating mutations, almost exclusively found in HPV + tumours [
      • Faden D.L.
      • Thomas S.
      • Cantalupo P.G.
      • Agrawal N.
      • Myers J.
      • DeRisi J.
      Multi-modality analysis supports APOBEC as a major source of mutations in head and neck squamous cell carcinoma.
      ,
      • Cannataro V.L.
      • Gaffney S.G.
      • Sasaki T.
      • Issaeva N.
      • Grewal N.K.S.
      • Grandis J.R.
      • et al.
      APOBEC-induced mutations and their cancer effect size in head and neck squamous cell carcinoma.
      ]. Consequently, positive correlation of APOBEC overexpression to increased immune signalling might influence IO effect on HPV + tumours [
      • Faden D.L.
      • Thomas S.
      • Cantalupo P.G.
      • Agrawal N.
      • Myers J.
      • DeRisi J.
      Multi-modality analysis supports APOBEC as a major source of mutations in head and neck squamous cell carcinoma.
      ]. Therefore, tumour genetic profiling can help identify these mutations and further illuminate their potential of response. Moreover, p16 encoding gene (CDKN2A) is known to downregulate the activity of cyclin D1 and cyclin-dependent kinases resulting in cell cycle control, whereas CDKN2A mutations lead to overexpression of cyclin D1 (CCDN1 gene) which is associated with poor prognosis [
      • Solomon B.
      • Young R.J.
      • Rischin D.
      Head and neck squamous cell carcinoma: Genomics and emerging biomarkers for immunomodulatory cancer treatments.
      ]. Smoking associated HNSCCs demonstrate poor immune infiltration while they are characterized by a high mutational load. Thus the overall effect on IO response cannot be predicted [
      • Mandal R.
      • Şenbabaoğlu Y.
      • Desrichard A.
      • Havel J.J.
      • Dalin M.G.
      • Riaz N.
      • et al.
      The head and neck cancer immune landscape and its immunotherapeutic implications.
      ].
      Regarding the use of HPV status (p16) as biomarker for ICI, SITC subcommittee recommends that HPV status per se should not dictate the use of immunotherapy as there is no strong data that p16 + patients benefit excessively [
      • Cohen E.E.W.
      • Bell R.B.
      • Bifulco C.B.
      • Burtness B.
      • Gillison M.L.
      • Harrington K.J.
      • et al.
      The society for immunotherapy of cancer consensus statement on immunotherapy for the treatment of squamous cell carcinoma of the head and neck (HNSCC).
      ].

      The microbiome

      Microbiome exerts critical effects on anti-cancer therapy response and toxicity and it has emerged as a promising therapeutic target [
      • Roy S.
      • Trinchieri G.
      Microbiota: a key orchestrator of cancer therapy.
      ]. The role of host gut microbiota regarding its influence on IO outcomes has been a field of investigation for recent studies combining both clinical and preclinical research data. Microbiota and the immune system are on a constantly fluctuating dynamic relationship with significant impact on local and systemic immune responses. T-cell activation, involvement of pattern recognition receptors and small metabolite recruitment constitute possible mechanisms by which gut bacteria participate in anti-cancer immunity [
      • Zitvogel L.
      • Ma Y.
      • Raoult D.
      • Kroemer G.
      • Gajewski T.F.
      The microbiome in cancer immunotherapy: diagnostic tools and therapeutic strategies.
      ]. Specific genera among gut microbiome have been identified as IO response and toxicity predictors and fecal microbiota transplant (FMT) in mice has further enlightened these findings [
      • Gopalakrishnan V.
      • Helmink B.A.
      • Spencer C.N.
      • Reuben A.
      • Wargo J.A.
      The influence of the gut microbiome on cancer, immunity, and cancer immunotherapy.
      ]. Research on lung, renal and urothelial cancer patients under treatment with ICI revealed that use of antibiotics right before or during therapy negatively affected median overall survival. Akkermansia muciniphila had the greatest association with response to ICI therapy and together with Enterococcus hirae increased responsiveness to ICI in previously non-responder mice following FMT. The immunological effect on these mice was depicted as increased CCR9 + CXCR3 + CD4 + T-cell tumour infiltration suggesting a stronger immune response [
      • Routy B.
      • Le Chatelier E.
      • Derosa L.
      • Duong C.P.M.
      • Alou M.T.
      • Daillère R.
      • et al.
      Gut microbiome influences efficacy of PD-1-based immunotherapy against epithelial tumors.
      ]. Two similar studies on melanoma associated the presence of certain microbiome members (Ruminococcaceae spp., Faecalibacterium spp., Bifidobacterium longum, Collinsella aerofaciens, Enterococcus faecius) with response to ICI. Microbiome diversity was also greater among responders while higher levels of Bacteriodales were noticed in non-responders [
      • Gopalakrishnan V.
      • Spencer C.N.
      • Nezi L.
      • Reuben A.
      • Andrews M.C.
      • Karpinets T.V.
      • et al.
      Gut microbiome modulates response to anti-PD-1 immunotherapy in melanoma patients.
      ]. FMT of these microbes to previously germ-free mice lead to augmentation of IO efficacy, possibly achieved by increased CD8 + T cell and decreased Foxp3 + CD4 + T cell TME infiltration [
      • Gopalakrishnan V.
      • Spencer C.N.
      • Nezi L.
      • Reuben A.
      • Andrews M.C.
      • Karpinets T.V.
      • et al.
      Gut microbiome modulates response to anti-PD-1 immunotherapy in melanoma patients.
      ,
      • Matson V.
      • Fessler J.
      • Bao R.
      • Chongsuwat T.
      • Zha Y.
      • Alegre M.L.
      • et al.
      The commensal microbiome is associated with anti-PD-1 efficacy in metastatic melanoma patients.
      ].
      Oral cavity microbiome has been shown to have clear implication in HNSCC development and progression. Fusobacteria species have been found in abundancy in both primary and metastatic cancerous tissues whereas Streptococcus presence was limited [
      • Shin J.M.
      • Luo T.
      • Kamarajan P.
      • Fenno J.C.
      • Rickard A.H.
      • Kapila Y.L.
      Microbial communities associated with primary and metastatic head and neck squamous cell carcinoma – a high fusobacterial and low streptococcal signature.
      ]. Alcohol consumption and tobacco, the two major risk factors for HNC, as well as periodontitis and poor oral hygiene have been shown to modify oral microbiome composition [
      • Fan X.
      • Peters B.A.
      • Jacobs E.J.
      • Gapstur S.M.
      • Purdue M.P.
      • Freedman N.D.
      • et al.
      Drinking alcohol is associated with variation in the human oral microbiome in a large study of American adults.
      ,
      • Chen J.
      • Domingue J.C.
      • Sears C.L.
      Microbiota dysbiosis in select human cancers: evidence of association and causality.
      ]. In addition, certain bacterial species have been associated with late stage oral squamous cell carcinoma, specifically F.periodonticum abundance and S.mitis and P.pasteri scarcity commonly found in stage 4 OSCC [
      • Yang C.Y.
      • Yeh Y.M.
      • Yu H.Y.
      • Chin C.Y.
      • Hsu C.W.
      • Liu H.
      • et al.
      Oral microbiota community dynamics associated with oral squamous cell carcinoma staging.
      ]. Accordingly, the potential use of microbiome as an IO predictive biomarker for HNSCC remains to be elucidated with further research pointed to this direction.
      Strategies to optimize response to immunotherapy are illustrated in Fig. 1.
      Figure thumbnail gr1
      Fig. 1Strategies to optimize response to immunotherapy depicted in the cancer immunity cycle.

      Conclusions

      Immunotherapy is a complex and rapidly evolving field that has the potential to provide substantial clinical benefit to patients with a variety of cancers. However, it becomes increasingly clear that a significant proportion of patients do not respond to widely used immunotherapies, such as immune checkpoint inhibitors. Currently, only PD-L1 is a validated biomarker used in clinical practice to guide treatment selection. Research should focus on improvement of patient selection, by implementing PD-L1 and by identifying new predictive biomarkers. In this context, tumor mutational burden, INF-γ signature, HPV status and the host’s microbiome have emerged as potential predictors of immune response and are currently being evaluated in clinical trials. In addition, the tumor microenvironment represents an interesting source for the development of novel biomarkers. On the other hand, combination therapies will be required to increase treatment efficacy. Investigating cancer immunology by reverse translating to the laboratory from clinical studies is needed to bring benefit to a greater number of patients. Development of strategies for patients who lack preexisting immunity is also necessary. Such strategies will be able to fulfill the promise that immunotherapy brings to the advancement of oncology. Indeed, we are at the beginning of an exciting journey for patients and for scientific investigation.

      Declaration of Competing Interest

      AP has received honoraria and research funding from BMS, MSD, Merck Serono, Pfizer, KURA, Astrazeneca

      References

        • Chen D.S.
        • Mellman I.
        Oncology meets immunology: the cancer-immunity cycle.
        Immunity. 2013; 39: 1-10
        • Kareemaghay S.
        • Tavassoli M.
        Clinical immunotherapeutic approaches for the treatment of head and neck cancer.
        Int J Oral Maxillofac Surg. 2018;
        • Burtness B.
        • Harrington K.J.
        • Greil R.
        • Soulières D.
        • Tahara M.
        • de Castro G.
        • et al.
        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.
        Lancet. 2019; 394: 1915-1928
        • Seiwert T.Y.
        • Burtness B.
        • Mehra R.
        • Weiss J.
        • Berger R.
        • Eder J.P.
        • et al.
        Safety and clinical activity of pembrolizumab for treatment of recurrent or metastatic squamous cell carcinoma of the head and neck (KEYNOTE-012): an open-label, multicentre, phase 1b trial.
        Lancet Oncol. 2016; 17: 956-965
        • Cohen E.E.W.
        • Bell R.B.
        • Bifulco C.B.
        • Burtness B.
        • Gillison M.L.
        • Harrington K.J.
        • et al.
        The society for immunotherapy of cancer consensus statement on immunotherapy for the treatment of squamous cell carcinoma of the head and neck (HNSCC).
        J Immunother Cancer. 2019; 7: 184
        • Freeman G.J.
        • Long A.J.
        • Iwai Y.
        • Bourque K.
        • Chernova T.
        • Nishimura H.
        • et al.
        Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation.
        J Exp Med. 2000; 192: 1027-1034
        • Dong H.
        • Strome S.E.
        • Salomao D.R.
        • Tamura H.
        • Hirano F.
        • Flies D.B.
        • et al.
        Tumor-associated B7–H1 promotes T-cell apoptosis: a potential mechanism of immune evasion.
        Nat Med. 2002; 8: 793-800
        • Topalian S.L.
        • Drake C.G.
        • Pardoll D.M.
        Targeting the PD-1/B7-H1(PD-L1) pathway to activate anti-tumor immunity.
        Curr Opin Immunol. 2012; 24: 207-212
        • Tumeh P.C.
        • Harview C.L.
        • Yearley J.H.
        • Shintaku I.P.
        • Taylor E.J.
        • Robert L.
        • et al.
        PD-1 blockade induces responses by inhibiting adaptive immune resistance.
        Nature. 2014; 515: 568-571
        • Herbst R.S.
        • Soria J.C.
        • Kowanetz M.
        • Fine G.D.
        • Hamid O.
        • Gordon M.S.
        • et al.
        Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients.
        Nature. 2014; 515: 563-567
        • Topalian S.L.
        • Hodi F.S.
        • Brahmer J.R.
        • Gettinger S.N.
        • Smith D.C.
        • McDermott D.F.
        • et al.
        Safety, activity, and immune correlates of anti-PD-1 antibody in cancer.
        N Engl J Med. 2012; 366: 2443-2454
        • Brahmer J.R.
        • Tykodi S.S.
        • Chow L.Q.
        • Hwu W.J.
        • Topalian S.L.
        • Hwu P.
        • et al.
        Safety and activity of anti-PD-L1 antibody in patients with advanced cancer.
        N Engl J Med. 2012; 366: 2455-2465
        • Cohen E.E.W.
        • Soulières D.
        • Le Tourneau C.
        • Dinis J.
        • Licitra L.
        • Ahn M.J.
        • et al.
        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.
        Lancet. 2019; 393: 156-167
        • Ferris R.L.
        • Blumenschein G.
        • Fayette J.
        • Guigay J.
        • Colevas A.D.
        • Licitra L.
        • et al.
        Nivolumab for recurrent squamous-cell carcinoma of the head and neck.
        N Engl J Med. 2016; 375: 1856-1867
        • Bauml J.
        • Seiwert T.Y.
        • Pfister D.G.
        • Worden F.
        • Liu S.V.
        • Gilbert J.
        • et al.
        Pembrolizumab for platinum- and cetuximab-refractory head and neck cancer: results from a single-arm phase II study.
        J Clin Oncol. 2017; 35: 1542-1549
        • Mehra R.
        • Seiwert T.Y.
        • Gupta S.
        • Weiss J.
        • Gluck I.
        • Eder J.P.
        • et al.
        Efficacy and safety of pembrolizumab in recurrent/metastatic head and neck squamous cell carcinoma: pooled analyses after long-term follow-up in KEYNOTE-012.
        Br J Cancer. 2018; 119: 153-159
        • Ferris R.L.
        • Blumenschein G.
        • Fayette J.
        • Guigay J.
        • Colevas A.D.
        • Licitra L.
        • et al.
        Nivolumab vs investigator's choice in recurrent or metastatic squamous cell carcinoma of the head and neck: 2-year long-term survival update of CheckMate 141 with analyses by tumor PD-L1 expression.
        Oral Oncol. 2018; 81: 45-51
        • Rasmussen J.H.
        • Lelkaitis G.
        • Hakansson K.
        • Vogelius I.R.
        • Johannesen H.H.
        • Fischer B.M.
        • et al.
        Intratumor heterogeneity of PD-L1 expression in head and neck squamous cell carcinoma.
        Br J Cancer. 2019; 120: 1003-1006
      1. M.J. Ratcliffe AS, M. Rebelatto, M. Scott, C. Barker, P. Scorer, J. Walker. A comparative study of PD-L1 diagnostic assays in squamous cell carcinoma of the head and neck (SCCHN). Annals Oncol. 2016 27 (6): 328–350 101093/annonc/mdw376. 2016.

        • Robbins P.F.
        • Lu Y.C.
        • El-Gamil M.
        • Li Y.F.
        • Gross C.
        • Gartner J.
        • et al.
        Mining exomic sequencing data to identify mutated antigens recognized by adoptively transferred tumor-reactive T cells.
        Nat Med. 2013; 19: 747-752
        • Linnemann C.
        • van Buuren M.M.
        • Bies L.
        • Verdegaal E.M.
        • Schotte R.
        • Calis J.J.
        • et al.
        High-throughput epitope discovery reveals frequent recognition of neo-antigens by CD4+ T cells in human melanoma.
        Nat Med. 2015; 21: 81-85
        • Brown S.D.
        • Warren R.L.
        • Gibb E.A.
        • Martin S.D.
        • Spinelli J.J.
        • Nelson B.H.
        • et al.
        Neo-antigens predicted by tumor genome meta-analysis correlate with increased patient survival.
        Genome Res. 2014; 24: 743-750
        • Schumacher T.N.
        • Schreiber R.D.
        Neoantigens in cancer immunotherapy.
        Science. 2015; 348: 69-74
        • Rizvi N.A.
        • Hellmann M.D.
        • Snyder A.
        • Kvistborg P.
        • Makarov V.
        • Havel J.J.
        • et al.
        Cancer immunology. Mutational landscape determines sensitivity to PD-1 blockade in non-small cell lung cancer.
        Science. 2015; 348: 124-128
        • Van Allen E.M.
        • Miao D.
        • Schilling B.
        • Shukla S.A.
        • Blank C.
        • Zimmer L.
        • et al.
        Genomic correlates of response to CTLA-4 blockade in metastatic melanoma.
        Science. 2015; 350: 207-211
        • Snyder A.
        • Makarov V.
        • Merghoub T.
        • Yuan J.
        • Zaretsky J.M.
        • Desrichard A.
        • et al.
        Genetic basis for clinical response to CTLA-4 blockade in melanoma.
        N Engl J Med. 2014; 371: 2189-2199
        • McGranahan N.
        • Furness A.J.
        • Rosenthal R.
        • Ramskov S.
        • Lyngaa R.
        • Saini S.K.
        • et al.
        Clonal neoantigens elicit T cell immunoreactivity and sensitivity to immune checkpoint blockade.
        Science. 2016; 351: 1463-1469
        • Cristescu R.
        • Mogg R.
        • Ayers M.
        • Albright A.
        • Murphy E.
        • Yearley J.
        • et al.
        Pan-tumor genomic biomarkers for PD-1 checkpoint blockade-based immunotherapy.
        Science. 2018; 362
        • Hanna G.J.
        • Lizotte P.
        • Cavanaugh M.
        • Kuo F.C.
        • Shivdasani P.
        • Frieden A.
        • et al.
        Frameshift events predict anti-PD-1/L1 response in head and neck cancer.
        JCI Insight. 2018; 3
        • Allen C.T.
        • Judd N.P.
        • Bui J.D.
        • Uppaluri R.
        The clinical implications of antitumor immunity in head and neck cancer.
        Laryngoscope. 2012; 122: 144-157
        • Gajewski T.F.
        • Fuertes M.B.
        • Woo S.R.
        Innate immune sensing of cancer: clues from an identified role for type I IFNs.
        Cancer Immunol Immunother. 2012; 61: 1343-1347
        • Corrales L.
        • Gajewski T.F.
        Molecular Pathways: targeting the stimulator of interferon genes (STING) in the immunotherapy of cancer.
        Clin Cancer Res. 2015; 21: 4774-4779
        • Woo S.R.
        • Corrales L.
        • Gajewski T.F.
        Innate immune recognition of cancer.
        Annu Rev Immunol. 2015; 33: 445-474
        • Woo S.R.
        • Fuertes M.B.
        • Corrales L.
        • Spranger S.
        • Furdyna M.J.
        • Leung M.Y.
        • et al.
        STING-dependent cytosolic DNA sensing mediates innate immune recognition of immunogenic tumors.
        Immunity. 2014; 41: 830-842
        • Mackenzie K.J.
        • Carroll P.
        • Martin C.A.
        • Murina O.
        • Fluteau A.
        • Simpson D.J.
        • et al.
        cGAS surveillance of micronuclei links genome instability to innate immunity.
        Nature. 2017; 548: 461-465
        • Ayers M.
        • Lunceford J.
        • Nebozhyn M.
        • Murphy E.
        • Loboda A.
        • Kaufman D.R.
        • et al.
        IFN-γ-related mRNA profile predicts clinical response to PD-1 blockade.
        J Clin Invest. 2017; 127: 2930-2940
        • Chen D.S.
        • Mellman I.
        Elements of cancer immunity and the cancer-immune set point.
        Nature. 2017; 541: 321-330
        • Spranger S.
        • Spaapen R.M.
        • Zha Y.
        • Williams J.
        • Meng Y.
        • Ha T.T.
        • et al.
        Up-regulation of PD-L1, IDO, and T(regs) in the melanoma tumor microenvironment is driven by CD8(+) T cells.
        Sci Transl Med. 2013; 5: 200ra116
        • Goltz D.
        • Gevensleben H.
        • Vogt T.J.
        • Dietrich J.
        • Golletz C.
        • Bootz F.
        • et al.
        CTLA4 methylation predicts response to anti-PD-1 and anti-CTLA-4 immunotherapy in melanoma patients.
        JCI Insight. 2018; 3
        • Spranger S.
        • Koblish H.K.
        • Horton B.
        • Scherle P.A.
        • Newton R.
        • Gajewski T.F.
        Mechanism of tumor rejection with doublets of CTLA-4, PD-1/PD-L1, or IDO blockade involves restored IL-2 production and proliferation of CD8(+) T cells directly within the tumor microenvironment.
        J ImmunoTher Cancer. 2014; 2: 3
        • Gajewski T.F.
        The next hurdle in cancer immunotherapy: overcoming the non-T-cell-inflamed tumor microenvironment.
        Semin Oncol. 2015; 42: 663-671
        • Spranger S.
        • Gajewski T.F.
        Impact of oncogenic pathways on evasion of antitumour immune responses.
        Nat Rev Cancer. 2018; 18: 139-147
        • Ager A.
        • May M.J.
        Understanding high endothelial venules: Lessons for cancer immunology.
        Oncoimmunology. 2015; 4e1008791
        • Sautès-Fridman C.
        • Petitprez F.
        • Calderaro J.
        • Fridman W.H.
        Tertiary lymphoid structures in the era of cancer immunotherapy.
        Nat Rev Cancer. 2019; 19: 307-325
        • Whiteside T.L.
        • Demaria S.
        • Rodriguez-Ruiz M.E.
        • Zarour H.M.
        • Melero I.
        Emerging opportunities and challenges in cancer immunotherapy.
        Clin Cancer Res. 2016; 22: 1845-1855
        • Davis R.J.
        • Van Waes C.
        • Allen C.T.
        Overcoming barriers to effective immunotherapy: MDSCs, TAMs, and Tregs as mediators of the immunosuppressive microenvironment in head and neck cancer.
        Oral Oncol. 2016; 58: 59-70
        • Chen Y.P.
        • Wang Y.Q.
        • Lv J.W.
        • Li Y.Q.
        • Chua M.L.K.
        • Le Q.T.
        • et al.
        Identification and validation of novel microenvironment-based immune molecular subgroups of head and neck squamous cell carcinoma: implications for immunotherapy.
        Ann Oncol. 2018;
        • de Ruiter E.J.
        • Ooft M.L.
        • Devriese L.A.
        • Willems S.M.
        The prognostic role of tumor infiltrating T-lymphocytes in squamous cell carcinoma of the head and neck: a systematic review and meta-analysis.
        Oncoimmunology. 2017; 6e1356148
        • Badoual C.
        • Hans S.
        • Rodriguez J.
        • Peyrard S.
        • Klein C.
        • NlH Agueznay
        • et al.
        Prognostic value of tumor-infiltrating CD4+ T-cell subpopulations in head and neck cancers.
        Clin Cancer Res. 2006; 12: 465-472
        • Badoual Cecile
        • Hans Stephane
        • Fridman Wolf H.
        • Brasnu Daniel
        • Erdman Susan
        • Tartour Eric
        Revisiting the prognostic value of regulatory T cells in patients with cancer.
        JCO. 2009; 27: e5-e6https://doi.org/10.1200/JCO.2009.23.0680
        • Mandal R.
        • Şenbabaoğlu Y.
        • Desrichard A.
        • Havel J.J.
        • Dalin M.G.
        • Riaz N.
        • et al.
        The head and neck cancer immune landscape and its immunotherapeutic implications.
        JCI Insight. 2016; 1e89829
        • Davis R.J.
        • Moore E.C.
        • Clavijo P.E.
        • Friedman J.
        • Cash H.
        • Chen Z.
        • et al.
        Anti-PD-L1 efficacy can be enhanced by inhibition of myeloid-derived suppressor cells with a selective inhibitor of PI3Kδ/γ.
        Cancer Res. 2017; 77: 2607-2619
        • Munn D.H.
        • Mellor A.L.
        IDO in the tumor microenvironment: inflammation, counter-regulation, and tolerance.
        Trends Immunol. 2016; 37: 193-207
        • Munn D.H.
        • Mellor A.L.
        Indoleamine 2,3-dioxygenase and tumor-induced tolerance.
        J Clin Invest. 2007; 117: 1147-1154
        • Holmgaard R.B.
        • Zamarin D.
        • Li Y.
        • Gasmi B.
        • Munn D.H.
        • Allison J.P.
        • et al.
        Tumor-expressed IDO recruits and activates MDSCs in a treg-dependent manner.
        Cell Rep. 2015; 13: 412-424
        • Garber K.
        A new cancer immunotherapy suffers a setback.
        Science. 2018; 360: 588
        • Holmgaard R.B.
        • Zamarin D.
        • Munn D.H.
        • Wolchok J.D.
        • Allison J.P.
        Indoleamine 2,3-dioxygenase is a critical resistance mechanism in antitumor T cell immunotherapy targeting CTLA-4.
        J Exp Med. 2013; 210: 1389-1402
        • Botticelli A.
        • Cerbelli B.
        • Lionetto L.
        • Zizzari I.
        • Salati M.
        • Pisano A.
        • et al.
        Can IDO activity predict primary resistance to anti-PD-1 treatment in NSCLC?.
        J Transl Med. 2018; 16: 219
        • Prince M.E.
        • Sivanandan R.
        • Kaczorowski A.
        • Wolf G.T.
        • Kaplan M.J.
        • Dalerba P.
        • et al.
        Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma.
        Proc Natl Acad Sci USA. 2007; 104: 973-978
        • Chikamatsu K.
        • Takahashi G.
        • Sakakura K.
        • Ferrone S.
        • Masuyama K.
        Immunoregulatory properties of CD44+ cancer stem-like cells in squamous cell carcinoma of the head and neck.
        Head Neck. 2011; 33: 208-215
        • Lee Y.
        • Shin J.H.
        • Longmire M.
        • Wang H.
        • Kohrt H.E.
        • Chang H.Y.
        • et al.
        CD44+ cells in head and neck squamous cell carcinoma suppress T-cell-mediated immunity by selective constitutive and inducible expression of PD-L1.
        Clin Cancer Res. 2016; 22: 3571-3581
        • Wood O.
        • Clarke J.
        • Woo J.
        • Mirza A.H.
        • Woelk C.H.
        • Thomas G.J.
        • et al.
        Head and neck squamous cell carcinomas are characterized by a stable immune signature within the primary tumor over time and space.
        Clin Cancer Res. 2017; 23: 7641-7649
        • Vassilakopoulou M.
        • Avgeris M.
        • Velcheti V.
        • Kotoula V.
        • Rampias T.
        • Chatzopoulos K.
        • et al.
        Evaluation of PD-L1 expression and associated tumor-infiltrating lymphocytes in laryngeal squamous cell carcinoma.
        Clin Cancer Res. 2016; 22: 704-713
        • Badr M.
        • Jöhrens K.
        • Allgäuer M.
        • Boxberg M.
        • Weichert W.
        • Tinhofer I.
        • et al.
        Morphomolecular analysis of the immune tumor microenvironment in human head and neck cancer.
        Cancer Immunol Immunother. 2019; 68: 1443-1454
        • Hanna G.J.
        • Liu H.
        • Jones R.E.
        • Bacay A.F.
        • Lizotte P.H.
        • Ivanova E.V.
        • et al.
        Defining an inflamed tumor immunophenotype in recurrent, metastatic squamous cell carcinoma of the head and neck.
        Oral Oncol. 2017; 67: 61-69
        • Bates A.M.
        • Lanzel E.A.
        • Qian F.
        • Abbasi T.
        • Vali S.
        • Brogden K.A.
        Cell genomics and immunosuppressive biomarker expression influence PD-L1 immunotherapy treatment responses in HNSCC-a computational study.
        Oral Surg Oral Med Oral Pathol Oral Radiol. 2017; 124: 157-164
        • Li B.
        • Cui Y.
        • Nambiar D.K.
        • Sunwoo J.B.
        • Li R.
        The immune subtypes and landscape of squamous cell carcinoma.
        Clin Cancer Res. 2019; 25: 3528-3537
        • Posner M.R.
        • Lorch J.H.
        • Goloubeva O.
        • Tan M.
        • Schumaker L.M.
        • Sarlis N.J.
        • et al.
        Survival and human papillomavirus in oropharynx cancer in TAX 324: a subset analysis from an international phase III trial.
        Ann Oncol. 2011; 22: 1071-1077
        • Lassen P.
        • Eriksen J.G.
        • Krogdahl A.
        • Therkildsen M.H.
        • Ulhøi B.P.
        • Overgaard M.
        • et al.
        The influence of HPV-associated p16-expression on accelerated fractionated radiotherapy in head and neck cancer: evaluation of the randomised DAHANCA 6&7 trial.
        Radiother Oncol. 2011; 100: 49-55
        • Vermorken J.B.
        • Psyrri A.
        • Mesía R.
        • Peyrade F.
        • Beier F.
        • de Blas B.
        • et al.
        Impact of tumor HPV status on outcome in patients with recurrent and/or metastatic squamous cell carcinoma of the head and neck receiving chemotherapy with or without cetuximab: retrospective analysis of the phase III EXTREME trial.
        Ann Oncol. 2014; 25: 801-807
        • Argiris A.
        • Li S.
        • Ghebremichael M.
        • Egloff A.M.
        • Wang L.
        • Forastiere A.A.
        • et al.
        Prognostic significance of human papillomavirus in recurrent or metastatic head and neck cancer: an analysis of eastern cooperative oncology group trials.
        Ann Oncol. 2014; 25: 1410-1416
        • Fakhry C.
        • Zhang Q.
        • Nguyen-Tan P.F.
        • Rosenthal D.
        • El-Naggar A.
        • Garden A.S.
        • et al.
        Human papillomavirus and overall survival after progression of oropharyngeal squamous cell carcinoma.
        J Clin Oncol. 2014; 32: 3365-3373
        • Partlová S.
        • Bouček J.
        • Kloudová K.
        • Lukešová E.
        • Zábrodský M.
        • Grega M.
        • et al.
        Distinct patterns of intratumoral immune cell infiltrates in patients with HPV-associated compared to non-virally induced head and neck squamous cell carcinoma.
        Oncoimmunology. 2015; 4e965570
        • Gameiro S.F.
        • Ghasemi F.
        • Barrett J.W.
        • Koropatnick J.
        • Nichols A.C.
        • Mymryk J.S.
        • et al.
        Treatment-naïve HPV+ head and neck cancers display a T-cell-inflamed phenotype distinct from their HPV- counterparts that has implications for immunotherapy.
        Oncoimmunology. 2018; 7e1498439
        • Wood O.
        • Woo J.
        • Seumois G.
        • Savelyeva N.
        • McCann K.J.
        • Singh D.
        • et al.
        Gene expression analysis of TIL rich HPV-driven head and neck tumors reveals a distinct B-cell signature when compared to HPV independent tumors.
        Oncotarget. 2016; 7: 56781-56797
        • Badoual C.
        • Hans S.
        • Merillon N.
        • Van Ryswick C.
        • Ravel P.
        • Benhamouda N.
        • et al.
        PD-1-expressing tumor-infiltrating T cells are a favorable prognostic biomarker in HPV-associated head and neck cancer.
        Cancer Res. 2013; 73: 128-138
        • Krishna S.
        • Ulrich P.
        • Wilson E.
        • Parikh F.
        • Narang P.
        • Yang S.
        • et al.
        Human papilloma virus specific immunogenicity and dysfunction of CD8.
        Cancer Res. 2018; 78: 6159-6170
        • Seiwert T.Y.
        • Zuo Z.
        • Keck M.K.
        • Khattri A.
        • Pedamallu C.S.
        • Stricker T.
        • et al.
        Integrative and comparative genomic analysis of HPV-positive and HPV-negative head and neck squamous cell carcinomas.
        Clin Cancer Res. 2015; 21: 632-641
        • Mori S.
        • Takeuchi T.
        • Ishii Y.
        • Kukimoto I.
        Identification of APOBEC3B promoter elements responsible for activation by human papillomavirus type 16 E6.
        Biochem Biophys Res Commun. 2015; 460: 555-560
        • Henderson S.
        • Fenton T.
        APOBEC3 genes: retroviral restriction factors to cancer drivers.
        Trends Mol Med. 2015; 21: 274-284
        • Faden D.L.
        • Thomas S.
        • Cantalupo P.G.
        • Agrawal N.
        • Myers J.
        • DeRisi J.
        Multi-modality analysis supports APOBEC as a major source of mutations in head and neck squamous cell carcinoma.
        Oral Oncol. 2017; 74: 8-14
        • Cannataro V.L.
        • Gaffney S.G.
        • Sasaki T.
        • Issaeva N.
        • Grewal N.K.S.
        • Grandis J.R.
        • et al.
        APOBEC-induced mutations and their cancer effect size in head and neck squamous cell carcinoma.
        Oncogene. 2019;
        • Solomon B.
        • Young R.J.
        • Rischin D.
        Head and neck squamous cell carcinoma: Genomics and emerging biomarkers for immunomodulatory cancer treatments.
        Semin Cancer Biol. 2018; 52: 228-240
        • Roy S.
        • Trinchieri G.
        Microbiota: a key orchestrator of cancer therapy.
        Nat Rev Cancer. 2017; 17: 271-285
        • Zitvogel L.
        • Ma Y.
        • Raoult D.
        • Kroemer G.
        • Gajewski T.F.
        The microbiome in cancer immunotherapy: diagnostic tools and therapeutic strategies.
        Science. 2018; 359: 1366-1370
        • Gopalakrishnan V.
        • Helmink B.A.
        • Spencer C.N.
        • Reuben A.
        • Wargo J.A.
        The influence of the gut microbiome on cancer, immunity, and cancer immunotherapy.
        Cancer Cell. 2018; 33: 570-580
        • Routy B.
        • Le Chatelier E.
        • Derosa L.
        • Duong C.P.M.
        • Alou M.T.
        • Daillère R.
        • et al.
        Gut microbiome influences efficacy of PD-1-based immunotherapy against epithelial tumors.
        Science. 2018; 359: 91-97
        • Gopalakrishnan V.
        • Spencer C.N.
        • Nezi L.
        • Reuben A.
        • Andrews M.C.
        • Karpinets T.V.
        • et al.
        Gut microbiome modulates response to anti-PD-1 immunotherapy in melanoma patients.
        Science. 2018; 359: 97-103
        • Matson V.
        • Fessler J.
        • Bao R.
        • Chongsuwat T.
        • Zha Y.
        • Alegre M.L.
        • et al.
        The commensal microbiome is associated with anti-PD-1 efficacy in metastatic melanoma patients.
        Science. 2018; 359: 104-108
        • Shin J.M.
        • Luo T.
        • Kamarajan P.
        • Fenno J.C.
        • Rickard A.H.
        • Kapila Y.L.
        Microbial communities associated with primary and metastatic head and neck squamous cell carcinoma – a high fusobacterial and low streptococcal signature.
        Sci Rep. 2017; 7: 9934
        • Fan X.
        • Peters B.A.
        • Jacobs E.J.
        • Gapstur S.M.
        • Purdue M.P.
        • Freedman N.D.
        • et al.
        Drinking alcohol is associated with variation in the human oral microbiome in a large study of American adults.
        Microbiome. 2018; 6: 59
        • Chen J.
        • Domingue J.C.
        • Sears C.L.
        Microbiota dysbiosis in select human cancers: evidence of association and causality.
        Semin Immunol. 2017; 32: 25-34
        • Yang C.Y.
        • Yeh Y.M.
        • Yu H.Y.
        • Chin C.Y.
        • Hsu C.W.
        • Liu H.
        • et al.
        Oral microbiota community dynamics associated with oral squamous cell carcinoma staging.
        Front Microbiol. 2018; 9: 862