The combination of a high Hsp70 expression (scores 3C4) and a low NK cell infiltration shows better negative prognostic value (p?=?0.0004; Fig. Hsp70 membrane\positive tumor cells.18 Safety and tolerability of these stimulated, autologous NK cells have been demonstrated in a phase I clinical trial.19 Presently a proof\of\concept phase II randomized clinical trial is ongoing to study the efficacy of Hsp70\stimulated NK cells in patients with squamous NSCLC in stage IIIA/B after RCT.8, 20 In our study, which is part of the DKTK\ROG initiative, which aims to identify and validate biomarkers for outcome of RCT in SCCHN,21, 22, 23, 24, 25, 26 we aim to study the role of Hsp70 and tumor\infiltrating NK cells as prognostic tumor biomarkers. Every year approximately 500,000 new cases are diagnosed worldwide with SCCHN with 4.8% of total cancer incidence and 4.6% cancer mortality.27 Apart from tobacco and alcohol, which are considered as main risk factors for the development of head and neck malignancy, infection with human papilloma computer virus (HPV) has been determined as causally connected to oropharyngeal cancer.28 Due to increasing numbers of HPV infections the incidence for SCCHN is rising especially in younger individuals.29 Patients with locally advanced SCCHN have a 5 year survival rate of 40C60%.27 Given the progress in medicine over the last decades these mortality rates are still not satisfying. Consequently, reliable biomarkers, which are able to predict outcome of therapy at an early time point are urgently needed to stratify patients with respect to prognosis and to guideline adaptations for the treatment. An early event in SCCHN carcinogenesis is related to somatic mutations of the protein p16 (chromosome 9p21) that exerts tumor suppressor function by binding to the cyclin D1 CDK4/CDK6 complex, which results in a G1 arrest.30 Silencing of p16 by homozygous deletion, CARMA1 methylation of the promoter and base pair mutations are associated with a more rapid tumor growth.31 However, with respect to SCCHN the role of p16, as a prognostic marker for outcome of RCT, remains a matter of debate.32 Between 40% and 70% of SCCHN contain mutations in the tumor suppressor gene p53.33 An accumulation of p53, which can be induced not only by mutations, but also by other mechanisms, results in invasive tumor growth and radioresistance.34 Although HPV infection is a risk factor for the development of SCCHN, HPV16 DNA\positive patients show a better clinical outcome compared to their HPV16 DNA\free counterparts.35 Part of this effect has been attributed to an HPV16\induced activation of the immune system. In line with this obtaining, infiltration of tumors with CD8+ cytotoxic T lymphocytes has been found to be associated with an HPV16 DNA\positive status and a favorable tumor prognosis.25 In addition, higher numbers of CD56+ tumor\infiltrating NK cells are associated with better prognosis in oropharyngeal squamous cell carcinoma.36 In our study, we aimed to assess the role of Hsp70 either alone or in relationship with HPV16 DNA, p16 and p53 status, and the infiltration of tumors with CD56+ NK cells, as prognostic markers in patients with SCCHN after surgery and RCT. Patients and Methods SCCHN patients Between 2004 and 2012, patients with histologically confirmed SCCHN of the oro\, hypopharynx and oral cavity were recruited into the study. Patient characteristics are summarized in Table 1. Apart from pN stage (and ?and11 and ?and33 and Table 5 clearly indicate that low numbers of CD56+ tumor\infiltrating NK cells have a negative prognostic value for a significantly decreased OS in patients with SCCHN (HR?=?0.29, and Table 7, distant metastases develop significantly earlier in patients with a low tumor\infiltration of CD56+ NK cells (HR?=?0.31, p?=?0.0001). Open in a separate window Figure 4 KaplanCMeier analysis of the prognostic value of Methoxsalen (Oxsoralen) infiltrating CD56+ NK cells in FFPE tumor sections of SCCHN patients with overall survival (OS) and distant metastases\free survival (DMFS). (a) Representative view of SCCHN sections with high (upper graph) and low numbers (lower graph) of infiltrating CD56+ NK cells. Selected singular as well as groups of CD56+ NK cells are marked with white arrows, scale bar, 100 m. (b) Significant correlation of infiltrating CD56+ NK cells in tumor sections of SCCHN patients (N?=?114) and Methoxsalen (Oxsoralen) OS. Black line represents high and gray lines represent low numbers of infiltrating CD56+ NK cells. Patients at risk with low and high numbers of infiltrating CD56+ NK cells at different time\points after start of therapy ranging from 0 to 96 months are indicated below the graph. (c) Significant correlation of infiltrating CD56+ NK cells in tumor sections of SCCHN patients (N?=?114) and DMFS. Black line represents Methoxsalen (Oxsoralen) high and gray lines represent low numbers of infiltrating CD56+ NK cells. Patients at risk with low and high numbers of infiltrating CD56+ NK cells at different time\points after start of therapy ranging from 0 to 96 months are indicated below the graph. (d) Significant correlation of infiltrating CD56+ NK cells in tumor sections with low (left graph; Methoxsalen (Oxsoralen) scores 0C2).