High-Risk Human Papillomavirus (HR-HPV) DNA Detection in Mouthwashes for Diagnosis of HPV-Driven Oropharynx Cancer and Its Curative Therapy-A Feasibility Study.

Detection of p16 through immunohistochemistry (IHC) is the standard for determining the HPV status of the tumor according the TNM eighth edition released in 2017 and has become crucial for determining the HPV status of oropharyngeal squamous cell carcinomas (OPSCC) with direct impact on staging and prognostication. In recent years, detection of HPV DNA in mouthwashes has been proposed as a noninvasive alternative, both for OPSCCs and for other head and neck squamous cell carcinomas (HNSCCs). However, the prospect of using the mouthwashes to monitor the response to therapy is unclear. To evaluate the effect of curative therapy on the detection of HPV DNA, we performed a prospective study comparing the detection frequency of high-risk HPV DNA (HR-HPV-DNA) in pre- and post-therapy mouthwashes. We collected 137 mouthwashes from 88 pathologically confirmed HNSCC patients for DNA isolation and HPV genotyping with the Inno-LiPA assay. We show that HPV DNA in pretherapeutic mouthwashes can detect HPV-driven HNSCCs with a sensitivity of 50.0% and specificity of 85.4%, alongside a high negative predictive value of 79.5% and an accuracy of 74.5%. Furthermore, we observed a notable decrease in the detection frequency of HR-HPV-DNA after successful treatment (pre-therapy 50.0% (9/18) versus post-therapy 9.7% (3/28)). However, the comparatively low sensitivity regarding detection of HPV-driven OPSCC argues against its use in clinical routine.

Cytokine Profiles of Head and Neck Squamous Cell Carcinoma Undergoing Dual Immunotherapy With Cetuximab and Pembrolizumab Identify Interferon Gamma-Induced Protein 10 as Novel Biomarker.

Background: Pembrolizumab and cetuximab are antibodies under investigation in head and neck squamous cell carcinoma (HNSCC) either as single agents or combined with cisplatin and other chemotherapeutic drugs, e.g., 5-fluorouracil and/or docetaxel. However, also the combination of both antibodies may have potential in recurrent/metastatic (R/M) HNSCC, in particular in cisplatin-resistant or -refractory cases or patients with comorbid disease, e.g. patients with impaired renal function. Methods: To clarify potential benefit that may result from such combination, we used the FLAVINO assay, a short-time ex vivo assay to compare responsiveness of HNSCC to pembrolizumab, cetuximab and both combined regarding colony formation of epithelial cells of biopsy-derived tumor samples and their cytokine production within three days either without or with stimulation with 10 ng/mL interferon gamma (IFN-γ). Vascular endothelial growth factor A (VEGF), monocyte chemoattractant protein 1 (MCP-1 or CCL2), interleukin 6 (IL-6), IL-8, IFN-γ, and interferon gamma-induced protein 10 (IP-10 or CXCL10) in supernatants were measured by ELISA. Results: We detected huge heterogeneity in response to cetuximab, pembrolizumab and both combined with and without IFN-γ stimulation. Moreover, we detected a link between IFN-γ induced IP-10 release and improved outcome in those HNSCC patients who were capable to respond to IFN-γ and pembrolizumab, cetuximab and both combined with a further increase in IP-10 production. We derived an "IP-10 score" that independent from clinical characteristics of HNSCC patients and therapy regimens applied was able to predict their outcome. Conclusions: The heterogeneity in the ex vivo response of cetuximab, pembrolizumab and both combined with and without IFN-γ stimulation identifies subgroups of HNSCC patients with deviating OS.

Pre-Therapeutic VEGF Level in Plasma Is a Prognostic Bio-Marker in Head and Neck Squamous Cell Carcinoma (HNSCC).

Vascular endothelial growth factor (VEGF) is centrally involved in cancer angiogenesis. We hypothesized that pre-therapeutic VEGF levels in serum and plasma differ in their potential as biomarkers for outcomes in head and neck squamous cell carcinoma (HNSCC) patients. As prospectively defined in the study protocols of TRANSCAN-DietINT and NICEI-CIH, we measured VEGF in pretreatment serum and plasma of 75 HNSCC test cohort (TC) patients. We analyzed the prognostic value of VEGF concentrations in serum (VEGFSerum) and plasma (VEGFPlasma) for event-free survival (EFS) utilizing receiver-operating characteristics (ROC). Mean VEGF concentrations in plasma (34.6, 95% CI 26.0-43.3 ng/L) were significantly lower (p = 3.35 × 10-18) than in serum (214.8, 95% CI 179.6-250.0 ng/L) but, based on ROC (area under the curve, AUCPlasma = 0.707, 95% CI 0.573-0.840; p = 0.006 versus AUCSerum = 0.665, 95% CI 0.528-0.801; p = 0.030), superiorly correlated with event-free survival (EFS) of TC patients. Youden indices revealed optimum binary classification with VEGFPlasma 26 ng/L and VEGFSerum 264 ng/L. Kaplan-Meier plots demonstrated superiority of VEGFPlasma in discriminating patients regarding outcome. Patients with VEGFPlasma < 26 ng/L had superior nodal (NC), local (LC) and loco-regional control (LRC) leading to significant prolonged progression-free survival (PFS) and EFS. We successfully validated VEGFPlasma according the cut-off <26 ng/L as predictive for superior outcome in an independent validation cohort (iVC) of 104 HNSCC patients from the studies DeLOS-II and LIFE and found better outcomes including prolonged tumor-specific (TSS) and overall survival (OS). Outcomes in TC and iVC combined again was related to VEGFPlasma, and multivariate Cox regression revealed that VEGFPlasma was an independent outcome predictor. In HNSCC, pre-therapeutic VEGFPlasma is prognostic for outcomes.