Independent validation of tumour volume, cancer stem cell markers and hypoxia-associated gene expressions for HNSCC after primary radiochemotherapy.

OBJECTIVE: To independently validate the impact of tumour volume, p16 status, cancer stem cell (CSC) marker expression and hypoxia-associated gene signatures as potential prognostic biomarkers for patients with locally advanced head and neck squamous cell carcinoma (HNSCC), who underwent primary radiotherapy or radiochemotherapy (RCTx). These markers have previously been reported in a study of the German Cancer Consortium Radiation Oncology Group (DKTK-ROG) (Linge et al., 2016). MATERIALS AND METHODS: In this retrospective monocentric study, 92 patients with locally advanced HNSCC were included. Univariable and multivariable logistic regressions and Cox models presented in the study of the DKTK-ROG were validated using the area under the curve (AUC) and the concordance index (ci), respectively. The primary endpoint of this study was loco-regional tumour control (LRC) after primary RCTx. RESULTS: Although both cohorts significantly differed in the proportion of the tumour subsites, the parameters tumour volume, p16 status and N stage could be validated regarding LRC and overall survival (OS) using multivariable Cox regression (LRC ci: 0.59, OS ci: 0.63). These models were slightly improved by combination with the putative CSC marker CD44 (LRC ci: 0.61, OS ci: 0.69). The logistic regression model for 2-year LRC based on tumour volume, p16 status and CD44 protein was validated with an AUC of 0.64. The patient stratification based on hypoxia-associated gene signatures status was similar to the original study but without significant differences in LRC and OS. CONCLUSIONS: In this validation study, the inclusion of the putative CSC marker CD44 slightly improved the prognostic performance of the baseline parameters tumour volume, p16 status and N stage. No improvement was observed when including expressions of the hypoxia-associated gene signatures. Prospective validation on a larger cohort is warranted to assess the clinical relevance of these markers.

Development and Validation of a Gene Signature for Patients with Head and Neck Carcinomas Treated by Postoperative Radio(chemo)therapy.

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

Independent validation of the prognostic value of cancer stem cell marker expression and hypoxia-induced gene expression for patients with locally advanced HNSCC after postoperative radiotherapy.

OBJECTIVE: To validate the impact of HPV status, cancer stem cell (CSC) marker expression and tumour hypoxia status in patients with locally advanced head and neck squamous cell carcinoma (HNSCC), who received postoperative radiotherapy. The results of the exploration cohort have previously been reported by the German Cancer Consortium Radiation Oncology Group (DKTK-ROG; Lohaus et al., 2014; Linge et al., 2016). MATERIALS AND METHODS: For 152 patients with locally advanced HNSCC the impact of HPV16 DNA status, CSC marker expression and hypoxia-associated gene signatures on outcome of postoperative radiotherapy were retrospectively analysed. Out of them, 40 patients received postoperative radiochemotherapy. Cox models presented in a previous study were validated using the concordance index as a performance measure. The primary endpoint of this study was loco-regional control. Results were compared to those previously reported by DKTK-ROG. RESULTS: Loco-regional control, freedom from distant metastases and overall survival were inferior to the previously reported cohort. Despite of this, the prognostic value of the combination of HPV infection status, CSC marker expression (SLC3A2) and tumour hypoxia status could be validated in univariate analyses using an independent validation cohort. For multivariate models, the concordance index was between 0.58 and 0.69 in validation, indicating a good prognostic performance of the models. The inclusion of CD44 and the 15-gene hypoxia signature moderately improved the performance compared to a baseline model without CSC markers or hypoxia classifiers. CONCLUSIONS: The HPV status, CSC marker expression of CD44 and SLC3A2 as well as hypoxia status are potential prognostic biomarkers for patients with locally advanced HNSCC treated by postoperative radiotherapy.