Metabolic pathway-based subtypes associate glycan biosynthesis and treatment response in head and neck cancer.

Head and Neck Squamous Cell Carcinoma (HNSCC) is a heterogeneous malignancy that remains a significant challenge in clinical management due to frequent treatment failures and pronounced therapy resistance. While metabolic dysregulation appears to be a critical factor in this scenario, comprehensive analyses of the metabolic HNSCC landscape and its impact on clinical outcomes are lacking. This study utilized transcriptomic data from four independent clinical cohorts to investigate metabolic heterogeneity in HNSCC and define metabolic pathway-based subtypes (MPS). In HPV-negative HNSCCs, MPS1 and MPS2 were identified, while MPS3 was enriched in HPV-positive cases. MPS classification was associated with clinical outcome post adjuvant radio(chemo)therapy, with MPS1 consistently exhibiting the highest risk of therapeutic failure. MPS1 was uniquely characterized by upregulation of glycan (particularly chondroitin/dermatan sulfate) metabolism genes. Immunohistochemistry and pilot mass spectrometry imaging analyses confirmed this at metabolite level. The histological context and single-cell RNA sequencing data identified the malignant cells as key contributors. Globally, MPS1 was distinguished by a unique transcriptomic landscape associated with increased disease aggressiveness, featuring motifs related to epithelial-mesenchymal transition, immune signaling, cancer stemness, tumor microenvironment assembly, and oncogenic signaling. This translated into a distinct histological appearance marked by extensive extracellular matrix remodeling, abundant spindle-shaped cancer-associated fibroblasts, and intimately intertwined populations of malignant and stromal cells. Proof-of-concept data from orthotopic xenotransplants replicated the MPS phenotypes on the histological and transcriptome levels. In summary, this study introduces a metabolic pathway-based classification of HNSCC, pinpointing glycan metabolism-enriched MPS1 as the most challenging subgroup that necessitates alternative therapeutic strategies.

DeepClassPathway: Molecular pathway aware classification using explainable deep learning.

OBJECTIVE: HPV-associated head and neck cancer is correlated with favorable prognosis; however, its underlying biology is not fully understood. We propose an explainable convolutional neural network (CNN) classifier, DeepClassPathway, that predicts HPV-status and allows patient-specific identification of molecular pathways driving classifier decisions. METHODS: The CNN was trained to classify HPV-status on transcriptome data from 264 (13% HPV-positive) and tested on 85 (25% HPV-positive) head and neck squamous carcinoma patients after transformation into 2D-treemaps representing molecular pathways. Grad-CAM saliency was used to quantify pathways contribution to individual CNN decisions. Model stability was assessed by shuffling pathways within 2D-images. RESULTS: The classification performance of the CNN-ensembles achieved ROC-AUC/PR-AUC of 0.96/0.90 for all treemap variants. Quantification of the averaged pathway saliency heatmaps consistently identified KRAS, spermatogenesis, bile acid metabolism, and inflammation signaling pathways as the four most informative for classifying HPV-positive patients and MYC targets, epithelial-mesenchymal transition, and protein secretion pathways for HPV-negative patients. CONCLUSION: We have developed and applied an explainable CNN classification approach to transcriptome data from an oncology cohort with typical sample size that allows classification while accounting for the importance of molecular pathways in individual-level decisions.

Integration of p16/HPV DNA Status with a 24-miRNA-Defined Molecular Phenotype Improves Clinically Relevant Stratification of Head and Neck Cancer Patients.

Human papillomavirus (HPV)-driven head and neck squamous cell carcinomas (HNSCC) generally have a more favourable prognosis. We hypothesized that HPV-associated HNSCC may be identified by an miRNA-signature according to their specific molecular pathogenesis, and be characterized by a unique transcriptome compared to HPV-negative HNSCC. We performed miRNA expression profiling of two p16/HPV DNA characterized HNSCC cohorts of patients treated by adjuvant radio(chemo)therapy (multicentre DKTK-ROG n = 128, single-centre LMU-KKG n = 101). A linear model predicting HPV status built in DKTK-ROG using lasso-regression was tested in LMU-KKG. LMU-KKG tumours (n = 30) were transcriptome profiled for differential gene expression and miRNA-integration. A 24-miRNA signature predicted HPV-status with 94.53% accuracy (AUC: 0.99) in DKTK-ROG, and 86.14% (AUC: 0.86) in LMU-KKG. The prognostic values of 24-miRNA- and p16/HPV DNA status were comparable. Combining p16/HPV DNA and 24-miRNA status allowed patient sub-stratification and identification of an HPV-associated patient subgroup with impaired overall survival. HPV-positive tumours showed downregulated MAPK, Estrogen, EGFR, TGFbeta, WNT signaling activity. miRNA-mRNA integration revealed HPV-specific signaling pathway regulation, including PD-L1 expression/PD-1 checkpoint pathway in cancer in HPV-associated HNSCC. Integration of clinically established p16/HPV DNA with 24-miRNA signature status improved clinically relevant risk stratification, which might be considered for future clinical decision-making with respect to treatment de-escalation in HPV-associated HNSCC.

Digital scoring of EpCAM and slug expression as prognostic markers in head and neck squamous cell carcinomas.

Head and neck squamous cell carcinomas (HNSCCs) have poor clinical outcome owing to therapy resistance and frequent recurrences that are among others attributable to tumor cells in partial epithelial-to-mesenchymal transition (pEMT). We compared side-by-side software-based and visual quantification of immunohistochemistry (IHC) staining of epithelial marker EpCAM and EMT regulator Slug in n = 102 primary HNSCC to assess optimal analysis protocols. IHC scores incorporated expression levels and percentages of positive cells. Digital and visual evaluation of membrane-associated EpCAM yielded correlating scorings, whereas visual evaluation of nuclear Slug resulted in significantly higher overall scores. Multivariable Cox proportional hazard analysis defined the median EpCAM expression levels resulting from visual quantification as an independent prognostic factor of overall survival. Slug expression levels resulting from digital quantification were an independent prognostic factor of recurrence-free survival, locoregional recurrence-free survival, and disease-specific survival. Hence, we propose to use visual assessment for the membrane-associated EpCAM protein, whereas nuclear protein Slug assessment was more accurate following digital measurement.

A Five-MicroRNA Signature Predicts Survival and Disease Control of Patients with Head and Neck Cancer Negative for HPV Infection.

PURPOSE: Human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) is associated with unfavorable prognosis, while independent prognostic markers remain to be defined. EXPERIMENTAL DESIGN: We retrospectively performed miRNA expression profiling. Patients were operated for locally advanced HPV-negative HNSCC and had received radiochemotherapy in eight different hospitals (DKTK-ROG; n = 85). Selection fulfilled comparable demographic, treatment, and follow-up characteristics. Findings were validated in an independent single-center patient sample (LMU-KKG; n = 77). A prognostic miRNA signature was developed for freedom from recurrence and tested for other endpoints. Recursive-partitioning analysis was performed on the miRNA signature, tumor and nodal stage, and extracapsular nodal spread. Technical validation used qRT-PCR. An miRNA-mRNA target network was generated and analyzed. RESULTS: For DKTK-ROG and LMU-KKG patients, the median follow-up was 5.1 and 5.3 years, and the 5-year freedom from recurrence rate was 63.5% and 75.3%, respectively. A five-miRNA signature (hsa-let-7g-3p, hsa-miR-6508-5p, hsa-miR-210-5p, hsa-miR-4306, and hsa-miR-7161-3p) predicted freedom from recurrence in DKTK-ROG [hazard ratio (HR) 4.42; 95% confidence interval (CI), 1.98-9.88, P < 0.001], which was confirmed in LMU-KKG (HR 4.24; 95% CI, 1.40-12.81, P = 0.005). The signature also predicted overall survival (HR 3.03; 95% CI, 1.50-6.12, P = 0.001), recurrence-free survival (HR 3.16; 95% CI, 1.65-6.04, P < 0.001), and disease-specific survival (HR 5.12; 95% CI, 1.88-13.92, P < 0.001), all confirmed in LMU-KKG data. Adjustment for relevant covariates maintained the miRNA signature predicting all endpoints. Recursive-partitioning analysis of both samples combined classified patients into low (n = 17), low-intermediate (n = 80), high-intermediate (n = 48), or high risk (n = 17) for recurrence (P < 0.001). CONCLUSIONS: The five-miRNA signature is a strong and independent prognostic factor for disease recurrence and survival of patients with HPV-negative HNSCC.See related commentary by Clump et al., p. 1441.

Postoperative (chemo) radiation in patients with squamous cell cancers of the head and neck - clinical results from the cohort of the clinical cooperation group "Personalized Radiotherapy in Head and Neck Cancer".

BACKGROUND: Postoperative (chemo) radiation improves tumor control and survival in high-risk patients with head and neck squamous cell carcinoma based on established risk factors. The clinical cooperation group "Personalized Radiotherapy in Head and Neck Cancer" focuses on the identification and validation of new biomarkers, which are aimed at eventually stratifying and personalizing the therapy concept. Hence, we reviewed all patients with head and neck squamous cell carcinoma of the oral cavity, oropharynx, hypopharynx, or larynx, treated with postoperative (chemo) radiation from 06/2008 until 06/2015 at the Department of Radiation Oncology in the University Hospital, LMU Munich. Here we report the clinical results of the cohort, laying the foundation for further research within the framework of a clinical cooperation group. METHODS: Patient data were retrospectively (until 2013) and prospectively (from 2013) collected and analyzed for outcome and treatment failures with regard to previously described and established risk factors. RESULTS: We identified 302 patients (median follow-up 45 months, average age 60.7 years), having received postoperative (chemo)radiation (median 64 Gy). Chemotherapy was added in 58% of cases, mostly Cisplatin/5- Fluorouracil in concordance with the ARO 96-3 study. The 3-year overall survival, local, locoregional and distant failure estimates were 70.5, 9.7, 12.2 and 13.5%, respectively. Human papillomavirus-associated oropharyngeal cancer was associated with a significant improved overall survival, locoregional, distant and overall tumor control rates in multivariate analysis. Additionally, in multivariate analysis, for local failure, resection status and perineural invasion, for locoregional and distant failure extracapsular extension and for overall survival the presence of nodal disease were significant adverse factors. Moreover, 138 patients have been treated in concordance with the ARO 96-3 protocol, corroborating the results of this study. CONCLUSIONS: Our cohort represents a large unselected cohort of patients with head and neck squamous cell carcinoma treated with postoperative (chemo)radiation. Tumor control rates and survival rates are consistent with the results of previously reported data.