Development of a Prognostic Pyroptosis-Related Gene Signature for Head and Neck Squamous Cell Carcinoma Patient

Objective: Head and neck squamous cell carcinoma (HNSCC) is a major threat to public health. Pyroptosis is a form of inflammatory programmed cell death that is still incompletely understood. The role of pyroptotic cell death in HNSCC remains to be fully defined. As such, the present study was developed to explore the potential prognostic utility of a pyroptosis-related gene (PRG) signature in HNSCC.Methods: PRG expression patterns and the associated mutational landscape in HNSCC were analyzed, after which a 6-gene prognostic model was constructed through least absolute shrinkage and selection operator (LASSO) and Cox regression analyses using the TCGA dataset, followed by validation with two GEO datasets (GSE41643 and GSE65858). Potential predictors of patient outcomes associated with this 6-gene model were identified through topological degree analyses of a protein-protein interaction network. Lastly, the prognostic value of NLRP3 as a predictor of HNSCC patient prognosis was established through immunohistochemical (IHC) analyses of samples from 176 HNSCC patients.Results: Differentially expressed PRGs were able to readily differentiate between HNSCC tumors and normal tissues. Risk scores derived from the 6-gene PRG model were independent predictors of HNSCC patient prognosis, and genes that were differentially expressed between low- and high-risk groups were associated with tumor immunity. IHC analyses further supported the value of NLRP3 as a predictor of HNSCC patient outcomes. Conclusions: Overall, these results highlight a novel prognostic gene signature that offers value in the context of HNSCC patient evaluation, although additional research will be essential to elucidate the mechanisms linking these PRGs to HNSCC outcomes.

Dose predictions for [177Lu]Lu-DOTA-panitumumab F(ab′)2 in NRG mice with HNSCC patient-derived tumour xenografts based on [64Cu]Cu-DOTA-panitumumab F(ab′)2 – implications for a PET theranostic strategy

Background Epidermal growth factor receptors (EGFR) are overexpressed on many head and neck squamous cell carcinoma (HNSCC). Radioimmunotherapy (RIT) with F(ab')2 of the anti-EGFR monoclonal antibody panitumumab labeled with the β-particle emitter, 177Lu may be a promising treatment for HNSCC. Our aim was to assess the feasibility of a theranostic strategy that combines positron emission tomography (PET) with [64Cu]Cu-DOTA-panitumumab F(ab')2 to image HNSCC and predict the radiation equivalent doses to the tumour and normal organs from RIT with [177Lu]Lu-DOTA-panitumumab F(ab')2. Results Panitumumab F(ab')2 were conjugated to DOTA and complexed to 64Cu or 177Lu in high radiochemical purity (95.6 ± 2.1% and 96.7 ± 3.5%, respectively) and exhibited high affinity EGFR binding (Kd = 2.9 ± 0.7 × 10− 9 mol/L). Biodistribution (BOD) studies at 6, 24 or 48 h post-injection (p.i.) of [64Cu]Cu-DOTA-panitumumab F(ab')2 (5.5–14.0 MBq; 50 μg) or [177Lu]Lu-DOTA-panitumumab F(ab')2 (6.5 MBq; 50 μg) in NRG mice with s.c. HNSCC patient-derived xenografts (PDX) overall showed no significant differences in tumour uptake but modest differences in normal organ uptake were noted at certain time points. Tumours were imaged by microPET/CT with [64Cu]Cu-DOTA-panitumumab F(ab')2 or microSPECT/CT with [177Lu]Lu-DOTA-panitumumab F(ab')2 but not with irrelevant [177Lu]Lu-DOTA-trastuzumab F(ab')2. Tumour uptake at 24 h p.i. of [64Cu]Cu-DOTA-panitumumab F(ab')2 [14.9 ± 1.1% injected dose/gram (%ID/g) and [177Lu]Lu-DOTA-panitumumab F(ab')2 (18.0 ± 0.4%ID/g) were significantly higher (P < 0.05) than [177Lu]Lu-DOTA-trastuzumab F(ab')2 (2.6 ± 0.5%ID/g), demonstrating EGFR-mediated tumour uptake. There were no significant differences in the radiation equivalent doses in the tumour and most normal organs estimated for [177Lu]Lu-DOTA-panitumumab F(ab')2 based on the BOD of [64Cu]Cu-DOTA-panitumumab F(ab')2 compared to those estimated directly from the BOD of [177Lu]Lu-DOTA-panitumumab F(ab')2 except for the liver and whole body which were modestly underestimated by [64Cu]Cu-DOTA-panitumumab F(ab')2. Region-of-interest (ROI) analysis of microPET/CT images provided dose estimates for the tumour and liver that were not significantly different for the two radioimmunoconjugates. Human doses from administration of [177Lu]Lu-DOTA-panitumumab F(ab')2 predicted that a 2 cm diameter HNSCC tumour in a patient would receive 1.1–1.5 mSv/MBq and the whole body dose would be 0.15–0.22 mSv/MBq. Conclusion A PET theranostic strategy combining [64Cu]Cu-DOTA-panitumumab F(ab')2 to image HNSCC tumours and predict the equivalent radiation doses in the tumour and normal organs from RIT with [177Lu]Lu-DOTA-panitumumab F(ab')2 is feasible. RIT with [177Lu]Lu-DOTA-panitumumab F(ab')2 may be a promising approach to treatment of HNSCC due to frequent overexpression of EGFR.

Evaluation of prognostic biomarkers in a population-validated Finnish HNSCC patient cohort

Introduction Prognostic biomarkers and novel therapeutic approaches have been slow to emerge in the treatment of head and neck squamous cell carcinoma (HNSCC). In this study, an HNSCC patient cohort is created and performance of putative prognostic biomarkers investigated in a population-validated setting. The overall goal is to develop a novel way to combine biomarker analyses with population-level clinical data on HNSCC patients and thus to improve the carryover of biomarkers into clinical practice. Materials and methods To avoid selection biases in retrospective study design, all HNSCC patients were identified and corresponding clinical data were collected from the Southwest Finland geographical area. A particular emphasis was laid on avoiding potential biases in sample selection for immunohistochemical staining analyses. Staining results were evaluated for potential prognostic resolution. Results After comprehensive evaluation, the patient cohort was found to be representative of the background population in terms of clinical characteristics such as patient age and TNM stage distribution. A negligible drop-out of 1.3% (6/476) was observed during the first follow-up year. By immunohistochemical analysis, the role of previously implicated HNSCC biomarkers (p53, EGFR, p16, CIP2A, Oct4, MET, and NDFIP1) was investigated. Discussion Our exceptionally representative patient material supports the use of population validation to improve the applicability of results to real-life situations. The failure of the putative prognostic biomarkers emphasizes the need for controlling bias in retrospective studies, especially in the heterogenous tumor environment of HNSCC. The resolution of simple prognostic examination is unlikely to be sufficient to identify biomarkers for clinical practice of HNSCC.

Identification of Potential Genes both Differentially Expressed and Regulated by Differential Methylation Regions in Oral Squamous Cell Carcinoma

Background: The aberrant methylation included hypermethylation and hypomethylation plays significant role in the progression of many kind of cancers but poorly investigated in oral squamous cell carcinoma (OSCC). Methods: GSE123781 and GSE87053 were used to identify the differential methylation regions (DMRs) by R software. the targeted genes regulated by DMRs were predicted by wANNOVAR. The biological process, cellular component, molecular function and cell pathways of common targeted genes between GSE123781 and GSE87053 were analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) enrichment analysis. The hub genes in common targeted genes both associated with cancer biological process and cancer cell pathways were identified by protein to protein interaction network (PPI). Finally, the expression level of hub genes and correspondence relationship with head and neck squamous cell carcinoma (HNSCC) patient survival were confirmed by The Cancer Genome Atlas (TCGA) dataset. Results: There are 372 common targeted genes regulated by DMRs between GSE123781 and GSE87053 and RUNX family transcript factor 1 (RUNX1), phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit gamma (PIK3CG), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit delta (PIK3CD), laminin subunit beta 1 (LAMB1), integrin subunit alpha M (ITGAM), interleukin 2 receptor subunit alpha (IL2RA), interleukin 10 (IL10), FYN proto-oncogene, Src family tyrosine kinase (FYN), cholinergic receptor muscarinic 1 (CHRM1) and C-C motif chemokine receptor 1 (CCR1) are hub genes in common targeted genes. Furthermore, RUNX1, PIK3R1, PIK3CD, LAMB1, ITGAM, IL2RA, IL10, FYN, CHRM1 and CCR1 differentially expressed in HNSCC tissues and the overexpressed genes PIK3CD, LAMB1 and IL10 could lead to the decrease of HNSCC patient survival. Conclusions: PIK3CD, LAMB1 and IL10 both differentially expressed and regulated by DMRs are quite associated with the progression of OSCC and the survival of HNSCC patients, which has much potential to be new biomarkers in targeted therapy of OSCC.

TMPRSS2, a SARS-CoV-2 internalization protease is downregulated in head and neck cancer patients

Background SARS-coronavirus-2 enters host cells through binding of the Spike protein to ACE2 receptor and subsequent S priming by the TMPRSS2 protease. We aim to assess differences in both ACE2 and TMPRSS2 expression in normal tissues from oral cavity, pharynx, larynx and lung tissues as well as neoplastic tissues from the same areas. Methods The study has been conducted using the TCGA and the Regina Elena Institute databases and validated by experimental model in HNSCC cells. We also included data from one COVID19 patient who went under surgery for HNSCC. Results TMPRSS2 expression in HNSCC was significantly reduced compared to the normal tissues. It was more evident in women than in men, in TP53 mutated versus wild TP53 tumors, in HPV negative patients compared to HPV positive counterparts. Functionally, we modeled the multivariate effect of TP53, HPV, and other inherent variables on TMPRSS2. All variables had a statistically significant independent effect on TMPRSS2. In particular, in tumor tissues, HPV negative, TP53 mutated status and elevated TP53-dependent Myc-target genes were associated with low TMPRSS2 expression. The further analysis of both TCGA and our institutional HNSCC datasets identified a signature anti-correlated to TMPRSS2. As proof-of-principle we also validated the anti-correlation between microRNAs and TMPRSS2 expression in a SARS-CoV-2 positive HNSCC patient tissues Finally, we did not find TMPRSS2 promoter methylation. Conclusions Collectively, these findings suggest that tumoral tissues, herein exemplified by HNSCC and lung cancers might be more resistant to SARS-CoV-2 infection due to reduced expression of TMPRSS2. These observations may help to better assess the frailty of SARS-CoV-2 positive cancer patients.

VAV2 signaling promotes regenerative proliferation in both cutaneous and head and neck squamous cell carcinoma

Regenerative proliferation capacity and poor differentiation are histological features usually linked to poor prognosis in head and neck squamous cell carcinoma (hnSCC). However, the pathways that regulate them remain ill-characterized. Here, we show that those traits can be triggered by the RHO GTPase activator VAV2 in keratinocytes present in the skin and oral mucosa. VAV2 is also required to maintain those traits in hnSCC patient-derived cells. This function, which is both catalysis- and RHO GTPase-dependent, is mediated by c-Myc- and YAP/TAZ-dependent transcriptomal programs associated with regenerative proliferation and cell undifferentiation, respectively. High levels of VAV2 transcripts and VAV2-regulated gene signatures are both associated with poor hnSCC patient prognosis. These results unveil a druggable pathway linked to the malignancy of specific SCC subtypes.

TMPRSS2, a SARS-CoV-2 internalization protease is downregulated in head and neck cancer patients.

Background: SARS-coronavirus-2 enters host cells through binding of the Spike protein to ACE2 receptor and subsequent S priming by the TMPRSS2 protease. We aim to assess differences in both ACE2 and TMPRSS2 expression in normal tissues from oral cavity, pharynx, larynx and lung tissues as well as neoplastic tissues from the same areas.Methods: The study has been conducted using the TCGA and the Regina Elena Institute databases and validated by experimental model in HNSCC cells. We also included data from one COVID19 patient who went under surgery for HNSCC.Results: TMPRSS2 expression in HNSCC was significantly reduced compared to the normal tissues. It was more evident in women than in men, in TP53 mutated versus wild TP53 tumors, in HPV negative patients compared to HPV positive counterparts. Functionally, we modeled the multivariate effect of TP53, HPV, and other inherent variables on TMPRSS2. All variables had a statistically significant independent effect on TMPRSS2. In particular, in tumor tissues, HPV negative, TP53 mutated status and elevated TP53-dependent Myc-target genes were associated with low TMPRSS2 expression. The further analysis of both TCGA and our institutional HNSCC datasets identified a signature anti-correlated to TMPRSS2. As proof-of-principle we also validated the anti-correlation between microRNAs and TMPRSS2 expression in a SARS-CoV-2 positive HNSCC patient tissues Finally, we did not find TMPRSS2 promoter methylation.Conclusions: Collectively, these findings suggest that tumoral tissues, herein exemplified by HNSCC and lung cancers might be more resistant to SARS-CoV-2 infection due to reduced expression of TMPRSS2. These observations may help to better assess the frailty of SARS-CoV-2 positive cancer patients.

Drug Vulnerabilities and Disease Prognosis Linked to the Stem Cell-Like Gene Expression Program Triggered by the RHO GTPase Activator VAV2 in Hyperplastic Keratinocytes and Head and Neck Cancer

We have recently shown that VAV2, a guanosine nucleotide exchange factor that catalyzes the stimulation step of RHO GTPases, is involved in a stem cell-like (SCL) regenerative proliferation program that is important for the development and subsequent maintenance of the tumorigenesis of both cutaneous (cSCC) and head and neck squamous cell carcinomas (hnSCC). In line with this, we have observed that the levels of the VAV2 mRNA and VAV2-regulated gene signatures are associated with poor prognosis in the case of human papillomavirus-negative hnSCC patients. These results suggest that the SCL program elicited by VAV2 in those cells can harbor therapeutically actionable downstream targets. We have addressed this issue using a combination of both in silico and wet-lab approaches. Here, we show that the VAV2-regulated SCL program does harbor a number of cell cycle- and signaling-related kinases that are essential for the viability of undifferentiated keratinocytes and hnSCC patient-derived cells endowed with high levels of VAV2 activity. Our results also show that the VAV2-regulated SCL gene signature is associated with poor hnSCC patient prognosis. Collectively, these data underscore the critical role of this VAV2-regulated SCL program for the viability of both preneoplastic and fully transformed keratinocytes.

TMPRSS2, a SARS-CoV-2 Internalization Protease is Downregulated in Head and Neck Cancer Patients.

Background: SARS-coronavirus-2 enters host cells through binding of the Spike protein to ACE2 receptor and subsequent S priming by the TMPRSS2 protease. We aim to assess differences in both ACE2 and TMPRSS2 expression in normal tissues from oral cavity, pharynx, larynx and lung tissues as well as neoplastic tissues from the same areas.Methods: The study has been conducted using the TCGA and the Regina Elena Institute databases and validated by experimental model in HNSCC cells. We also included data from one COVID19 patient who went under surgery for HNSCC.Results: TMPRSS2 expression in HNSCC was significantly reduced compared to the normal tissues. It was more evident in women than in men, in TP53 mutated versus wild TP53 tumors, in HPV negative patients compared to HPV positive counterparts. Functionally, we modeled the multivariate effect of TP53, HPV, and other inherent variables on TMPRSS2. All variables had a statistically significant independent effect on TMPRSS2. In particular, in tumor tissues, HPV negative, TP53 mutated status and elevated TP53-dependent Myc-target genes were associated with low TMPRSS2 expression. The further analysis of both TCGA and our institutional HNSCC datasets identified a signature anti-correlated to TMPRSS2. As proof-of-principle we also validated the anti-correlation between microRNAs and TMPRSS2 expression in a SARS-CoV-2 positive HNSCC patient tissues Finally, we did not find TMPRSS2 promoter methylation.Conclusions: Collectively, these findings suggest that tumoral tissues, herein exemplified by HNSCC and lung cancers might be more resistant to SARS-CoV-2 infection due to reduced expression of TMPRSS2. These observations may help to better assess the frailty of SARS-CoV-2 positive cancer patients.

NAB 2-Expressing Cancer-Associated Fibroblast Promotes HNSCC Progression

Cancer-associated fibroblast (CAF)-specific proteins serve as both prognostic biomarkers and targets for anticancer drugs. In this study, we investigated the role of NGFI-A-binding protein (NAB)2 derived from CAFs in the progression of head and neck squamous cell carcinoma (HNSCC). Patient-derived HNSCC and paired metastatic lymph node tissues were examined for NAB2 expression by immunohistochemistry. Primary CAF cultures were established from HNSCC patient tissue, with paired non-tumor fibroblasts (NTFs) serving as a control. CAF or NTF was used to evaluate the effect of NAB2 on HNSCC progression using FaDu cell spheroids and an in vivo mouse xenograft model. NAB2 was detected in interstitial CAFs in primary and metastatic lymph node tissues of HNSCC patients. NAB2 mRNA and protein levels were higher in CAFs as compared to paired NTFs. Conditioned medium (CM) of NAB2-overexpressing CAFs increased the invasion of FaDu spheroids in the Matrigel invasion assay as compared to CM of NTF. Co-injection of NAB2-overexpressing CAFs with FaDu spheroids into mice enhanced the growth of tumors. These data suggest that NAB2-overexpressing CAFs promotes HNSCC progression and is a potential therapeutic target for preventing HNSCC metastasis.