Head And Neck Squamous Cell Carcinoma Subtypes Research Articles

Single-Cell Transcriptome Analysis Reveals Different Immune Signatures in HPV- and HPV + Driven Human Head and Neck Squamous Cell Carcinoma.

Background Head and neck squamous cell carcinoma (HNSCC) is a significant health problem and related to poor long-term outcomes, indicating more research to be done to deeply understand the underlying pathways. Objective This current study aimed in the assessment of the viral- (especially human papilloma virus [HPV]) and carcinogen-driven head and neck squamous cell carcinoma (HNSCC) microenvironment based on single-cell sequencing analysis. Methods Data were downloaded from GEO database (GSE139324), including 131224 cells from 18 HP- HNSCC patients and 8 HPV+ HNSCC patients. Following data normalization, all highly variable genes in single cells were identified, and batch correction was applied. Differentially expressed genes were identified using Wilcoxon rank sum test. A gene enrichment analysis was performed in each cell cluster using KEGG analysis. Single-cell pseudotime trajectories were constructed with MONOCLE (version 2.6.4). Cell-cell interactions were analyzed with CellChat R package. Additionally, cell-cell communication patterns in key signal pathways were compared in different tissue groups. A hidden Markov model (HMM) was used to predict gene expression states (on or off) throughout pseudotime. Five-year overall survival outcomes were compared in both HPV+ and HPV- subsets. Results 20,978 high-quality individual cells passed quality control. RNA-seq data were used from 522 HNSCC primary tumor samples. 1,137 differentially expressed genes between HPV+ and HPV- HNSCC patients were investigated. 96 differentially expressed genes were associated with overall survival and highly enriched in B cell associated biological process. Cell composition differed between types of samples. MHC-I, MHC-II, and MIF signaling pathways were found to be most relevant. Within these pathways, some cells were either signal receiver or signal sender, depending on sample type, respectively. Six genes were obtained, AREG and TGFBI (upregulation), CD27, CXCR3, MS4A1, and CD19 (downregulation), whose expression and HPV types were highly associated with worse overall survival. AREG and TGFBI were pDC marker genes, CXCR3 and CD27 were significantly expressed in T cell-related cells, while MS4A1 and CD19 were mainly expressed in B naïve cells. Conclusions This study revealed dynamic changes in cell percentage and heterogeneity of cell subtypes of HNSCC. AREG, TGFBI, CD27, CXCR3, MS4A1, and CD19 were associated with worse overall survival in HPV-related HNSCC. Especially B-cell related pathways were revealed as particularly relevant in HPV-related HNSCC. These findings are a basis for the development of biomarkers and therapeutic targets in respective patients.

Pharmacogenomic landscape of head and neck squamous cell carcinoma informs precision oncology therapy.

Head and neck squamous cell carcinoma (HNSCC) is a common and frequently lethal cancer with few therapeutic options. In particular, there are few effective targeted therapies. Development of highly effective therapeutic strategies tailored to patients with HNSCC remains a pressing challenge. To address this, we present a pharmacogenomic study to facilitate precision treatments for patients with HNSCC. We established a large collection of 56 HNSCC patient-derived cells (PDCs), which recapitulated the molecular features of the original tumors. Pharmacological assessment of HNSCCs was conducted using a three-tiered high-throughput drug screening using 2248 compounds across these PDC models and an additional 18 immortalized cell lines. We integrated genomic, transcriptomic, and pharmacological analysis to predict biomarkers, gene-drug associations, and validated biomarkers. These results supported drug repurposing for multiple HNSCC subtypes, including the JAK2 inhibitor fedratinib, for low KRT18-expressing HNSCC cases, and the topoisomerase inhibitor mitoxantrone, for IL6R-activated HNSCC cases. Our results demonstrated concordance between susceptibility predictions from the PDCs and the matched patients' responses to standard clinical medication. Moreover, we identified and experimentally confirmed that high expression of ITGB1 elicited therapeutic resistance to docetaxel and high SOD1 expression conferred resistance to afatinib. We further validated ITGB1 as a predictive biomarker for the efficacy of docetaxel therapy in a phase 2 clinical trial. In summary, our study shows that this HNSCC cell resource, as well as the resulting pharmacogenomic profiles, is effective for biomarker discovery and for guiding precision oncology therapies in HNSCCs.

A novel anoikis-related gene signature predicts prognosis in patients with head and neck squamous cell carcinoma and reveals immune infiltration

Background: Head and neck squamous cell carcinoma (HNSCC) is a highly aggressive disease with a poor prognosis for advanced tumors. Anoikis play a key role in cancer metastasis, facilitating the detachment and survival of cancer cells from the primary tumor site. However, few studies have focused on the role of anoikis in HNSC, especially on the prognosis. Methods: Anoikis-related genes (ANRGs) integrated from Genecards and Harmonizome portals were used to identify HNSCC subtypes and to construct a prognostic model for HNSCC patients. Also, we explored the immune microenvironment and enrichment pathways between different subtypes. Finally, we provide clinical experts with a novel nomogram based on ANRGs, with DCA curves indicating the potential clinical benefit of the model for clinical strategies. Results: We identified 69 survival-related HNSCC anoikis-related DEGs, from which 7 genes were selected to construct prognostic models. The prognostic risk score was identified as an independent prognostic factor. Functional analysis showed that these high and low risk groups had different immune status and drug sensitivity. Next risk scores were combined with HNSCC clinicopathological features together to construct a nomogram, and DCA analysis showed that the model could benefit patients from clinical treatment strategies. Conclusion: The predictive seven-gene signature and nomogram established in this study can assist clinicians in selecting personalized treatment for patients with HNSCC.

Systematic Analysis of Molecular Subtypes and Immune Prediction Based on CD8 T Cell Pattern Genes Based on Head and Neck Cancer.

CD8+ T lymphocytes, also known as cytotoxic T lymphocytes, are the most powerful antitumour cells in the human body. Patients with head and neck squamous cell carcinoma (HNSCC) in whom CD8+ T lymphocyte infiltration is high have a better prognosis. However, the clinical significance and prognostic significance of CD8+ T cell-related regulatory genes in HNSCC remain unclear, and further research is required. In total, 446 CD8+ T cell-related genes were obtained using WGCNA. It was discovered that 111 genes included within the TCGA and GSE65858 datasets were intimately linked to the patient's prognosis. These genes were included in the subsequent analysis. According to consensus clustering analysis, HNSCC samples were classified into 3 subtypes (IC1, IC2, and IC3). There were substantial differences between the three subtypes in terms of immunological molecules, immune function, and the response to drug treatment. In addition, the 8-gene signature, which was generated premised on CD8+ T cell-related genes, exhibited stable prognostic prediction in the TCGA and GEO datasets and different HNSCC patient subgroups and independently served as a prognostic indicator for HNSCC. More importantly, the 8-gene signature effectively predicted immunotherapy response. We first constructed a molecular subtype of HNSCC based on CD8+ T cell-related genes. Between the three subtypes, there were significant differences in the prognosis, clinical features, immunological molecules, and drug treatment response. The 8-gene signature that was further constructed effectively predicted prognosis and immunotherapy response.

Novel prognostic matrisome-related gene signature of head and neck squamous cell carcinoma.

Background: Head and neck squamous cell carcinoma (HNSCC) is a common malignancy of the mucosal epithelium of the oral cavity, pharynx, and larynx. Laryngeal squamous cell carcinoma (LSCC) and oral squamous cell carcinoma are common HNSCC subtypes. Patients with metastatic HNSCC have a poor prognosis. Therefore, identifying molecular markers for the development and progression of HNSCC is essential for improving early diagnosis and predicting patient outcomes. Methods: Gene expression RNA-Seq data and patient clinical traits were obtained from The Cancer Genome Atlas-Head and Neck Squamous Cell Carcinoma (TCGA-HNSC) and Gene Expression Omnibus databases. Differentially expressed gene (DEG) screening was performed using the TCGA-HNSC dataset. Intersection analysis between the DEGs and a list of core matrisome genes obtained from the Matrisome Project was used to identify differentially expressed matrisome genes. A prognostic model was established using univariate and multivariate Cox regression analyses, least absolute shrinkage, and selection operator (LASSO) regression analysis. Immune landscape analysis was performed based on the single-sample gene set enrichment analysis algorithm, Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, prognostic value, receiver operating characteristic curve analysis, and gene mutation analyses. Immunohistochemical results regarding prognostic protein levels were obtained from the Human Protein Atlas. Single-gene RNA-sequencing data were obtained from GSE150321 and GSE172577 datasets. CCK-8 and Transwell assays were used to confirm cell proliferation and migration. Results: A total of 1,779 DEGs, including 939 upregulated and 840 downregulated genes, between tumor and normal samples were identified using the TCGA-HNSC microarray data. Intersection analysis revealed 52 differentially expressed matrisome-related genes. After performing univariate and multivariate Cox regression and LASSO analyses, a novel prognostic model based on six matrisome genes (FN1, LAMB4, LAMB3, DMP1, CHAD, and MMRN1) for HNSCC was established. This risk model can successfully predict HNSCC survival. The high-risk group had worse prognoses and higher enrichment of pathways related to cancer development than the low-risk group. Silencing LAMB4 in HNSCC cell lines promoted cell proliferation and migration. Conclusion: This study provides a novel prognostic model for HNSCC. Thus, FN1, LAMB4, LAMB3, DMP1, CHAD, and MMRN1 may be the promising biomarkers for clinical practice.

High enhancer activity is an epigenetic feature of HPV negative atypical head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous disease with significant mortality and frequent recurrence. Prior efforts to transcriptionally classify HNSCC into groups of varying prognoses have identified four accepted molecular subtypes of the disease: Atypical (AT), Basal (BA), Classical (CL), and Mesenchymal (MS). Here, we investigate the active enhancer landscapes of these subtypes using representative HNSCC cell lines and identify samples belonging to the AT subtype as having increased enhancer activity compared to the other 3 HNSCC subtypes. Cell lines belonging to the AT subtype are more resistant to enhancer-blocking bromodomain inhibitors (BETi). Examination of nascent transcripts reveals that both AT TCGA tumors and cell lines express higher levels of enhancer RNA (eRNA) transcripts for enhancers controlling BETi resistance pathways, such as lipid metabolism and MAPK signaling. Additionally, investigation of higher-order chromatin structure suggests more enhancer-promoter (E-P) contacts in the AT subtype, including on genes identified in the eRNA analysis. Consistently, known BETi resistance pathways are upregulated upon exposure to these inhibitors. Together, our results identify that the AT subtype of HNSCC is associated with higher enhancer activity, resistance to enhancer blockade, and increased signaling through pathways that could serve as future targets for sensitizing HNSCC to BET inhibition.

Abstract 3315: CRISPR-Cas9 screening reveals novel targets that govern HNSCC response to radiation therapy

Abstract Purpose: Although Head and Neck Squamous Cell Carcinoma (HNSCC) is the sixth most common cancer worldwide, the high failure rates for non-HPV associated tumors and the absence of new effective therapies has caused the survival rate for HNSCC to remain stagnant for the past decade. As radiation is a frontline treatment for HNSCC, we sought to uncover novel actionable targets that affect cellular responses to radiation therapy. Experimental Procedures: We performed a kinome-wide CRISPR-Cas9 knockout screen in UM-SCC47, FaDu, Cal27, and Detroit562 HNSCC cell lines using a multi-fraction radiation regimen as a selective pressure. These four cell lines were chosen to enhance molecular diversity as they represent the classical, atypical, basal, and mesenchymal transcriptional subtypes of HNSCC. sgRNAs from irradiated and unirradiated controls underwent Next Generation Sequencing, and the results were analyzed via MAGeCK analysis. This approach provided a systematic method to identify sgRNAs that were enriched (loss of gene causes radioresistance) or depleted (loss of gene causes radiosensitivity) following HNSCC cell radiation treatment. Results: Our results indicate that Cal27 and Detroit562 cell lines share multiple targets that govern cell responses to radiation despite the different mutational backgrounds and transcriptional subtypes. In particular, sgRNAs targeting ATM and PRKDC were significantly depleted in Cal27 and Detroit562 cells (FDR<0.01, p<0.01). ATM and PRKDC are regulators of the DNA damage response, and loss of function of these genes is known to radiosensitize cells. Therefore, depletion of sgRNAs targeting these genes serves as internal validation of our screening methodology. sgRNAs targeting JAK1 were enriched in the Cal27 and Detroit562 cell lines (FDR<0.01, p<0.01), suggesting that loss of JAK1 caused decreased radiation-induced cell death. As a role for JAK1 in the cell response to DNA damage has not been described, we sought to validate and characterize the role of JAK1 in HNSCC cell radiation treatment using JAK1 knockout (KO) cell lines. Clonogenic assays revealed that JAK1 KO cells were radioresistant when treated with both single and multi-fraction radiation regimens, validating the results of the CRISPR screen. We observed cell signaling changes consistent with loss of JAK1 including decreased phospho-STAT3 and IRF9 signaling. To uncover pathways that may contribute to JAK1 KO cell radioresistance, we performed bulk RNA sequencing of JAK1 KO and control cell lines. Our analysis revealed that JAK1 KO cells may undergo an epithelial-mesenchymal transition among other signaling changes. Conclusion: The use of a CRISPR-Cas9 screen with radiation as a selective pressure revealed both known and novel targets that govern HNSCC cell line responses to radiation therapy. Loss of JAK1 causes radioresistance, and its role in HNSCC DNA damage response warrants further investigation. Citation Format: Vanessa M. Kelley, Marta Baro, Aanchal Katoch, Chatchai Phoomak, Hojin Lee, Joseph Contessa. CRISPR-Cas9 screening reveals novel targets that govern HNSCC response to radiation therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3315.

P63 Directs Subtype-Specific Gene Expression in HPV+ Head and Neck Squamous Cell Carcinoma.

The complex heterogeneity of head and neck squamous cell carcinoma (HNSCC) reflects a diverse underlying etiology. This heterogeneity is also apparent within Human Papillomavirus-positive (HPV+) HNSCC subtypes, which have distinct gene expression profiles and patient outcomes. One aggressive HPV+ HNSCC subtype is characterized by elevated expression of genes involved in keratinization, a process regulated by the oncogenic transcription factor ΔNp63. Furthermore, the human TP63 gene locus is a frequent HPV integration site and HPV oncoproteins drive ΔNp63 expression, suggesting an unexplored functional link between ΔNp63 and HPV+ HNSCC. Here we show that HPV+ HNSCCs can be molecularly stratified according to ΔNp63 expression levels and derive a ΔNp63-associated gene signature profile for such tumors. We leveraged RNA-seq data from p63 knockdown cells and ChIP-seq data for p63 and histone marks from two ΔNp63high HPV+ HNSCC cell lines to identify an epigenetically refined ΔNp63 cistrome. Our integrated analyses reveal crucial ΔNp63-bound super-enhancers likely to mediate HPV+ HNSCC subtype-specific gene expression that is anchored, in part, by the PI3K-mTOR pathway. These findings implicate ΔNp63 as a key regulator of essential oncogenic pathways in a subtype of HPV+ HNSCC that can be exploited as a biomarker for patient stratification and treatment choices.

Head and Neck Squamous Cell Carcinoma Subtypes Based on Immunologic and Hallmark Gene Sets in Tumor and Non-tumor Tissues.

BackgroundNon-tumor tissue has a significant impact on the prognosis of head and neck squamous cell carcinoma (HNSCC). Previous studies for HNSCC have mainly focused on tumor tissue, greatly neglecting the role of non-tumor tissue. This study aimed to identify HNSCC subtypes and prognostic gene sets based on activity changes of immunologic and hallmark gene sets in tumor and adjacent non-tumor tissues to improve patient prognosis.MethodsIn the study, we used gene set variation analysis (GSVA) to estimate the relative enrichment of gene sets over the sample population, and identified relevant subtypes of HNSCC by Cox regression analysis and the non-negative matrix factorization (NMF) method. The representative gene sets were identified by calculating the differential enrichment score of gene sets between each of the two subgroups, intersecting them, and screening them using univariate Cox regression analysis. The least absolute shrinkage and selection operator (LASSO) regression analysis was used to screen out potential prognostic gene sets and establish a risk model. Finally, genes encompassed in each prognostic gene set were obtained and subjected to enrichment analysis and protein–protein interaction (PPI) in tumor and non-tumor tissues.ResultsWe identified three subtypes of HNSCC based on gene sets in tumor and non-tumor tissues, and patients with subtype 1 had a higher survival rate than subtypes 2 and 3. The subtypes were related to the survival status, pathological stage, and T stage of HNSCC patients. In total 450 differentially gene sets and 39 representative gene sets were obtained by calculating the differential enrichment score of gene sets between each of the two subgroups, intersecting them, and screening them using univariate Cox regression analysis. The prognostic model was constructed by LASSO regression analysis, including five prognostic gene sets. Kaplan-Meier analysis indicated that different risk groups and the five prognostic gene sets were associated with survival status in the model. Finally, enrichment analysis and PPI indicated that non-tumor and tumor tissues affect the prognosis of HNSCC patients in different ways.ConclusionIn conclusion, we provide a novel insight for rational treatment strategies and precise prognostic assessments based on tumor and adjacent non-tumor tissues, suggesting that more emphasis should be placed on changes in adjacent non-tumor and tumor tissues, rather than just the tumor itself.

Identification of Molecular Subtypes in Head and Neck Squamous Cell Carcinoma Based on Dysregulated Immune LncRNAs.

Long noncoding RNAs (lncRNAs) perform indispensable functions in cancer pathologies and are involved in the onset and progression of multiple cancers. Multiple platforms were performed to comprehensively analyze the head and neck squamous cell carcinoma (HNSCC) for determining molecular subtypes. Molecular subtypes were clustered and analyzed by the “ConsensusClusterPlus” R package. The Limma software was utilized to screen for differentially expressed genes (DEGs). Functional enrichment analyses, including Gene Set Enrichment Analysis (GSEA), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Ontology (GO), were performed on the three database resources. Seventeen lncRNAs were determined as HNSCC-specific immune lncRNAs that were dysregulated. Our research identified and redefined two distinct molecular subtypes, C1 (230 samples) and C2 (269 samples). Moreover, the C1 subtype had a higher survival rate than the C2 subtype in HNSCC samples, as well as a prolonged median survival duration with activated immune response. 1531 DEGs, including 529 upmodulated genes and 1002 downmodulated genes, were identified in the above two subtypes. Functional enrichment analysis revealed that upmodulated genes in C2 were associated with tumorigenesis and development, while downregulated genes in C2 were associated with immune response. By comparing with the existing immunophenotyping group, it found that C1 had more overlaps with the existing Atypical and Basal, and C2 and Classical and Mesenchymal had a high degree of coincidence. On the basis of lncRNA, there were significant differences in the aspect of prognostic and immunological characteristics in the two identified molecular subtypes of HNSCC.

Immune-related lncRNA classification of head and neck squamous cell carcinoma

BackgroundLong noncoding RNAs (lncRNAs) play a critical role in innate and adaptive immune responses. Thus, we aimed to identify ideal subtypes for head and neck squamous cell carcinoma (HNSCC) based on immune-related lncRNAs.MethodsTCGA HNSCC cohort was divided into two datasets (training and validation dataset), and 960 previously characterized immune-related lncRNAs were extracted for non-negative matrix factorization analysis. We characterized our HNSCC subtypes based on biological behaviors, immune landscape and response to immunotherapy in both training and validation cohort. A lncRNA-signature was generated to predict our HNSCC subtypes, and essential lncRNAs involved in tumor microenvironment (TME) were identified.ResultsWe developed and validated two HNSCC subtypes (C1 and C2) based on the 70 lncRNAs in the training and validation cohort. C2 subtype displayed good prognosis, high immune cell infiltration, immune-related genes expression and sensitivity to PD-1 blockade. C1 subtype was associated with high activity of mTORC1 signaling and glycolysis as well as high fraction of inactive immune cells. Finally, we generated a 31-lncRNA signature that could predict our above subtypes with high accurate. Additionally, TRG-AS1 was identified as the essential lncRNA involving TME formation. Knockdown of TRG-AS1 inhibited the expression of HLA-A, HLA-B, HLA-C, CXCL9, CXCL10 and CXCL11. High expression of TRG-AS1 indicated a favorable prognosis in HNSCC and anti-PD-L1 cohort (IMvigor210).ConclusionsOur study establishes a novel HNSCC classification on the basis of 31-lncRNA, helping to identify beneficiaries for anti-PD-1 treatment. In addition, a critical lncRNA TRG-AS1 is identified as a new potential prognosis biomarker as well as therapeutic target.

Characterization of Molecular Subtypes in Head and Neck Squamous Cell Carcinoma With Distinct Prognosis and Treatment Responsiveness.

Head and neck squamous cell carcinoma (HNSCC) is one of the most aggressive malignancies with complex phenotypic, etiological, biological, and clinical heterogeneities. Previous studies have proposed different clinically relevant subtypes of HNSCC, but little is known about its corresponding prognosis or suitable treatment strategy. Here, we identified 101 core genes from three prognostic pathways, including mTORC1 signaling, unfold protein response, and UV response UP, in 124 pairs of tumor and matched normal tissues of HNSCC. Moreover, we identified three robust subtypes associated with distinct molecular characteristics and clinical outcomes using consensus clustering based on the gene expression profiles of 944 HNSCC patients from four independent datasets. We then integrated the genomic information of The Cancer Genome Atlas (TCGA) HNSCC cohort to comprehensively evaluate the molecular features of different subtypes and screen for potentially effective therapeutic agents. Cluster 1 had more arrested oncogenic signaling, the highest immune cell infiltration, the highest immunotherapy and chemotherapeutic responsiveness, and the best prognosis. By contrast, Cluster 3 showed more activated oncogenic signaling, the lowest immune cell infiltration, the lowest immunotherapy and chemotherapy responsiveness, and the worst prognosis. Our findings corroborate the molecular diversity of HNSCC tumors and provide a novel classification strategy that may guide for prognosis and treatment allocation.

Identification and Confirmation of the miR-30 Family as a Potential Central Player in Tobacco-Related Head and Neck Squamous Cell Carcinoma.

Constituents of tobacco that can cause DNA adduct formation and oxidative stress are implicated in the development of head and neck squamous cell carcinoma (HNSCC). However, there are few studies on the mechanism(s) that underlie tobacco-associated HNSCC. Here, we used a model in which tumors were induced in rats using 4-nitroquinoline 1-oxide (4NQO), which mimicked tobacco-related HNSCC, and analyzed the expression profiles of microRNAs and mRNAs. Our results indicated that 57 miRNAs and 474 mRNA/EST transcripts exhibited differential expression profiles between tumor and normal tongue tissues. In tumor tissue, the expression levels of rno-miR-30 family members (rno-miR-30a, rno-miR-30a-3p, rno-miR-30b-5p, rno-miR-30c, rno-miR-30d, rno-miR-30e and rno-miR-30e-3p) were only 8% to 37% of those in the control group. The GO terms enrichment analysis of the differentially expressed miRNAs indicated that oxidation reduction was the most enriched process. Low expression of miR-30 family members in human HNSCC cell lines and tissues was validated by qPCR. The results revealed that the expression of miR-30b-5p and miR-30e-5p was significantly decreased in the TCGA HNSCC dataset and validation datasets, and this decrease in expression further distinguishes HNSCC associated with tobacco use from other subtypes of HNSCC. CCK8, colony formation, transwell migration and HNSCC xenograft tumor assays indicated that miR-30b-5p or miR-30e-5p inhibited proliferation, migration and invasion in vitro, and miR-30b-5p suppressed tumor growth in vivo. Moreover, we uncovered that KRAS might be the potential target gene of miR-30e-5p or miR-30b-5p. Thus, our data clearly showed that decreased expression of miR-30e-5p or miR-30b-5p may play a crucial role in cancer development, especially that of tobacco-induced HNSCC, and may be a novel candidate biomarker and target for this HNSCC subtype.

Immune Infiltrates of m6A RNA Methylation-Related lncRNAs and Identification of PD-L1 in Patients With Primary Head and Neck Squamous Cell Carcinoma.

Background: The purpose of this study was to determine the association between m6A-modified lncRNAs, immune infiltration, and PD-L1 expression in patients with primary head and neck squamous cell carcinoma (HNSCC) and the prognostic value of m6A RNA methylation-related lncRNAs in HNSCC.Methods: We downloaded the RNA-seq transcriptome data and the clinical information for HNSCC from the TCGA databases and used consensus clustering analysis to divide the samples into two groups. To identify a risk signature, least absolute shrinkage and selection operator (LASSO) analyses were conducted. the association between m6A-modified lncRNAs, immune infiltration, and PD-L1 expression were detected by using the R packages. What is more, we used cBioPortal tools to identify genomic alterations and PD-L1 mutations and Gene set enrichment analysis (GSEA) was utilized to predict downstream access of two clusters.Results: Notably, lncRNAs play significant roles in tumorigenesis and development. In total, we identified two subtypes of HNSCC according to consensus clustering of the m6A RNA methylation-related lncRNAs, and the T, grade and age were proven to be related to the subtypes. The Cox regression and LASSO analyses identified a risk signature including GRHL3-AS1, AL121845.4, AC116914.2, AL513190.1. The prognostic value of the risk signature was then proven. The selected gene PD-L1 mutations and the immune infiltration in both groups were further explored.Conclusion: Collectively, our study elucidated the important role of m6A RNA methylation- related lncRNAs in tumor microenvironment of HNSCC. The proposed m6A RNA methylation- related lncRNAs might serve as crucial mediators of tumor microenvironment of HNSCC, representing promising therapeutic targets in improving immunotherapeutic efficacy.

Molecular landscape of head and neck cancer and implications for therapy.

Head and neck squamous cell carcinomas (HNSCC) arising from the oral cavity, pharynx, and larynx constitute the 6th most common human cancer. Human papillomavirus (HPV)-positive tumours are distinct from HPV-negative counterparts, with HPV status affording clear clinical utility, prognostic benefit and better treatment outcomes. In contrast to their HPV-positive counterparts, HPV-negative tumours are characterized by high mutational load and chromosomal aberrations, with varying copy number alteration (CNA) profiles. HNSCC are distinct tumours at the chromosomal, gene and expression levels, with additional insight gained from immune profiling. Based on mutational analyses, HNSCC are categorized as HPV-positive, HPV-negative CNA-silent, and HPV-negative CNA-high tumours. Furthermore, gene expression profiling segregates these tumours into atypical, classical, basal, and mesenchymal, with clear differences observed between tumours of the oral cavity, oropharynx, hypopharynx and larynx. Additional immune profiling further classifies tumours as either immune-active or immune-exhausted. The clinical utility and impact of these tumour molecular subtypes however remains to be determined. HNSCC harbor high levels of somatic mutations. They display loss at 3p and 18q and gain at 3q and 8q, with mutations in CDKN2A, TP53, CCND1, EGFR, PIK3CA, PTEN, NOTCH1, NSD1, FAT1, AJUBA and KMT2D. Important pathways include the p53 and RB pathways which are involved in cell cycle control and are frequently lost in HPV-negative tumours, the WNT-β-catenin pathway related to the mesenchymal subtype and smoking etiology, and the PI3K pathway which includes the most common genetic alteration in HPV-positive HNSCC. Understanding the mutational, genomic and transcriptomic landscape of HNSCC has leveraged better therapeutic approaches to manage this group of diseases, and it is hoped that additional insight into the molecular subtypes of HNSCC and its specific subsites will further drive improved strategies to stratify and treat patients with this debilitating disease.

Identification of Immune Subtypes for Predicting the Prognosis of Patients in Head and Neck Squamous Cell Carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a common malignancy with poor prognosis and immune response, which plays an important role in tumor progression. Recently, immunotherapies have revolutionized the therapeutic means of malignancies including HNSCC. However, the relationship between immunophenotypes of HNSCC and its clinical response to immune-checkpoint inhibitors remains unclear. We aim to identify molecular subtyping related to distinct immunophenotypes in HNSCC. Consensus clustering algorithm was conducted for subtyping. Immunophenotypes between subtypes were compared according to infiltrating immunocytes, immune reactions, major histocompatibility complex (MHC) family, immunoinhibitory, immunostimulatory and immune scores. The relationship between immunophenotype and genotype was investigated from gene mutation and tumor mutation burden. The potential response of Immune-checkpoint blockade (ICB) therapy was estimated with TIDE and ImmuCellAI algorithms, and immune-checkpoint genes. The immune characteristics were also investigated. Biological functions were annotated by the gene-set enrichment analysis (GSEA) algorithm. Two distinct immune subtypes of HNSCC with different survival outcomes, biological characteristics, immunophenotype, and ICB response were identified. The subtype-1 was featured with better prognosis, more infiltrated immunocytes, stronger immune reaction, higher immune-related gene expression, higher immune-checkpoint gene expression (PD-1, PD-L1, and CTLA-4), and better ICB response. A higher immune response in subtype-1 was also revealed by GSEA. Subtype-1 possessed a higher immune response and more sensitivity to ICB therapy leading to a better prognosis. These findings may shed promising light on the immunotherapy strategy in HNSCC

A HPV16-related prognostic indicator for head and neck squamous cell carcinoma.

BackgroundThe human papillomavirus (HPV) is emerging as an important risk factor in head and neck squamous cell carcinoma (HNSCC) patients. This has been observed particularly in the case of HPV16. The HPV16+ HNSCC subtype has distinct pathological, clinical, molecular, and prognostic characteristics. This study aimed to identify potential microRNAs (miRNAs) and their roles in HPV16+ HNSCC progression.MethodmiRNA, mRNA and the clinical data of 519 HNSCC and 44 HNSCC-negative samples were obtained from The Cancer Genome Atlas (TCGA) database. Differentially expressed miRNAs (DEMs) in HPV16-related HNSCC tissues with prognostic value were selected. DEM levels were assessed based on clinicopathological parameters and overall survival (OS). Target genes were also predicted and functional analysis based on Gene Set Enrichment Analysis (GSEA) were then performed.ResultsIn HPV16+ HNSCC tissues, miR-99a-3p and miR-4746-5p were significantly upregulated. In contrast, miR-411-5p was shown to be downregulated. miR-99a-3phighmiR-411-5plowmiR-4746-5phigh expression could estimate improved OS and low frequent perineural invasion (PNI). Predicted target genes were enriched in cell growth, neuroepithelial cell differentiation, MAPK and FoxO signaling pathways. Epithelial mesenchymal transition (EMT) gene set and invasion related genes were downregulated in miR-99a-3phighmiR-411-5plowmiR-4746-5phigh HNSCC patients.ConclusionmiR-99a-3p, miR-411-5p and miR-4746-5p might participate in HPV16+ HNSCC progression through EMT related pathways and affect prognosis.

EGFR overexpression increases radiotherapy response in HPV-positive head and neck cancer through inhibition of DNA damage repair and HPV E6 downregulation.

High-risk Human Papillomavirus (HPV) infections have recently emerged as an independent risk factor in head and neck squamous cell carcinoma (HNSCC). There has been a marked increase in the incidence of HPV-induced HNSCC subtype, which demonstrates different genetics with better treatment outcome. Despite the favourable prognosis of HPV-HNSCC, the treatment modality, consisting of high dose radiotherapy (RT) in combination with chemotherapy (CT), remains similar to HPV-negative tumours, associated with toxic side effects. Epidermal growth factor receptor (EGFR) is overexpressed in over 80% of HNSCC and correlates with RT resistance. EGFR inhibitor Cetuximab is the only FDA approved targeted therapy for both HNSCC subtypes, however the response varies between HNSCC subtypes. In HPV-negative HNSCC, Cetuximab sensitises HNSCC to RT improving survival rates. To reduce adverse cytotoxicity of CT, Cetuximab has been approved for treatment de-escalation of HPV-positive HNSCC. The results of several recent clinical trials have concluded differing outcome to HPV-negative HNSCC. Here we investigated the role of EGFR in HPV-positive HNSCC response to RT. Remarkably, in HPV-positive HNSCC cell lines and in vivo tumour models, EGFR activation was strongly indicative of increased RT response. In response to RT, EGFR activation induced impairment of DNA damage repair and increased RT response. Furthermore, EGFR was found to downregulate HPV oncoproteinE6 expression and induced p53 activity in response to RT. Collectively, our data uncovers a novel role for EGFR in virally induced HNSCC and highlights the importance of using EGFR-targeted therapies in the context of the genetic makeup of cancer.

The clinical significance of apolipoprotein L1 in head and neck squamous cell carcinoma.

Approximately 500,000 new head and neck squamous cell carcinoma (HNSCC) cases are detected every year around the world, and its incidence ranks sixth among all cancer types globally. Among these cases, oral squamous cell carcinoma (OSCC) and laryngeal squamous cell carcinoma (LSCC) are HNSCC subtypes with high incidence rates, especially in China. The present study examines the association between the apolipoprotein L1 (APOL1) mRNA and protein expression and clinical parameters in HNSCC. The two most common types (oral and larynx) of HNSCC were selected for subgroup analyses. Immunohistochemistry (IHC) was used to detect APOL1 protein expression levels in HNSCC clinical specimens. It was demonstrated that APOL1 protein expression in 221 cases of HNSCC was higher compared with that in normal tissues. Consistent upregulation of APOL1 protein was also found in subgroups of OSCC and LSCC. Through mining the ArrayExpress, The Cancer Genome Atlas and the Gene Expression Omnibus databases, microarrays and RNA sequencing data for HNSCC were retrieved, which were used to analyze APOL1 mRNA expression levels. The results showed that APOL1 expression was higher in both OSCC and LSCC subtypes, as well as in HNSCC, compared with that in non-cancerous squamous epithelium. The summary receiver operating characteristic analysis showed that APOL1 had potential as a diagnostic biomarker for HNSCC, OSCC and LSCC. Thus, upregulation of APOL1 may contribute to the tumorigenesis of HNSCC.

Identification of Prognosis Associated microRNAs in HNSCC Subtypes Based on TCGA Dataset.

Background and Objectives: Head and Neck Squamous Cell Carcinoma (HNSCC) includes cancers from the oral cavity, larynx, and oropharynx and is the sixth-most common cancer worldwide. MicroRNAs are small non-coding RNAs for which altered expression has been demonstrated in pathological processes, such as cancer. The objective of our study was to evaluate the different expression profile in HNSCC subtypes and the prognostic value that one or several miRNAs may have. Materials and Methods: Data from The Cancer Genome Atlas Program-Head and Neck Squamous Cell Carcinoma (TCGA-HNSCC) patients were collected. Differential expression analysis was conducted by edge R-powered TCGAbiolinks R package specific function. Enrichment analysis was developed with Diana Tool miRPath 3.0. Kaplan-Meier survival estimators were used, followed by log-rank tests to compute significance. Results: A total of 127 miRNAs were identified with differential expression level in HNSCC; 48 of them were site-specific and, surprisingly, only miR-383 showed a similar deregulation in all locations studied (tonsil, mouth, floor of mouth, cheek mucosa, lip, tongue, and base of tongue). The most probable affected pathways based on miRNAs interaction levels were protein processing in endoplasmic reticulum, proteoglycans in cancer (p < 0.01), Hippo signaling pathway (p < 0.01), and Transforming growth factor-beta (TGF-beta) signaling pathway (p < 0.01). The survival analysis highlighted 38 differentially expressed miRNAs as prognostic biomarkers. The miRNAs with a greater association between poor prognosis and altered expression (p < 0.001) were miR-137, miR-125b-2, miR-26c, and miR-1304. Conclusions: In this study we have determined miR-137, miR-125b-2, miR-26c, and miR-1304 as novel powerful prognosis biomarkers. Furthermore, we have depicted the miRNAs expression patterns in tumor patients compared with normal subjects using the TCGA-HNSCC cohort.