Head And Neck Squamous Cell Carcinoma Cases Research Articles

The two-faced role of RNA methyltransferase METTL3 on cellular response to cisplatin in head and neck squamous cell carcinoma in vitro model.

RNA methyltransferase-like 3 (METTL3) is responsible for methyl group transfer in the progression of N 6-methyladenosine (m6A) modification. This epigenetic feature contributes to the structural and functional regulation of RNA and consequently may promote tumorigenesis, tumor progression, and cellular response to anticancer treatment (chemo-, radio-, and immunotherapy). In head and neck squamous cell carcinoma (HNSCC), the commonly used chemotherapy is cisplatin. Unfortunately, cisplatin resistance is still a major cause of tumor relapse and patients' death. Thus, this study aimed to investigate the role of METTL3 on cellular response to cisplatin in HNSCC in vitro models. HNSCC cell lines (H103, FaDu, and Detroit-562) with stable METTL3 knockdown (sgMETTL3) established with CRISPR-Cas9 system were treated with 0.5 tolerable plasma level (TPL) and 1 TPL of cisplatin. Further, cell cycle distribution, apoptosis, CD44/CD133 surface marker expression, and cell's ability to colony formation were analyzed in comparison to controls (cells transduced with control sgRNA). The analyses of cell cycle distribution and apoptosis indicated a significantly higher percentage of cells with METTL3 knockdown 1) arrested in the G2/S phase and 2) characterized as a late apoptotic or death in comparison to control. The colony formation assay showed intensified inhibition of a single cell's ability to grow into a colony in FaDu and Detroit-562 METTL3-deficient cells, while a higher colony number was observed in H103 METTL3 knockdown cells after cisplatin treatment. Also, METTL3 deficiency significantly increased cancer stem cell markers' surface expression in all studied cell lines. Our findings highlight the significant influence of METTL3 on the cellular response to cisplatin, suggesting its potential as a promising therapeutic target for addressing cisplatin resistance in certain cases of HNSCC.

Analysis of differentially expressed genes in dysplastic oral keratinocyte cell line and their role in the development of HNSCC

Dysplasia is the presence of abnormal types of cells in a tissue precipitated by over or diminished expression of certain genes. These cells act as a precursor to cancer. Dysplastic oral keratinocyte (DOK) cell lines have an aneuploid complex karyotype. They provide an opportunity to study the action of specific carcinogens on malignant transformations. This study aimed to identify the differentially expressed genes in dysplastic cells and their possible association with head and neck squamous cell carcinoma (HNSCC). These genes can be developed as diagnostic, prognostic, or therapeutic leads.The list of genes related to oral keratinocyte dysplasia and head and neck cancer was accessed from the GEO (Gene Expression Omnibus) database. Gene expression profiling was done between dysplastic oral keratinocytes and normal human oral keratinocytes. Gene expression and Kaplan Meier survival analysis were performed using the UALCAN database to assess the correlations between dysregulated genes identified in dysplastic keratinocytes and primary tumors of HNSCC. The GEO omnibus dataset identified numerous differentially expressed genes of which the top 10 up and downregulated genes in dysplastic oral keratinocytes were curated for further analysis. The expression profile of these genes was assessed using the HNSCC dataset (TCGA, Firehose Legacy). Among all the genes assessed, only one gene, the OLR1 gene encoding oxidized low-density lipoprotein, was found to be overexpressed in both the groups viz., dysplastic keratinocytes and HNSCC cases with a strong correlation with the survival status of patients. There was significant correlation between the gene expression pattern observed in dysplastic keratinocytes and the primary tumor of the HNSCC group, with an exotic gene that was seldom discussed in association with cancer, viz., OLR1. Exploration into other top-ranking differentially expressed genes in dysplastic cases would aid in identifying the candidate gene associated with both phenotypes.

Prognostic value of CDKN2A in head and neck squamous cell carcinoma via pathomics and machine learning.

This study aims to enhance the prognosis prediction of Head and Neck Squamous Cell Carcinoma (HNSCC) by employing artificial intelligence (AI) to analyse CDKN2A gene expression from pathology images, directly correlating with patient outcomes. Our approach introduces a novel AI-driven pathomics framework, delineating a more precise relationship between CDKN2A expression and survival rates compared to previous studies. Utilizing 475 HNSCC cases from the TCGA database, we stratified patients into high-risk and low-risk groups based on CDKN2A expression thresholds. Through pathomics analysis of 271 cases with available slides, we extracted 465 distinctive features to construct a Gradient Boosting Machine (GBM) model. This model was then employed to compute Pathomics scores (PS), predicting CDKN2A expression levels with validation for accuracy and pathway association analysis. Our study demonstrates a significant correlation between higher CDKN2A expression and improved median overall survival (66.73 months for high expression vs. 42.97 months for low expression, p = 0.013), establishing CDKN2A's prognostic value. The pathomic model exhibited exceptional predictive accuracy (training AUC: 0.806; validation AUC: 0.710) and identified a strong link between higher Pathomics scores and cell cycle activation pathways. Validation through tissue microarray corroborated the predictive capacity of our model. Confirming CDKN2A as a crucial prognostic marker in HNSCC, this study advances the existing literature by implementing an AI-driven pathomics analysis for gene expression evaluation. This innovative methodology offers a cost-efficient and non-invasive alternative to traditional diagnostic procedures, potentially revolutionizing personalized medicine in oncology.

Abstract LB414: Novel ALK/PIK3CA cooperativity in head and neck squamous cell carcinoma (HNSCC) progression

Abstract Recent genomic characterization studies have demonstrated the genetic heterogeneity of head and neck squamous cell carcinoma (HNSCC). Yet, most of these genetic aberrations remain undruggable. Anaplastic Lymphoma Kinase (ALK) is somatically mutated in ~3.6% of HNSCC (18/504 cases, TCGA-HNSCC Firehose cohort, USA), and the majority of these ALK-mutated HNSCC cases are of advanced stage diseases. Here, we hypothesized that ALK mutations might plausibly contribute to HNSCC progression and could be potentially druggable. Unexpectedly, analysis of HNSCC genomic events revealed a novel co-occurrence relationship between ALK and PIK3CA genomic aberrations in TCGA-HNSCC Firehose cohort (Odds Ratio=2.41; N=522 samples with CNV data), with subsequent validation in an independent metastatic MSK-MetTropism HNSCC cohort (Odds Ratio=5.12; N=412). Consistent with this newly identified co-occurrence relationship, we showed that all 7 HNSCC-relevant ALK point mutations were potent drivers for HNSCC invasiveness and clonogenic growth only in corroboration with PIK3CA aberrations in HNSCC cell models, highlighting the functional importance of this novel ALK/PIK3CA cooperativity in HNSCC, likely reminiscent of ALK/MYCN cooperativity in neuroblastoma. Further, by clonogenic assay, we demonstrated pharmacologic vulnerabilities of all 7 ALK point mutations (including extracellular domain mutations) to ALK targeting by ceritinib in FaDu cell background with endogenous PIK3CA amplification. Strikingly, we were able to capture, for the first time, spontaneous emergence of a druggable ALK somatic mutation directly from a PIK3CA-altered HNSCC patient-derived culture (PDC) upon forced acquisition of anoikis-resistance ex vivo (PDC-25-AR), demonstrating co-evolution of ALK/PIK3CA aberrations in HNSCC likely associated with cancer progression. Patient-derived culture-xenograft (PDCx) of PDC-25-AR displayed remarkable sensitivity to ALK targeting, PI3K targeting, as well as their combination, with induction of apoptosis in these tumors compared to vehicle control. In conclusion, ALK aberrations corroborate with PIK3CA aberrations to promote HNSCC progression, yet, these events are potentially druggable in advanced HNSCC. Citation Format: Lan Wang, Helen Hoi-Yin Chan, Angel Yee Lok Fung, Yuen-Keng Ng, Patrick Kwok-Shing Ng, Yuxiong Su, Jason Ying Kuen Chan, Peony Hiu Yan Poon, Thomas Chun Kit Yeung, Chi Man Tsang, Hoi-Lam Ngan, Wenying Piao, Yuchen Liu, Jack Nimitz Smith, Tin Yu Samuel Law, Ee Ling Kong, Melissa Sue Ann Chan, Chun Ho Law, Sze-Man Chan, Zoey Wing Yee Liu, Joshua Chung-Hymn Ng, Gordon B. Mills, Vivian W.Y. Lui. Novel ALK/PIK3CA cooperativity in head and neck squamous cell carcinoma (HNSCC) progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB414.

Evaluation of the significance of tumor stromal patterns and peri-tumoral inflammation in head and neck squamous cell carcinoma with special reference to the Yamamoto-Kohama classification.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide with 878,348 new cases. Cancer-associated fibroblasts (CAFs) are the predominant cell type in tumor stroma and are important promoters of tumor progression. The aim of the study was to evaluate the pattern of desmoplastic stromal reaction and peri-tumoral inflammatory infiltrate with the histological grade and clinical data. A total of 60 cases of HNSCC were included in the study. The hematoxylin and eosin (H and E)-stained sections from all cases were examined by two experienced pathologists for the grade, nature of stomal reaction (SR), peri-tumoral inflammatory infiltration, Yamamoto-Kohama classification grade, worst pattern of invasion (WPOI), depth of invasion (DOI), and other histopathological parameters. Correlation analysis was conducted using the Chi-square test. P- value less than 0.05 was considered statistically significant. Immature SR was not observed in any of the well-differentiated squamous cell carcinoma (SCC) cases. However, one (3.7%) case of moderately differentiated SCC and two (28.6%) cases of poorly differentiated SCC showed signs of immature SR. In the case of the higher grades of the YK classification, specifically grades 4C and 4D, a more profound depth of tumor cell invasion, equal to or exceeding 10 mm, was evident in six (66.67%) and two (28.57%) cases, respectively. Additionally, among the seven (11.7%) cases classified as poorly differentiated carcinoma, three (42.85%) displayed a WPOI score of 5. SR and the tumor invasive pattern in HNSCC are related to prognosis and may indicate tumor aggressiveness.

Abstract 2419: Personalized longitudinal ctDNA monitoring is useful for biomarker in head and neck squamous cell carcinoma

Abstract Introduction: There is no established and useful tumor biomarker to evaluate treatment response and predict early locoregional and distant metastatic relapse in head and neck squamous cell carcinoma (HNSCC). Circulating tumor DNA (ctDNA) has a potential for detecting minimal residual diseases (MRDs). However, the absence of specific hotspot mutations in most HNSCCs makes ctDNA-based studies difficult in HNSCC. As a novel biomarker, we investigated whether individualized ctDNA analysis could help monitor and predict treatment response and relapse in HNSCC. Methods: Thirty-two HNSCC patients were enrolled in this study. Twenty-two patients received radical resection and 10 patients were received radical irradiation as initial curative treatment. We collected tumor tissues and paired PBMCs (peripheral blood mononuclear cells) before treatment. Furthermore, we collected serial plasma samples for ctDNA analysis. To identify tumor specific individualized mutation, we isolated DNA from tumor tissue and PBMC and performed mutation analysis using custom SCC panel with frequent altered 31 genes (all exon coverage). We defined tumor-specific mutated genes per patient with variant allele frequency (VAF) >10 % and total coverage >100 as ctDNA candidate genes and performed ctDNA analysis using dPCR. We generated mutation specific primer-probe for droplet digital PCR (ddPCR), quantified the amount of ctDNA in plasma and compared ctDNA level with clinical data. Results: For mutation analysis, most frequent mutated gene was TP53 (21/32, 65.6%). Longitudinal ctDNA was monitored in a total of 24 cases. In 13 cases, ctDNA tested positive again or did not test negative, and all 13 cases had a residual tumor or relapsed after initial treatment. In 11 cases, after initial treatment, ctDNA remained negative and patients were alive without recurrence. Our cohort included 6 patients in whom it was possible to monitor ctDNA during chemotherapy in recurrent metastatic HNSCC cases. ctDNA alterations correlated well with clinical treatment response in four cases, but not in two cases. Patients who remained negative for ctDNA during follow-up after initial treatment (n=11) had a significantly better prognosis than those who reverted to ctDNA positivity (n=13, P < 0.0001). Conclusion: Custom SCC panel is useful to detect more tumor specific mutation in HNSCC patients. Detection of ctDNA using next generation sequencing and digital PCR is promising strategy capable of monitoring in HNSCC patients. However, this method is not useful in all HNSCC, and there is room for improvement in the selection of ctDNA candidate genes. Citation Format: Ryunosuke Kogo, Tomomi Manako, Takashi Nakagawa. Personalized longitudinal ctDNA monitoring is useful for biomarker in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2419.

Involvement of TGFBI-TAGLN axis in cancer stem cell property of head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) is a significant healthcare burden globally. Previous research using single-cell transcriptome analysis identified TGFBI as a crucial marker for the partial-epithelial-mesenchymal transition (partial-EMT) program. However, the precise role of TGFBI in HNSCC progression remains unclear. Therefore, our study aimed to clarify the impact of TGFBI on the malignant behavior of HNSCC cells. Through RNA-sequencing data from the TCGA database, we validated that increased TGFBI expression correlates with a higher occurrence of lymph node metastasis and unfavorable prognosis in HNSCC cases. Functional experiments demonstrated that TGFBI overexpression enhances the ability of sphere formation, indicating stem-cell-like properties. Conversely, TGFBI depletion reduces sphere formation and suppresses the expression of cancer stem cell (CSC) markers. RNA-sequencing analysis of TGFBI-overexpressing and control HNSCC cells revealed TAGLN as a downstream effector mediating TGFBI-induced sphere formation. Remarkably, TAGLN depletion abolished TGFBI-induced sphere formation, while its overexpression rescued the suppressed sphere formation caused by TGFBI depletion. Moreover, elevated TAGLN expression showed correlations with the expression of TGFBI and partial-EMT-related genes in HNSCC cases. In conclusion, our findings suggest that TGFBI may promote CSC properties through the upregulation of TAGLN. These novel insights shed light on the involvement of the TGFBI-TAGLN axis in HNSCC progression and hold implications for the development of targeted therapies.

Polygenic risk score-based phenome-wide association study of head and neck cancer across two large biobanks

BackgroundNumerous observational studies have highlighted associations of genetic predisposition of head and neck squamous cell carcinoma (HNSCC) with diverse risk factors, but these findings are constrained by design limitations of observational studies. In this study, we utilized a phenome-wide association study (PheWAS) approach, incorporating a polygenic risk score (PRS) derived from a wide array of genomic variants, to systematically investigate phenotypes associated with genetic predisposition to HNSCC. Furthermore, we validated our findings across heterogeneous cohorts, enhancing the robustness and generalizability of our results.MethodsWe derived PRSs for HNSCC and its subgroups, oropharyngeal cancer and oral cancer, using large-scale genome-wide association study summary statistics from the Genetic Associations and Mechanisms in Oncology Network. We conducted a comprehensive investigation, leveraging genotyping data and electronic health records from 308,492 individuals in the UK Biobank and 38,401 individuals in the Penn Medicine Biobank (PMBB), and subsequently performed PheWAS to elucidate the associations between PRS and a wide spectrum of phenotypes.ResultsWe revealed the HNSCC PRS showed significant association with phenotypes related to tobacco use disorder (OR, 1.06; 95% CI, 1.05–1.08; P = 3.50 × 10−15), alcoholism (OR, 1.06; 95% CI, 1.04–1.09; P = 6.14 × 10-9), alcohol-related disorders (OR, 1.08; 95% CI, 1.05–1.11; P = 1.09 × 10−8), emphysema (OR, 1.11; 95% CI, 1.06–1.16; P = 5.48 × 10−6), chronic airway obstruction (OR, 1.05; 95% CI, 1.03–1.07; P = 2.64 × 10−5), and cancer of bronchus (OR, 1.08; 95% CI, 1.04–1.13; P = 4.68 × 10−5). These findings were replicated in the PMBB cohort, and sensitivity analyses, including the exclusion of HNSCC cases and the major histocompatibility complex locus, confirmed the robustness of these associations. Additionally, we identified significant associations between HNSCC PRS and lifestyle factors related to smoking and alcohol consumption.ConclusionsThe study demonstrated the potential of PRS-based PheWAS in revealing associations between genetic risk factors for HNSCC and various phenotypic traits. The findings emphasized the importance of considering genetic susceptibility in understanding HNSCC and highlighted shared genetic bases between HNSCC and other health conditions and lifestyles.

Genetic alterations in CDKN2A interacting network and their putative association with head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) is a complex, polygenic disorder involving the host, microbial, and environmental factors. The present study investigated the CDKN2A (cyclin-dependent kinase inhibitor 2 A) gene interacting network. This gene is a key player in the cell cycle pathway, and any abnormalities in this gene have been linked to abnormal cell cycle processes. The present study followed an observational study design wherein computational tools were used to demonstrate the genetic alterations, expression profile, and survival status of head and neck cancer patients. The head and neck squamous cell carcinoma and oral squamous cell carcinoma datasets (The Cancer Gene Atlas (TCGA), Firehose Legacy) were analyzed. The cBioportal and UALCAN [University of ALabama at Birmingham CANcer data analysis Portal] were used to demonstrate the genetic alterations and gene expression profile alongside the survival of patients. The oncoprint data analysis of the CDKN2A gene network showed 72% and 48% of aberrations in the TP53 gene among HNSCC and OSCC (Oral Squamous Cell Carcinoma) cases, respectively. The gene expression profile demonstrated over-expression of CDKN2A in primary tissues. The survival analysis showed a statistically significant difference between the low/medium and high expression groups. The deep deletion was the common alteration observed among head and neck cancer patients of the CDKN2A gene. The gene expression profile showed significant upregulation of CDKN2A in primary tissues [p = 〈10−12]. The genetic alterations observed in the CDKN2A gene were found to correlate well with the survival status of head and neck cancer patients. The functional validation of the mutations and alterations observed in this gene would aid us in understanding the vital role of this gene network in the process of oral carcinogenesis.

The Effect of Tumor Resection and Radiotherapy on the Expression of Stem Cell Markers (CD44 and CD133) in Patients with Squamous Cell Carcinoma.

Background: Head and Neck Squamous Cell Carcinomas (HNSCCs) are heterogeneous malignancies that comprise 90% of the head and neck cancers. HNSCCs originate from the mucosal lining epithelium of the upper aerodigestive tract. Cancer stem cells (CSCs) that generate HNSCCs with the CD44, CD133, and ALDH phenotype and are resistant to radiotherapy and chemotherapy. In the current, the quantitative alteration in CD44 and CD133 expression pre- and post-tumor resection and radiotherapy was evaluated in HNSCC patients. Moreover, the alterations in the expression of Bax, Bak, Bcl-2, ALDH, and PTEN genes were measured. Materials and Methods: Flow cytometry was performed to evaluate the alterations in CD44 and CD133 surface markers pre- and posttumor resection and radiotherapy. Quantitative real-time RT-PCR (qRT-PCR) was conducted to investigate the mRNA expression levels of Bax, Bak, Bcl-2, ALDH, and PTEN. Results: The results indicated that the cancer stem cell CD44 surface marker significantly decreased after tumor resection and radiotherapy in HNSCC cases, while the decrease was insignificant for CD133 marker expression. mRNA expression level of Bcl-2 and ALDH was increased, but Bax and Bak gene expressions were reduced significantly Conclusion: The results also indicated that the expression of CD44 significantly decreased after tumor resection and radiotherapy. The upregulation of mRNA level of Bcl-2 and ALDH, and the downregulation of Bax and Bak gene expression were noted in these cases when compared to the healthy control group.

Nicotinic acetylcholine receptor CHRNA5 is overexpressed in head and neck squamous cell carcinoma patients with a recent tobacco smoking history.

Tobacco smoking is a major driver of head and neck squamous cell carcinoma (HNSCC) occurrence, and previous studies have shed light on the precise molecular alterations in tobacco-related HNSCCs when compared to HNSCCs associated with other risk factors (ex: human papillomavirus/HPV status). In this study, we analyzed the gene expression differences in HNSCC cases with a recent smoking history and revealed that the nicotinic acetylcholine receptor CHRNA5 is differentially overexpressed in smoking-related HNSCCs. CHRNA5 overexpression in these HNSCCs corresponds with a worse prognosis and is inversely correlated with an immune expression signature commonly associated with better prognosis. From these results, our study highlights the potential role of the nicotine-activated CHRNA5 receptor in HNSCC progression and corresponds with other recent reports highlighting the potential role of nicotine induction in promoting cancer progression.

Clinicopathologic characteristics of HPV-associated head and neck squamous cell carcinoma in Southern China: long-term retrospective study of 400 cases.

Human papillomavirus (HPV)-associated head and neck squamous cell carcinoma (HNSCC) is an evolving and growing disease, especially in developing countries. However, the clinical characteristics of HPV-associated HNSCC in regard to HPV infection rates, patient features, and prognosis are under-reported in the Asian population. In this study, we retrospectively enrolled a 400-case cohort of HNSCC with p16 immunochemistry and analyzed with long-term follow-up. We investigate the current HPV prevalence of HNSCC, unique HPV-associated patient clinical characteristics, and patient prognosis in the southern China population. HPV infection exhibited a 15% prevalence in all HNSCC cases, notably higher in oropharyngeal cases (30.7%), followed by oral cavity (11.8%), laryngeal (10.1%), and hypopharyngeal (2.5%). HPV status, gender, old age, and location of tumor were significantly associated with the patient's survival. Tonsil invasion was found more frequent in HPV-positive oropharyngeal HNSCC than in HPV-negative cases. HPV-associated HNSCC patients tend to possess stronger tobacco and alcohol habits, which were correlated to poor survival. HPV status's correlation with gender, age, and anatomical location is associated intricately with patient survival. The secondary primary tumor rate was found higher within the HPV-negative group, compared to the HPV-positive group (9.12% versus 1.67%). Our study provided a current picture of HPV-associated HNSCC in the southern China population and elaborated the understanding of key factors that correlate to HNSCC prognosis. Our findings indicated a strong susceptibility of HPV-associated oropharyngeal HNSCC in the tonsil and the difference in secondary primary tumor rates associated with HPV status.

Immunohistochemical study of Programmed Cell Death Ligand 1 (PDL1) expression by combined positive score using 22C3 clone in head and neck squamous cell carcinomas, its correlation with clinicopathological features and outcome.

Programmed cell death ligand 1 (PD L1) is a transmembrane protein that is highly expressed in neoplastic cells. Therapy with immune checkpoint inhibitors target PD-1/PD-L1 blockade-inducing tumour regression. Immunohistochemistry (IHC) for PD-L1 expression enables patient selection for immunotherapy and can be considered as a potential predictive biomarker for immunotherapy in head and neck squamous cell carcinoma (HNSCC). To determine the PDL1 expression in HNSCC, to correlate with clinicopathological features and outcome. We retrospectively analysed 59 cases of HNSCC at our Tertiary Hospital between January 2017 and November 2018 and followed up until death/Nov 2022 for Overall survival. IHC analysis of PD-L1 using Combined Positive Score (CPS) with antibody clone 22C3 in 59 cases of HNSCC was performed. PD-L1 expression was correlated with clinicopathological features and outcomes. Pearson Chi-square test was used to analyse the correlation between PD-L1 expression and clinicopathological parameters using SPSS20.0. Survival curves were calculated by Kaplan-Meier method, and differences were analysed by log-rank test. A total of 25 cases (42.4%) had positive PDL expression (CPS ≥1). 16/25 cases (27.1%) belonged to CPS (≥1, <10). An almost-perfect interobserver agreement was noted by two pathologists for PD-L1 IHC expression. No statistically significant correlation was noted between PD-L1 score and clinicopathologic features. Detection of PD-L1 status gives further insight into frequency of PD-L1 expression in Indian HNSCC patients to possibly improve clinical treatment strategies, ensuring that our patients get the maximum therapeutic benefit of immunotherapy.

Programmed Cell Death Ligand 1 Immunoexpression in Head and Neck Squamous Cell Carcinoma: A Narrative Review of Considerations for the Histopathologist

Head and Neck Squamous Cell Carcinoma (HNSCC) is one of the most common malignancies worldwide with most of the patients presenting in advanced stage. The associated high mortality and poor prognosis of the tumour has rendered the search for better therapeutic options than the traditional modalities of surgery, radiotherapy and chemotherapy. Immunotherapy targeting Programmed Cell Death Protein 1 / Programmed Cell Death Ligand 1 (PD-1/PD-L1) has been approved in recurrent/ metastatic HNSCC cases. PD-L1 expressing HNSCCs are likely to respond to immunotherapy. PD-L1 expression has been studied in peripheral blood utilising Circulating Tumour Cells (CTC) and soluble exosomes. Despite these non invasive techniques, PD-L1 expression has been most frequently assessed by immunohistochemistry (IHC) application on the Formalin Fixed Paraffin Embedded (FFPE) tissue of the tumour. Currently, many antibody clones and various IHC platforms are available for PD-L1, but only 22c3 has been approved by the Food and Drug Administration (FDA). Although, the availability of multiple options has made PD-L1 assessment possible and affordable at many centres worldwide, but various procedural, pre-analytical, and analytic issues have to be considered prior to the interpretation of immunoexpression of PD-L1. In this review, the authors aim to highlight the problem areas, and to understand the implications of PD-L1 expression in HNSCC. The authors propose recommendations for the optimal assessment of PD-L1 expression in HNSCC on IHC.

Bioinformatics analysis of immune characteristics in tumors with alternative carcinogenesis pathways induced by human papillomaviruses

BackgroundHuman papillomaviruses (HPVs) induce a subset of head and neck squamous cell carcinomas (HNSCC) and anogenital cancers, particularly cervical cancer (CC). The major viral proteins that contribute to tumorigenesis are the E6 and E7 oncoproteins, whose expression is usually enhanced after the integration of viral DNA into the host genome. Recently, an alternative tumorigenesis pathway has been suggested in approximately half of HNSCC and CC cases associated with HPV infection. This pathway is characterized by extrachromosomal HPV persistence and increased expression of the viral E2, E4, and E5 genes. The E6, E7, E5, and E2 proteins have been shown to modify the expression of numerous cellular immune-related genes. The antitumor immune response is a critical factor in the prognosis of HPV-driven cancers, and its characterization may contribute to the prediction and personalization of the increasingly used cancer immunotherapy.MethodsWe analyzed the immune characteristics of HPV-dependent tumors and their association with carcinogenesis types. Transcriptomic HNSCC and CC datasets from The Cancer Genome Atlas were used for this analysis.ResultsClustering with immune-related genes resulted in two clusters of HPV16-positive squamous cell carcinomas in both tumor types: cluster 1 had higher activation of immune responses, including stimulation of the antigen processing and presentation pathway, which was associated with higher immune cell infiltration and better overall survival, and cluster 2 was characterized by keratinization. In CC, the distribution of tumor samples into clusters 1 and 2 did not depend on the level of E2/E5 expression, but in HNSCC, most E2/E5-high tumors were localized in cluster 1 and E2/E5-low tumors in cluster 2. Further analysis did not reveal any association between the E2/E5 levels and the expression of immune-related genes.ConclusionsOur results suggest that while the detection of immune responses associated with preserved expression of genes encoding components of antigen processing and presentation machinery in HPV-driven tumors may be markers of better prognosis and an important factor in therapy selection, the type of carcinogenesis does not seem to play a decisive role in the induction of antitumor immunity.

A PRISMA meta-analysis for diagnostic value of microRNA-21 in head and neck squamous cell carcinoma along with bioinformatics research.

The use of miR-21 expression remains vague in diagnosis of head and neck squamous cell carcinoma (HNSCC). This study aimed to systematically evaluate the diagnostic potential of the miR-21 expression in patients with HNSCCs through investigating and summarizing the results reported in the literature. Extant medical databases were examined for articles of clinical study assessing the miR-21 expression in HNSCC cases, published in the past 20 years. Bioinformatics research was also performed for finding miR-21 targets differentially expressed in HNSCC so as to present their biological behaviors. Our meta-analysis comprised 11 studies including 622/450 cases in HNSCC/control group. Forest plots displayed miR-21 which possessed significantly good specificity (0.76, p<0.001) and sensitivity (0.80, p<0.001). Diagnostic odds ratio was 2.46 (95% CI 1.87-3.24). Positive and negative likelihood ratio was 3.40 (95% CI 1.94-5.97) and 0.26 (95% CI 0.18-0.38), respectively. Area under the receiver operating characteristic curve was 0.85. This study is the highest level of evidence presently available in diagnosing HNSCC. This PRISMA meta-analysis indicated that the pooled results were robust, confirming the oncogenic potential of miR-21 that could be used successfully as a screening biomarker in HNSCC patients. Specifically, the overexpression of miR-21 in these patients presents a worse survival outcome.

A comprehensive analysis of LINC00958 as a prognostic biomarker for head and neck squamous cell carcinoma

This work focused on exploring whether the long intergenic non-protein coding RNA LINC00958 is associated with the prognosis of head and neck squamous cell carcinoma (HNSCC). Associations of the LINC00958 expression level with clinicopathological features of HNSCC were investigated by logistic regression and Wilcoxon signed-rank test. The Kaplan–Meier method was applied to evaluate patient survival. Clinical data and expression profiles were obtained from The Cancer Genome Atlas (TCGA). Associations of patient clinical characteristics with overall survival (OS), progression-free interval (PFI), and disease-specific survival (DSS) were assessed by univariate and multivariate analysis using the Cox proportional hazard model. Immune cell infiltration analysis and gene set enrichment analysis (GSEA) were applied to determine any significant effects of LINC00958. High LINC00958 expression was related to early pT stage (P < 0.01), primary therapy outcome (P < 0.01), HPV status (P < 0.001), lymphovascular invasion (P < 0.001), and perineural invasion (P < 0.01). The receiver operating characteristic curve showed strong prognostic power for LINC00958 (area under curve = 0.886). High LINC00958 expression predicted poor OS (P = 0.007), DSS (P = 0.036), and PFI (P = 0.040). LINC00958 was related to signalling pathways and the infiltration of certain immune cells. miR-27b-5p was significantly associated with LINC00958, and downstream NT5E predicted poor survival in HNSCC cases. LINC00958 may affect the prognosis by regulating NT5E via miR-27b-5p, and could serve as a possible factor to predict the prognosis of HNSCC, especially oral squamous cell carcinoma.

Tumor-Infiltrating CD103+ Tissue-Resident Memory T Cells and CD103-CD8+ T Cells in HNSCC Are Linked to Outcome in Primary but not Metastatic Disease.

High numbers of tumor-infiltrating lymphocytes (TIL) are linked to better survival in patients with cancer. Tissue-resident memory T cells (TRM; CD8+CD103+) are recognized as a key player of anticancer immune response. To assess TRM cells in primary, metastatic, and recurrent head and neck squamous cell carcinoma (HNSCC), we developed a tissue microarray (TMA) and used multiplex IHC (MxIHC). Samples from primary tumors of 379 HNSCC cases treated at Southampton Hospitals between 2000 and 2016 were collected and analyzed. Of these, 105 cases had lymph node metastases and 82 recurrences. A TMA was generated with triplicate cores for each sample. MxIHC with a stain-and-strip approach was performed using CD8, CD103, and TIM3. Scanned slides were analyzed (digital image analysis) and quality checked (QC). After QC, 194 primary tumors, 76 lymph node metastases, and 65 recurrences were evaluable. Alcohol consumption was statistically significantly correlated with a reduction of TRM cells in primary tumors (nondrinker vs. heavy drinker: P = 0.0036). The known survival benefit of TRM cell infiltration in primary tumors was not found for lymph node metastasis. In recurrences, a high TRM cell number led to a favorable outcome after 12 months. The checkpoint molecule TIM3, was expressed significantly higher on TRM and non-TRM cells in the lymph node compared with primary tumors (P < 0.0001), which was also seen in recurrences (P = 0.0134 and P = 0.0007, respectively). We confirm the prognostic impact of TIL in primary tumors and in recurrences. TRM cell density in lymph node metastases was not linked to outcome. The role of TIM3, as a therapeutic target remains to be defined.

Influence of Medicaid Expansion on Head and Neck Squamous Cell Carcinoma Presentation and Survival.

To compare head and neck squamous cell carcinoma stage at presentation and survival in Medicaid-expanded states versus nonexpanded states. Retrospective cohort. Northwestern University Feinberg School of Medicine. The Head and Neck with human papillomavirus Status Database within the Surveillance, Epidemiology, and End Results (SEER) Program was queried for cases of head and neck squamous cell carcinoma (HNSCC) diagnosed in the years 2010 to 2016. Cases were grouped according to their respective state Medicaid expansion status. Multivariable logistic regressions and multivariable Cox proportional hazards models were used to evaluate associations with stage IV disease and survival. Compared to nonexpanded states, Medicaid-expanded states had a significantly larger proportion of Medicaid patients (20.3% vs 16.7%, P = .0009) and a significantly smaller proportion of uninsured patients (1.7% vs 10.1%, P < .0001). The case selection process resulted in 2215 patients meeting inclusion criteria. In multivariable analysis, cases under Medicaid expansion were 31% less likely to present with stage IV disease compared to cases in nonexpanded states (odds ratio: 0.69, 95% confidence interval [CI]: 0.51-0.93). In the multivariable Cox proportional hazards model, cases under Medicaid expansion had significantly better mortality outcomes and were 32% less likely to die compared to cases in nonexpanded states (hazard ratio: 0.68, 95% CI: 0.55-0.84). Medicaid expansion is associated with fewer stage IV cases and improved survival of HNSCC cases. These findings support continued efforts to expand Medicaid coverage.

Abstract PO-032: Association of a polymorphic variant in JMJD1C with tumor recurrence after adjuvant chemoradiation therapy in head and neck squamous cell carcinoma

Abstract Background: Adjuvant chemoradiation therapy (CRT) is the standard treatment for locally advanced head and neck squamous cell carcinomas (HNSCCs). Over 50% of HNSCC cases are diagnosed at the locally advanced stage, with in-field persistence and recurrence major causes of morbidity and poor survival outcome. Apart from human papillomavirus (HPV) status, there are no predictive biomarkers for CRT outcomes. CRT lethality arises from unrepaired double strand breaks (DSBs) in DNA. Recent work has suggested that dynamic changes in lysine modification of chromatin regulate DSB repair. This study investigated the association of germline single nucleotide polymorphisms (SNPs) in lysine-modifying genes with DSB repair and CRT outcomes in HNSCC. Methods: Blood specimens and associated retrospective clinical data from HNSCC patients (n=90; age range=41-89, median age 64; males=69, females=21) were obtained under IRB# 21-9921. The patients had HPV-negative tumors in the larynx or oral cavity, and had received radiation, with some receiving concurrent chemotherapy. Patients receiving concurrent chemotherapy were: 40% in disease-free group, 21% in recurrence disease group, and 38% in never disease-free group. The patients were segregated into 3 groups: disease-free (n=30), recurrent disease (n=28), and never-disease free (n=32). DNA from blood was genotyped for specific germline SNPs in lysine-modifying genes. Isogenic HNSCC lines (Cal-27, SCC9) carrying either the wild type (WT) or homozygous SNP were generated using CRISPR/Cas9 technology, and bulk RNA sequencing and immunofluorescence (IF)-based studies were performed to assess cellular pathways affected. Results: A homozygous variant SNP in the coding region of JMJD1C was significantly enriched in the disease-free group (n=21/30) versus the recurrent disease (n=3/28) and never disease-free (n=2/32) groups (p=0.0003, OR=0, Fisher’s Exact test). Analysis of SNP frequency in large population datasets found that while it was abundant in European Americans (EAs), it is relatively rare in African Americans (AAs) (EA= 0.3139 vs. AA=0.072; gnomAD population). Cal-27 JMJD1C SNP cells were significantly enriched for altered mRNA levels of genes associated with DNA repair, replication, cell cycle, oxidative phosphorylation, and histone kinase activity versus CAL-27 with WT JMJD1C (q-value&amp;lt;0.05). The SCC9 JMJD1C SNP cells were significantly enriched in extracellular matrix organization, cytokine-cytokine receptor interaction, and humoral immune response versus SCC9 with WT JMJD1C (q-value&amp;lt;0.05). On irradiation, the JMJD1C SNP cells had robust activation of the DSB repair pathway (BRCA1- mediated repair foci) versus the WT cell lines. Conclusion and future work: This work provides novel insight into a germline SNP in a lysine-modifying gene effecting DSB repair and associating with CRT outcomes in HNSCC. It may serve as a predictive biomarker for recurrence. Future research will explore racial disparities in tumor recurrence and confirm results in larger HNSCC datasets. Citation Format: Adria Hasan, Elena V. Demidova, Shreya M. Shah, Philip Czyzewicz, Karthik Devarajan, Thomas Galloway, Barbara Burtness, Erica A. Golemis, Joshua E. Meyer, Sanjeevani Arwesd. Association of a polymorphic variant in JMJD1C with tumor recurrence after adjuvant chemoradiation therapy in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-032.