Amphiregulin promotes activated regulatory T cell-suppressive function via the AREG/EGFR pathway in laryngeal squamous cell carcinoma.

BACKGROUND: Activated regulatory T cells (aTregs) play a vital role in promoting a tumor immunosuppressive microenvironment in laryngeal squamous cell carcinoma (LSCC). However, the regulatory factors that induce the generation of aTregs are not clear. Herein, we investigated the effect of amphiregulin (AREG) on the production of aTregs in the tumor microenvironment of LSCC. METHODS: Immunohistochemical (IHC) analysis was conducted to examine the expression of AREG and FOXP3, and their association with clinical parameters and patient outcomes was demonstrated. The expression level of EGFRs in three functional subsets of Tregs was assessed, and the induction of CD4+ T cells into aTregs in the presence or absence of AREG or Gefitinib was analyzed using flow cytometry. RESULTS: Our results showed a higher expression level of AREG was significantly related to advanced clinical stage and worse survival, particularly with increased infiltration of Tregs in LSCC tumor tissue. The in vitro study showed that AREG significantly promoted the differentiation of aTregs, and enhanced the inhibitory effect of Tregs on T cell proliferation, which could be reversed by epidermal growth factor receptor (EGFR) inhibitors. In addition, we found that EGFR was highly expressed in aTregs, but not in other subsets of Tregs. It is suggested that AREG might induce aTregs, and enhance the immunosuppressive function of Tregs via the AREG/EGFR signal pathway. CONCLUSIONS: Collectively, this study revealed the role and mechanism of AREG in negative immune regulation, and targeting AREG might be a novel immunotherapy for LSCC.

Immunomodulatory role of tumor microenvironment on oncological outcomes in advanced laryngeal cancer.

BACKGROUND: The study evaluated the prognostic impact of the immune microenvironment in LSCC with markers of major immune cells to identify the key determinants of short-term disease-free survival (ST DFS) and reveal factors related to disease progression. METHODS: The study cohort included 61 patients who underwent total laryngectomy, 83.6% of whom were male with a mean age of 64.3 years at the time of surgery. Twenty-five patients had long term DFS (over 5 years), 8 - had moderate DFS (between 2 and 5 years), and 28 had short-term DFS (less than 2 years). Immunohistochemical staining and evaluation were performed on samples collected after the laryngectomy. RESULTS: The samples' assessment revealed that the mean expression of all analysed markers was the highest both in stroma and the tumor compartment for short term DFS (ST DFS) patients. Analysis confirmed that a high stromal density of CD8 cells (p = 0.038) significantly correlated with DFS, and that the increased presence of CD57 cells (p = 0.021) was significantly associated with ST DFS. Moreover, the high density of CD68 cells in the tumor epithelial compartment had a negative prognostic impact on DFS (p = 0.032). Analysis of overall survival in the studied cohort with Kaplan-Meyer curves revealed that a high stromal density of CD68 cells was a significant negative predictor of OS (p = 0.008). CONCLUSIONS: The observed associations of CD68 cells infiltration with progression and prognosis in patients with LSCC provide potential screening and therapeutic opportunities for patients with unfavourable outcomes.

Unlocking the potential of HHLA2: identifying functional immune infiltrating cells in the tumor microenvironment and predicting clinical outcomes in laryngeal squamous cell carcinoma.

BACKGROUND: HHLA2 (human endogenous retrovirus-H long terminal repeat-associating protein 2) represents a recently identified member of the B7 immune checkpoint family, characterized by limited expression in normal tissues but notable overexpression in various cancer types. Nevertheless, the precise function and interaction with immune cells remain poorly understood, particularly in laryngeal squamous cell carcinoma (LSCC). This investigation endeavored to elucidate the biological significance of HHLA2 within the tumor microenvironment of human LSCC tissues and delineate the clinical relevance and functional roles of HHLA2 in LSCC pathogenesis. METHODS: Through multiplexed immunohistochemistry analyses conducted on tissue microarrays sourced from LSCC patients (n = 72), the analysis was executed to assess the expression levels of HHLA2, density and spatial patterns of CD68+HLA-DR+CD163- (M1 macrophages), CTLA-4+CD4+FoxP3+ (CTLA-4+Treg cells), CTLA-4+CD4+FoxP3- (CTLA-4+Tcon cells), exhausted CD8+T cells, and terminally exhausted CD8+T cells in LSCC tissues. Survival analysis was conducted to evaluate the prognostic significance of HHLA2 and these immune checkpoints or immune cell populations, employing COX regression analysis to identify independent prognostic factors. RESULTS: Kaplan-Meier (K-M) survival curves revealed a significant association between HHLA2 expression and overall survival (OS) in LSCC. Elevated levels of HHLA2 were linked to reduced patient survival, indicating its potential as a prognostic marker (HR: 3.230, 95%CI 0.9205-11.34, P = 0.0067). Notably, increased infiltration of CD68+ cells (total macrophages), STING+CD68+HLA-DR+CD163- (STING+M1 macrophages), CTLA-4+CD4+FoxP3+, CTLA-4+CD4+FoxP3-, PD-1+LAG-3+CD8+T cells, and PD-1+LAG-3+TIM-3+CD8+T cells strongly linked to poorer survival outcomes (P < 0.05). A discernible trend was observed between the levels of these immune cell populations, STING+CD68+ (STING+ total macrophages), CD68+HLA-DR+CD163-, STING+CD68+CD163+HLA-DR- (STING+M2 macrophages), PD-1+LAG-3-CD8+T cells, PD-1+TIM-3+CD8+T cells, and PD-1+LAG-3+TIM-3-CD8+T cells and prognosis. Importantly, multivariate COX analysis identified HHLA2 as an independent predictive factor for OS in LSCC patients (HR = 3.86, 95% CI 1.08-13.80, P = 0.038). This underscored the potential of HHLA2 as a critical marker for predicting patient outcomes in LSCC. CONCLUSIONS: HHLA2 emerged as a detrimental prognostic biomarker for assessing OS in LSCC patients. Relative to other immune checkpoints, HHLA2 exhibited heightened predictive efficacy for the prognosis of LSCC patients.

M2 Macrophage exosomal HOXC13-AS in laryngeal cancer immunity via targeting miR-485-5p/IGF2BP2/PD-L1.

This study investigates the role of M2-exo-mediated HOXC13-AS in laryngeal squamous cell carcinoma (LSCC) by examining its transmission to tumor microenvironment (TME) macrophages. Exosomes from M2 macrophages were isolated and characterized using transmission electron microscopy, nanoparticle tracer analysis and western blot. Expression of HOXC13-AS, miR-485-5p, IGF2BP2, and PD-L1 was analyzed. Different interventions on LSCC cell function and immune escape were detected using molecular biological techniques. The study found that elevated HOXC13-AS were present in LSCC, and M2-exo expression was significantly increased in LSCC cells. Silencing HOXC13-AS in M2-exo inhibited LSCC malignant progression and immune escape in vivo and in vitro. M2-exo-mediated HOXC13-AS also regulated IGF2BP2 expression, impacting cellular biological function and immune escape process. The study concludes that M2-exo-mediated HOXC13-AS promotes LSCC malignancy and immune escape.

Cancer and immune response: The role of PD-1/PD-L1 checkpoint in laryngeal carcinoma. Preliminary results.

PURPOSE: The overall survival of laryngeal squamous cell carcinoma (LSCC) hasn't changed significantly in the last decades, leading to a negative prognosis in advanced stages. So, the immunotherapy takes space with the inhibition of PD-1/PD-L1 checkpoint, involved in suppression of immune response. METHODS: A prospective study was conducted on LSCC patients, selected according to strict criteria. The study was approved by the ethics committee of our Hospital. Parameters were: sex, age, smoking and alcohol habits, C-reactive protein (CRP) level in the serum, laryngeal subsite involved, differentiation-based histopathologic grading of tumor, neck node involvement, tumor stage, expression levels of PD-L1 (as Combined Positive Score - CPS). P-value < 0.05 was statistically significant. RESULTS: 58 patients were included: 31.03% were females, mean age 63.55±10.09. A statistically significant correlation between CPS and smoking habits and N stage was found. CRP resulted increased in 44.83% of patients with a statistically significant correlation with CPS. The most cases were glottic cancers (46.55%). 75.86% of tumors were moderately-differentiated, without correlation with CPS. CONCLUSION: PD-L1 expression levels are variables independent of sex, age, alcohol consumption, differentiation degree of LSCC. A statistically significant correlation between PD-L1 expression levels and smoking habits, neck node status and CRP was found. This last finding demonstrates the involvement of PD-1/PD-L1 checkpoint in immune response in case of cancer. However, these results need further studies to detect the best patients tailored for treatment with anti-PD-1/PD-L1 blocking antibodies.

The impact of the tumor immune microenvironment and tumor-infiltrating lymphocyte subgroups on laryngeal cancer prognosis.

The absence of improvement in survival rates across various cancers, including laryngeal cancer, has led to an increasing interest in understanding the immune response to cancer. In head and neck cancers, immune modulatory mechanisms such as immune microenvironment and immune infiltration are important in cancer pathogenesis. This study aims to explore the distribution of tumor-infiltrating lymphocyte (TIL) subgroups in the immune microenvironment and evaluate their impact on tumor histopathological characteristics and prognosis. The study included 50 patients who underwent laryngectomy for laryngeal squamous cell carcinoma, in Istanbul University - Cerrahpasa, Faculty of Medicine Department of Otorhinolaryngology, between January 2016 and January 2018. Pathology specimens were evaluated using immunohistochemistry to assess the expressions of the CD3, CD20, CD8, CD4, CD25, and FoxP3 markers, identifying subgroups of TILs. The investigation aimed to uncover how these subgroups influence tumor histopathological features and survival outcomes. The high infiltration of CD3, CD20, and CD4 had a positive impact on disease-specific survival, disease-free survival, and recurrence-free survival. In addition, overall survival was positively affected by high CD3 and CD4 infiltrations. However, no significant relationship was observed between the expressions of CD8, FoxP3, and CD25 and any of the survival parameters. The infiltration of CD3, CD20, and CD4 positive cells indicative of a robust antitumoral immune response-emerged as favorable prognostic factors in laryngeal cancer. These findings suggest that enhancing the infiltration of CD3, CD20, and CD4 lymphocytes could be a therapeutic strategy worth exploring in clinical trials.

Analysis of Th1/Th2 cytokine profile and clinical characteristics of patients with head and neck squamous cell carcinoma.

Immune system biomarkers in cancer pathogenesis offer new therapeutic avenues, as seen in cytokine (CK) profiles of laryngeal and hypopharyngeal tumors. A retrospective analysis was conducted on 58 patients with laryngeal and hypopharyngeal tumors and 27 patients with benign vocal cord lesions to explore the role of serum CKs in these diseases' characteristics and immunotherapy. The differences in the levels of 12 cytokines were measured. Additionally, the study examined the correlation between T helper cells (Th)1/Th2 cytokine levels and the clinical characteristics and immunotherapy efficacy of laryngeal and hypopharyngeal cancers. The results show that the balance of Th1/Th2 is biased towards Th2 in patients with laryngeal and hypopharyngeal tumors. Among these, interleukin (IL)-6 (P = 0.021) was highly expressed in laryngeal tumors, and the expression levels of IL-1ß (P = 0.008), IL-6 (P = 0.005), and IL-8 (P = 0.05) were higher in patients with poorly differentiated laryngeal tumors. The level of IL-4 (P = 0.0048) was significantly correlated with tumor location. The expression levels of IL-2 (P = 0.010), IL-4 (P = 0.028), IL-10 (P = 0.011), IL-12p70 (P = 0.034), IL-17 (P = 0.024), tumor necrosis factor (TNF)-α (P = 0.003), and interferon (IFN)-γ (P = 0.007) were related to lymph node metastasis. The level of IFN-γ (P = 0.016) was correlated with the efficacy of neoadjuvant therapy, while the level of IFN-α (P = 0.013) was significantly correlated with programmed death ligand 1 (PD-L1) expression. The Principal Component Analysis (PCA) results showed that patients with tumors, poor differentiation, and lymph node metastasis had higher levels of Th1 and Th2 cytokine separation. In conclusion, the shift in the balance of Th1 and Th2 cytokine expression indicates higher tumor invasiveness, and IFN has potential as a circulating molecular marker for immunotherapy of head and neck squamous cell carcinoma.

Exploring the protective association between COVID-19 infection and laryngeal cancer: insights from a Mendelian randomization study.

Introduction: Viral infections have been implicated as a risk factor for laryngeal cancer. Given the possible effects of Corona virus disease 2019 (COVID-19) on the laryngeal tissue, we investigated the causal link between COVID-19 and laryngeal cancer using a two-sample Mendelian randomization (MR) approach. Methods: We utilized genetic data from the 5th Genome-wide association studies (GWAS) edition of the COVID-19 Host Genetics Initiative (published on January 18, 2021) and a large-scale laryngeal cancer GWAS comprising 180 cases and 218,612 controls of European ancestry. We applied inverse variance weighting, MR Egger, and weighted median methods to infer causality. We performed sensitivity analysis using the "leave-one-out" method to verify robustness. Results: We found no evidence of a causal association between gene-predicted COVID-19 and laryngeal cancer [Odds ratio (OR)=0.24 (95% Confidence intervals (CI), 0.05-1.26), P=0.09]. However, we observed significant inverse associations between gene-predicted COVID-19 hospitalization [OR=0.51 (95% CI, 0.28-0.95), P=0.03] and severe patients [OR=0.62 (95% CI, 0.43-0.90), P=0.01] and laryngeal cancer. Notably, the study detected important genetic variants, such as rs13050728, that modulate the expression of interferon alpha receptor 2 (IFNAR2), indicating possible roles for immune response pathways in both COVID-19 and cancer. Discussion: This study reveals a potential interaction between COVID-19 severity, genetic factors, and laryngeal cancer, underscoring the importance of investigating the immune response mechanisms in both conditions. These findings contribute to the understanding of the complex interactions between COVID-19 and laryngeal cancer and may guide future research on the role of immune response, particularly involving IFNAR2.

Expression and clinical significance of NKG2A and HLA-E in advanced laryngeal carcinoma.

OBJECTIVES: The purpose was to detected features of the expression levels of NKG2A and its ligand HLA-E, a new member of the immune checkpoints, in advanced laryngeal carcinoma and their clinicopathologic significance. MATERIAL AND METHODS: We analyzed the expression levels of HLA-E and NKG2A in multiple types of tumors utilizing the Tumor Immune Estimation Resource (TIMER) database and immunohistochemistry and qRT-PCR analysis of paraffin embedded tissue samples to reveal the correlations of the clinicopathological factors with the expression of these two proteins in advanced laryngeal carcinoma as well as their prognostic significance. RESULTS: KLRC1 (the coding gene of NKG2A) and HLA-E are substantially overexpressed in various human cancers than normal tissues. HNSCC is also included. KLRC1 is differentially expressed in different HPV subgroups of patients, with higher expression in the HPV-positive group. Consistent with this, immunohistochemical results also revealed the high expression of these two proteins in tumor tissue. In addition, immunohistochemical staining also displayed a preference for the distribution of NKG2A-positive cells in tumor tissue. Clinicopathological analyses also displayed that the density of NKG2A-positive cells of the HPV-positive group infiltrating laryngeal carcinoma tissue was larger than that in the HPV-negative group. Prognostic analyses indicated that the expression of this immune checkpoint does not affect the overall survival length of patients, but the highly expressed HLA-E is significantly correlated with local recurrence in the patients. CONCLUSIONS: The findings suggest that the expression levels of HLA-E and NKG2A is upregulated in advanced laryngeal carcinoma. The NKG2A-positive cells infiltrating the tumor are mainly distributed in the cancer nest, while infiltrating cell number may be regulated by HPV. The highly expressed HLA-E may promote local recurrence in patients with advanced laryngeal carcinoma.

Identification of Regulatory RNA-Binding Proteins Associated with Immune Infiltration in Laryngeal Squamous Cell Carcinoma.

We analyzed bulk RNA sequencing and single-cell RNA sequencing (scRNA-seq) data to identify alternative splicing (AS) events and regulatory RNA-binding proteins (RBPs) associated with immune infiltration in human laryngeal squamous cell carcinoma (LSCC). Whole-transcriptome sequencing data of 20 human laryngeal cancer and paracancerous tissues were downloaded from the Gene Expression Omnibus public database, using newly published splicing-site usage variation analysis software to obtain highly conserved regulated AS (RAS) events, and scientific reverse convolution algorithm analysis was used to identify significantly different immune cells and perform a correlation analysis between the two. The software package edgeR was used to identify differentially expressed RBPs and the immune infiltration-related LSCC-RAS they may regulate. Finally, we present the expression profiles and survival curves of 117 human laryngeal cancer samples from The Cancer Genome Atlas dataset for the identified RBPs and LSCC-RAS. We also downloaded the gene set enrichment 150321 scRNA-seq data for two human LSCC tissue samples. The RBP expression pattern and the expression of prophase RBP genes were analyzed in different LSCC cell populations. RNA-binding motif protein 47 (RBM47) and filamin A, as well as the RBP-RAS events that were screened in both the fibulin 2 and fibronectin 1 genes, were all significantly associated with the prognosis, and the RBM47 gene was upregulated in myeloid cells. Because the prognosis was significantly associated with two RBP regulators and two LSCC-RAS events, they may be critical regulators of immune cell survival during laryngeal cancer progression, and RBM47 may regulate macrophage-associated AS and affect immunity.

Comprehensive analysis of the immune implication of EPHX4 gene in laryngeal squamous cell carcinoma.

OBJECTIVES: The role of Epoxide Hydrolase-4 (EPHX4), a member of epoxide hydrolase family, has not been investigated in cancer. The purpose of this article is to explore the application value of EPHX4 in laryngeal cancer and its relationship with immune infiltration. METHODS: We observed that EPHX4 expression and its survival assays in laryngeal cancer specimens based on The Cancer Genome Atlas (TCGA) cohorts. We also analyzed the correlation between immune cell infiltration levels and EPHX4 gene copy number in laryngeal cancer. Finally, we conducted in vitro assay to evaluate the functions of EPHX4 in laryngeal cancer cell line. RESULTS: EPHX4 is highly expressed in laryngeal cancer specimens and has a poor prognosis. EPHX4 related immune cell analysis showed that it participated in NK Natural killer cell mediated cytotoxicity. Finally, Cell experiments indicate that EPHX4 could promote laryngeal cancer cell line proliferation, colony formation and invasion. CONCLUSIONS: Our research results suggest that EPHX4 may be a potential immunotherapy target for laryngeal cancer. The nominated immune signature is a helpful and promising prognostic indicator in laryngeal cancer. LEVELS OF EVIDENCE: Level 3.

Quantification and Functional Studies of Neutrophilic Cells Identifies Distinct Papilloma Phenotypes.

OBJECTIVES: To characterize the distribution of immune cell subsets within laryngeal papillomas and to study the function of potentially immunosuppressive neutrophilic and regulatory T cells (Tregs). METHODS: Fresh clinical papilloma specimens were collected at the time of surgery and studied with multiparameter flow cytometry. Papilloma infiltrating neutrophilic cells and Tregs were sorted and studied functionally with ex vivo T cell suppression assays. RESULTS: Flow cytometric analysis of fresh laryngeal papillomas samples from 18 adult patients with recurrent respiratory papillomatosis revealed patterns in immune constituency between patients. Clearly divergent phenotypes based primarily on the degree of neutrophilic and T cell infiltration were identified. Relative neutrophilic cell enrichment and T cell depletion were observed in 50% of samples and neutrophilic cell depletion and T cell enrichment were observed in the others. Greater papilloma neutrophilic cell enrichment was positively associated with the number of clinically indicated interventions required in the 12 months prior to sample collection, linking papilloma neutrophil inflammation to disease severity. Functional assays revealed the ability of both papilloma infiltrating neutrophilic and Tregs to suppress T cell function at roughly equal magnitudes, but substantially increased infiltration of neutrophilic cells compared to Tregs across samples. CONCLUSION: Neutrophilic cells are an important contributor to immunosuppression within the respiratory papilloma microenvironment. Given these data and the association between greater neutrophilic cell infiltration and lack of clinical response to therapeutic vaccination, additional study of strategies aimed at limiting neutrophilic cell infiltration or function within papillomas is warranted. LEVEL OF EVIDENCE: 4 Laryngoscope, 134:3238-3244, 2024.

ANXA3-Rich Exosomes Derived from Tumor-Associated Macrophages Regulate Ferroptosis and Lymphatic Metastasis of Laryngeal Squamous Cell Carcinoma.

Tumor-associated macrophages (TAM) induce immunosuppression in laryngeal squamous cell carcinoma (LSCC). The interaction between LSCC cells and TAMs affects the progression of laryngeal cancer through exosomes, but the underlying molecular mechanism remains unclear. Proteomics analysis of TAMs isolated from human laryngeal tumor tissues obtained from patients with confirmed lymphatic metastasis revealed an upregulation of annexin A3 (ANXA3). In TAMs, ANXA3 promoted macrophages to polarize to an M2-like phenotype by activating the AKT-GSK3ß-ß-catenin pathway. In addition, ANXA3-rich exosomes derived from TAMs inhibited ferroptosis in laryngeal cancer cells through an ATF2-CHAC1 axis, and this process was associated with lymphatic metastasis. Mechanistically, ANXA3 in exosomes inhibited the ubiquitination of ATF2, whereas ATF2 acted as a transcription factor to regulate the expression of CHAC1, thus inhibiting ferroptosis in LSCC cells. These data indicate that abnormal ANXA3 expression can drive TAM reprogramming and promote an immunosuppressive microenvironment in LSCC. Meanwhile, ANXA3-rich exosomes inhibit ferroptosis of LSCC cells and promote lymphatic metastasis, thus promoting tumor progression.

Autoantibodies in laryngeal cancer: detection and role as a biomarker.

OBJECTIVE: Diagnostic role of autoantibodies (AAb) as serological biomarkers has not been specifically investigated in laryngeal cancer (LC) previously. The study investigates the presence of anti-LC AAbs and their potential as a biomarker for early diagnosis of LC, to improve patient outcome. METHOD: Anti-LC AAb levels were investigated in LC patients (n = 30) and healthy individuals (n = 30) by indirect enzyme-linked immunosorbent assay (ELISA). Patient AAb levels were analyzed with various clinical factors, primarily tumor stage. RESULTS: AAb levels were significantly higher in LC patients than in the control group (P = .019). The diagnostic performance of AAb-level testing for LC detection presented a sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of 70% each. The positive likelihood (LR+) and negative likelihood (LR-) ratios were 2.33 and 0.43, respectively. AAb levels were independent of cancer stage (P = .708), duration since first appearance of symptoms (P = .228), duration of medical attention (P = .231), and degree of risk-factor exposure (P = .478). CONCLUSION: Significant level of AAbs could be detected among LC patients with good diagnostic performance, irrespective of stage. Thus, anti-LC AAbs reflect potential to be utilized as predictive biomarkers in early diagnostics of LC.

Long Noncoding RNA Hotair Promotes the Progression and Immune Escape in Laryngeal Squamous Cell Carcinoma through MicroRNA-30a/GRP78/PD-L1 Axis.

Homeobox (HOX) transcript antisense RNA (Hotair) is elevated in many cancers significantly. However, the oncogenic role of Hotair in human laryngeal squamous cell carcinoma (LSCC) is still unknown. Thus, we explored the expression profile of Hotair and its function in LSCC. We observed high expression levels of Hotair in six LSCC cell lines compared to the human nasopharyngeal epithelial cell line. Knockdown of Hotair inhibited proliferation and enhanced apoptosis of Tu212 and Hep-2 cell lines in vitro. Moreover, the overexpression of hsa-miR-30a-5p inhibited the expression of GRP78 and PD-L1, but Hotair overexpression in LSCC cells rescues both proteins. Furthermore, the impacts of hsa-miR-30a-5p upregulation on the apoptosis and proliferation of LSCC cells were rescued by overexpression of Hotair. Finally, we combined si-Hotair and a VEGF inhibitor to treat LSCC cells in vitro or in vivo and surprisingly observed a significant inhibition of LSCC growth. In summary, these results indicate that Hotair displays an oncogenic role in both malignancy and immune escape in LSCC related to hsa-miR-30a-5p/GRP78/PD-L1 signaling. Therefore, Hotair may be a potential target for treating LSCC.

Development of Antibodies against HPV-6 and HPV-11 for the Study of Laryngeal Papilloma.

Laryngeal papilloma (LP), which is associated with infection by human papillomavirus (HPV)-6 or -11, displays aggressive growth. The precise molecular mechanism underlying the tumorigenesis of LP has yet to be uncovered. Building on our earlier research into HPV-6, in this study, the viral gene expression of HPV-11 was investigated by quantitative PCR and DNA/RNA in situ hybridization. Additionally, newly developed antibodies against the E4 protein of HPV-6 and HPV-11 were evaluated by immunohistochemistry. The average viral load of HPV-11 in LP was 1.95 ± 0.66 × 105 copies/ng DNA, and 88% of HPV mRNA expression was found to be E4, E5a, and E5b mRNAs. According to RNA in situ hybridization, E4 and E5b mRNAs were expressed from the middle to upper part of the epithelium. E4 immunohistochemistry revealed a wide positive reaction in the upper cell layer in line with E4 mRNA expression. Other head and neck lesions with HPV-11 infection also showed a positive reaction in E4 immunohistochemistry. The distribution pattern of HPV DNA, viral mRNA, and E4 protein in LP with HPV-11 infection was quite similar to that of HPV-6. Therefore, it might be possible to apply these E4-specific antibodies in other functional studies as well as clinical applications, including targeted molecular therapies in patients with HPV-6 and HPV-11 infection.

Infiltration of CD1a-positive dendritic cells in advanced laryngeal cancer correlates with unfavorable outcomes post-laryngectomy.

BACKGROUND: The prognosis of advanced laryngeal cancer is unfavorable despite advances in multidisciplinary therapy. Dendritic cells (DCs) play a central role in antitumor immunity. Tumor-infiltrating CD1a+ DCs have been reported to be associated with clinical outcomes in carcinomas of various organs, but the clinical impact of CD1a+ DCs in laryngeal cancer remains to be unequivocally established. METHODS: We retrospectively analyzed the cases of 57 patients with Stage III or IV laryngeal cancer who underwent a total laryngectomy. Immunohistochemistry detection of CD1a, S100 and CD8 was performed on representative resected specimens. CD1a+ DCs, S100+ DCs and CD8+ cytotoxic T-lymphocytes (CTLs) were evaluated, and the cases divided into high and low groups according to the cut-off of the median values for each of these 3 parameters. RESULTS: Compared to the CD1a-low group, the CD1a-high group had more advanced cases and showed significantly worse disease-specific survival (DSS) (P = 0.008) and overall survival (OS) (P = 0.032). The analyses of S100 DCs and CD8+ CTLs revealed no significant impact on clinical outcomes. However, multivariate analysis revealed that infiltration of CD1a+ DCs was an independent unfavorable prognostic factor for both DSS (P = 0.009) and OS (P = 0.013). CONCLUSIONS: Our results demonstrated that the infiltration of CD1a+ DCs was associated with unfavorable clinical outcomes in patients with advanced laryngeal cancer who underwent a total laryngectomy as the initial treatment.

APE1 facilitates PD-L1-mediated progression of laryngeal and hypopharyngeal squamous cell carcinoma.

Laryngeal squamous cell carcinoma (LSCC) and hypopharyngeal squamous cell carcinoma (HSCC) seriously affect the life quality of patients. Nowadays, immunotherapy is widely used in the treatment of cancer. Tumor-infiltrating lymphocytes (TILs), programmed cell death 1 (PD-1) and its ligand programmed cell death ligand 1 (PD-L1) play key roles in the immunotherapy of cancer. Moreover, study has reported that the upregulation of PD-L1 and apurinic/apyrimidinic endonuclase 1 (APE1) are associated with tumorigenesis and poor prognosis of gastric cancer. In the present study, the number of CD3+ T lymphocytes and the expressions of PD-1 and PD-L1 in LSCC and HSCC were detected in clinical samples. In addition, the expressions of PD-L1 and APE1 and their correlation were explored. The results showed that PD-1+ T lymphocytes were wildly infiltrated and PD-L1 was overexpressed in LSCC and HSCC tissues. PD-1 had a positive correlation with cancer progression, and glottic and subglottic LSCC tissues might have a more active immune microenvironment. Moreover, the results showed that upregulated co-expression of PD-L1 and APE1 was a biomarker of LSCC, and APE1 could regulate the expression of PD-L1 through NF-κB signaling pathway. In conclusion, the combine detection of the expressions of PD-1, PD-L1 and APE1 will provide predictive value for the treatment of LSCC and HSCC via immune checkpoint inhibitors, which will help us to identify the patient population more likely to benefit from the immune checkpoint inhibitors based on the tumor immune microenvironment.

Anti-CD47 antibody synergizes with cisplatin against laryngeal cancer by enhancing phagocytic ability of macrophages.

Cisplatin is mainly used in late-stage or recurrent laryngeal cancer patients. However, the effect of the chemotherapy is limited due to cisplatin resistance. Therefore, we explored the synergized role of immunosuppressive mediator with cisplatin in laryngeal cancer. Cancer cells isolated from tissues of patients with laryngeal cancer were treated with cisplatin to screen the potential immunosuppressive mediator, whose synergized effects with cisplatin were explored both in vivo and in vitro. CD47 was selected for its high expression in cisplatin-treated laryngeal cancer cells. Blocking CD47 expression using its neutralizing antibody (aCD47) synergized with cisplatin to increase macrophage phagocytosis in a co-culture system of human epithelial type 2 (Hep-2) cancer cells with tumor-associated macrophages (TAMs). Moreover, aCD47 together with cisplatin prevented tumor growth by inhibiting proliferation of cancer cells and the secretion of proinflammatory cytokines, as well as by inducing the apoptosis of cancer cells and phagocytosis of TAMs in a Hep-2-implanted mouse tumor model. aCD47 synergized with cisplatin against laryngeal cancer by enhancing the phagocytic ability of TAMs, and the combined therapy of cisplatin and aCD47 might serve as a novel therapeutic strategy against laryngeal cancer.

High endothelial venules are present in pharyngeal and laryngeal carcinomas and they are associated with better prognosis.

BACKGROUND: Tumors lymphocytic infiltration has prognostic and predictive value. However, the mechanisms involved in lymphocyte recruitment remain poorly characterized. High endothelial venules (HEV) are blood vessels specialized in lymphocyte recruitment, recently showing prognostic significance in some types of cancer. Their implications in laryngeal or pharyngeal cancer is largely unknown. AIM OF THE STUDY: To investigate the possible presence of HEVs in head and neck cancer. MATERIAL AND METHODS: Oropharyngeal (n = 61), hypopharyngeal (n = 53) and laryngeal (n = 21) squamous cell carcinomas were immunohistochemically studied with the MECA-79 antibody, which specifically recognizes HEVs. Histological and clinical factors were correlated with HEVs' presence. RESULTS: HEVs were present in 34% of tumors, showing significant correlations with oropharyngeal localization, higher lymphocytic response, lower tumor budding, lower T status, absence of distant metastases and better overall and progression-free survival. CONCLUSION: HEVs represent an important prognostic factor in head and neck cancer.