Human Papillomavirus Negative Head And Neck Squamous Cell Carcinoma Research Articles

Clinical implications of activation of the LIMD1-VHL-HIF1α pathway during head-&-neck squamous cell carcinoma development.

Background & objectives Given the importance of the role of hypoxia induced pathway in different cancers including head-and-neck squamous cell carcinoma (HNSCC), this study delved into elucidating the molecular mechanism of hypoxia-inducible factor-1α (HIF1α) activation in HNSCC. Additionally, it analyzes the alterations of its regulatory genes [von Hippel-Lindau (VHL) and LIM domain containing 1 (LIMD1)] and target gene vascular endothelial growth factor (VEGF) in head-and-neck lesions at different clinical stages in relation with human papillomavirus (HPV) infection. Methods Global mRNA expression profiles of HIF1α, VHL, LIMD1 and VEGF were evaluated from public datasets of HNSCC, followed by validation of their expression (mRNA/protein) in an independent set of HPV+ve/-ve HNSCC samples of different clinical stages. Results A diverse expression pattern of the HIF1α pathway genes was observed, irrespective of HPV infection, in the datasets. In validation in an independent set of HNSCC samples, high mRNA expressions of HIF1α/VEGF were observed particularly in HPV positive samples. However, VHL/LIMD1 mRNA expression was low in tumours regardless of HPV infection status. In immunohistochemical analysis, high/medium (H/M) expression of HIF1α/VEGF was observed in basal/parabasal layers of normal epithelium, with significantly higher expression in tumours, especially in HPV-positive samples. Conversely, high cytoplasmic VHL expression in these layers gradually decreased with the progression of HNSCC, regardless of HPV infection. A similar trend was noted in LIMD1 expression (nuclear/cytoplasmic) during the disease development. The methylation pattern of VHL and LIMD1 promoters in the basal/parabasal layers of normal epithelium correlated with their expression, exhibiting a gradual increase with the progression of HNSCC. The H/M expression of HIF1α/VEGF proteins and reduced VHL expression was associated with poor clinical outcomes. Interpretation & conclusions The results of this study showed differential regulation of the LIMD1-VHL-HIF1α pathway in HPV positive and negative HNSCC samples, illustrating the molecular distinctiveness of these two groups.

Personalized ctDNA for Monitoring Disease Status in Head and Neck Squamous Cell Carcinoma.

Many patients with locoregionally advanced human papillomavirus-negative head and neck squamous cell carcinoma (HNSCC) relapse. ctDNA has the potential to identify minimal residual disease, but its clinical utility for virus-negative HNSCC is not well understood. We retrospectively evaluated a personalized, commercial ctDNA assay (Signatera, Natera) during clinical care of patients treated for predominantly newly diagnosed human papillomavirus-negative HNSCC. Signatera utilizes 16-plex PCR from matched tumor and blood. Objectives were to understand ctDNA detectability and correlate changes posttreatment with disease outcomes. Testing was successful in 100/116 (86%) patients (median age: 65 years, 68% male, 65% smokers); testing failed in 16 (14%) because of insufficient tissue. Oral cavity (55, 47%) tumors were most common; most had stage III to IV disease (82, 71%), whereas 17 (15%) had distant metastases. Pretreatment, 75/100 patients with successful testing (75%) had detectable ctDNA (range: 0.03-4049.69 mean tumor molecules/mL). No clinical features predicted ctDNA detectability or levels (multivariate analysis). At a median follow-up of 5.1 months (range: 0.2-15.1), 55 (55%) had >1 test result (range: 1-7; 194 samples). Of 55 patients, 17 (31%) remained ctDNA positive after starting treatment. Progression-free survival was significantly worse for patients who were ctDNA positive versus ctDNA negative posttreatment (HR, 7.33; 95% confidence interval, 3.12-17.2; P < 0.001); 1-year overall survival was 89.1% versus 100%, respectively (HR, 7.46; 95% confidence interval, 0.46-119.5; P = 0.155). Tumor-informed ctDNA testing is feasible in nonviral HNSCC. ctDNA positivity is an indicator of disease progression and associated with inferior survival. Further research is warranted to understand whether ctDNA may be leveraged to guide therapy in HNSCC.

LncRNA SELL/L-selectin promotes HPV-positive HNSCC progression and drives fucoidan-mediated therapeutic strategies

Positive human papillomavirus (HPV+) head and neck squamous cell carcinoma (HNSCC) presents a higher risk of lymph node metastasis and poor prognosis. Here, advanced microarray analysis of clinically collected HNSCC tissues revealed significant upregulation of the lncRNA SELL in HPV+ HNSCC, and its overexpression was obviously associated with lymph node metastasis. The lncRNA SELL could function as a promigratory and proinvasive mediator as well as an inducer of M1-like tumour-associated macrophages (TAM) by increasing the level of L-selectin. Furthermore, fucoidan, as an L-selectin inhibitor, obviously weakened the formation of tongue lesions induced by 4-Nitroquinoline N-oxide (4-NQO) in HPV16 E6/E7 transgenic mice. This result drove us to synchronously develop a nanodelivery platform to verify fucoidan-mediated anti-growth and anti-metastasis effects. This work highlighted the important influence of the lncRNA SELL/L-selectin on promoting HPV+ HNSCC progression and proposed a potential fucoidan-mediated therapeutic strategy. STATEMENT OF SIGNIFICANCE: Head and neck squamous cell carcinoma (HNSCC) patients with human papillomavirus (HPV) involvement present a greater risk of lymph node metastasis than HPV negative HNSCC patients. However, treatment protocols, including surgery and platinum-based chemo- and radiotherapy, have not improved the 5-year overall survival due to the high tendency of lymphatic metastasis. Here, microarray of clinical HNSCC samples confirms the oncogenic significance of lncRNA SELL, which acts as an M1-like TAM inducer and promotes tumorigenesis by upregulating L-selectin. Fucoidan, as an L-selectin inhibitor, suppresses tongue lesions in transgenic mice, and a fucoidan-mediated nanodelivery platform inhibits HPV+ HNSCC growth. The present study highlights lncRNA SELL/L-selectin on promoting HPV+ HNSCC progression and proposes a potential fucoidan-mediated therapeutic.

Identification and validation of a prognostic signature of autophagy, apoptosis and pyroptosis-related genes for head and neck squamous cell carcinoma: to imply therapeutic choices of HPV negative patients.

An effective tool is needed to predict the prognosis of head and neck squamous cell carcinoma (HNSCC). Human papillomavirus (HPV) positive HNSCC patients generally have a favorable survival and a promising responsiveness to radiotherapy, chemoradiotherapy and checkpoint blockades. However, HPV negative patients, the majority of HNSCC patients, have been largely overlooked. Cell death has been involved in the therapeutic resistance of cancers. To this end, we aimed to identify the association of autophagy, apoptosis and pyroptosis-related genes with the prognosis of HNSCC, and construct a prognostic signature to predict the prognosis for HNSCC, especially for HPV negative HNSCC. Autophagy and apoptosis-related genes were obtained from Gene Set Enrichment Analysis (GSEA) website, and pyroptosis-related genes were obtained from GSEA and Gene Ontology (GO) database. We established the cell death index (CDI) based on RNA sequencing (RNA-seq) data and clinicopathological information from The Cancer Genome Atlas (TCGA) dataset. The prognostic value of CDI was verified by Kaplan-Meier, receiver operating characteristic (ROC) and univariate and multivariate Cox regression analyses in TCGA dataset, and validated with the datasets from Gene Expression Omnibus (GEO) and Qilu Hospital of Shandong University. We further assessed the immune microenvironment of patients with high and low CDI scores. Moreover, the expression of the signature genes in HNSCC cell lines were explored. We found that CDI was an independent prognostic indicator for overall survival (hazard ratio 3.80, 95% confidential interval: 2.70-5.40, P < 0.001). Furthermore, HNSCC patients with high CDI scores obtained increased overall survival post radiation indicating benefits from radiotherapy of this subgroup. On the other hand, HPV negative HNSCC patients with low CDI exhibited increased checkpoint gene expressions, an inflamed tumor microenvironment and an enriched immune response-related functions, suggesting the potential benefits from checkpoint immunotherapies of this subgroup. Moreover, we validated the baseline and induced expressions of above 16 genes in two HPV negative HNSCC cell lines, CAL27 and SCC-15. We established a prognostic signature and emphasized its implements in the therapeutic choices of HPV negative HNSCC patients, the majority and the poor outcome population of HNSCC.

HRAS Mutations Define a Distinct Subgroup in Head and Neck Squamous Cell Carcinoma

In head and neck squamous cell carcinoma (HNSCC), HRAS mutation is a new actionable oncogene driver. We aimed to evaluate HRAS mutational variants, comutation profile, and survival outcomes of this molecularly defined population. We leveraged four deidentified patient data sets with HRAS-mutant HNSCC, MD Anderson Cancer Center, Kura Oncology, Inc trial, Foundation Medicine, and American Association for Cancer Research GENIE v.12. Patient demographic information and clinical courses were extracted, when available, in addition to HRAS mutation type and co-occurring mutations. Survival outcomes were analyzed (Kaplan-Meier method). Two hundred forty-nine patients with HRAS-mutant HNSCC were identified from the four data sets. Median age ranged from 55 to 65 years, with a higher frequency in male patients (64%); the majority of HRAS-mutant HNSCC occurred in human papillomavirus-negative HNSCC. HRAS mutation patterns were similar across data sets; G12S was the most common (29%). Treatment responses to tipifarnib were not codon-specific. Compared with wild-type, significantly co-occurring mutations with HRAS were Casp8 (Fisher's exact test, P < .00013), TERT (P < .0085), and NOTCH1 (P < .00013). Analysis of clinical courses from the MD Anderson Cancer Center and Kura Oncology, Inc data sets demonstrated poor clinical outcomes with a high rate of recurrence following primary definitive treatment (50%-67% relapse < 6 months) and short disease-free survival (4.0 months; 95% CI, 1.0 to 36.0) and overall survival (OS; 15.0 months; 95% CI, 6.0 to 52.0). Use of tipifarnib in this data set demonstrated improved OS (25.5 months; 95% CI, 18.0 to 48.0). Oncogenic mutations in HRAS occur in 3%-4% of HNSCC, with G12S being the most frequent. Without targeted therapy, patients with HRAS-mutant HNSCC had poor clinic outcomes; observable trend toward improvement in OS has been noted in cohorts receiving treatments such as tipifarnib. The comutation pattern of HRAS-mutant in HNSCC is distinct, which may provide insight to future therapeutic combination strategies.

Doublecortin-like kinase 1 is a therapeutic target in squamous cell carcinoma.

Doublecortin like kinase 1 (DCLK1) plays a crucial role in several cancers including colon and pancreatic adenocarcinomas. However, its role in squamous cell carcinoma (SCC) remains unknown. To this end, we examined DCLK1 expression in head and neck SCC(HNSCC) and anal SCC(ASCC). We found that DCLK1 is elevated in patient SCC tissue, which correlated with cancer progression and poorer overall survival. Furthermore, DCLK1 expression is significantly elevated in human papilloma virusnegative HNSCC, which are typically aggressive with poor responses to therapy. To understand the role of DCLK1 in tumorigenesis, we used specific shRNA to suppress DCLK1 expression. This significantly reduced tumor growth, spheroid formation, and migration of HNSCC cancer cells. To further the translational relevance of our studies, we sought to identify a selective DCLK1 inhibitor. Current attempts to target DCLK1 using pharmacologic approaches have relied on nonspecific suppression of DCLK1 kinase activity. Here, we demonstrate that DiFiD (3,5-bis [2,4-difluorobenzylidene]-4-piperidone)binds to DCLK1 with high selectivity. Moreover, DiFiD mediated suppression of DCLK1 led to G2/M arrest and apoptosis and significantly suppressed tumor growth of HNSCC xenografts and ASCC patient derived xenografts, supporting that DCLK1 is critical for SCC growth.

Evolutionary Action Score of TP53 Analysis in Pathologically High-Risk Human Papillomavirus-Negative Head and Neck Cancer From a Phase 2 Clinical Trial: NRG Oncology Radiation Therapy Oncology Group 0234

An evolutionary action scoring algorithm (EAp53) based on phylogenetic sequence variations stratifies patients with head and neck squamous cell carcinoma (HNSCC) bearing TP53 missense mutations as high-risk, associated with poor outcomes, or low-risk, with similar outcomes as TP53 wild-type, and has been validated as a reliable prognostic marker. We performed this study to further validate prior findings demonstrating that EAp53 is a prognostic marker for patients with locally advanced HNSCC and explored its predictive value for treatment outcomes to adjuvant bio-chemoradiotherapy. Eighty-one resection samples from patients treated surgically for stage III or IV human papillomavirus-negative HNSCC with high-risk pathologic features, who received either radiation therapy+cetuximab+cisplatin (cisplatin) or radiation therapy+cetuximab+docetaxel (docetaxel) as adjuvant treatment in a phase 2 study were subjected to TP53 targeted sequencing and EAp53 scoring to correlate with clinical outcomes. Due to the limited sample size, patients were combined into 2 EAp53 groups: (1) wild-type or low-risk; and (2) high-risk or other. At a median follow-up of 9.8 years, there was a significant interaction between EAp53 group and treatment for overall survival (P=.008), disease-free survival (P=.05), and distant metastasis (DM; P=.004). In wild-type or low-risk group, the docetaxel arm showed significantly better overall survival (hazard ratio [HR] 0.11, [0.03-0.36]), disease-free survival (HR 0.24, [0.09-0.61]), and less DM (HR 0.04, [0.01-0.31]) than the cisplatin arm. In high-risk or other group, differences between treatments were not statistically significant. The docetaxel arm was associated with better survival than the cisplatin arm for patients with wild-type or low-risk EAp53. These benefits appear to be largely driven by a reduction in DM.

Lachnoclostridium and Immune Inflamed Gene Expression Signature Association in Head & Neck Cancers

<h3>Purpose/Objective(s)</h3> The immune microenvironment plays an essential role in the progression of head and neck squamous cell carcinoma (HNSCC). The association of the tumor microbiota and immune microenvironment is not well defined. The Cancer Genome Atlas Project (TCGA) analysis provided the diversity of microbiota in different cancer types. We analyzed the TCGA microbiome database to better understand the role of the tumor microbiome in HNSCC. <h3>Materials/Methods</h3> Human papillomavirus (HPV) positive and HPV negative HNSCC samples in the TCGA database were analyzed. Data were accessed using cBioPortal. Spearman's test was used to correlate Lachnoclostridium microbiome signature with immune-related gene expressions. <h3>Results</h3> Microbiome signatures were compared in PDL1 low and PDL1 high HNSCC. Lachnoclostridium was among the top genus enriched in the PDL1 high expression group in both HPV+ and HPV- HNSCC. Lachnoclostridium was correlated with immune inflamed gene expression profile. To evaluate the differential gene expressions in Lachnoclostridium enriched HNSCC, the gene expression profile of Lachnoclostridium high and low HNSCC samples were compared. MHC class I genes; HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, TAP1, and TAP2 were among the top differentially expressed genes. Then we evaluated the correlation of MHC-I genes with Lachnoclostridium enrichment. Lachnoclostridium signature was significantly correlated with expression of these MHC I genes, with Spearman's correlation coefficient ranging between 0.51-0.59. <h3>Conclusion</h3> Lachnoclostridium is a recently defined genus found in the human gut microbiome. Here we, for the first time, identify the enrichment of this genus in high PDL1 expressing HNSCC. The association of Lachnoclostridium signature with the immune inflamed gene expression profile and MHC-I gene signature suggests its potential role in modulating the immune microenvironment in HNSCC.

4NQO enhances differential activation of DNA repair proteins in HPV positive and HPV negative HNSCC cells

Tobacco exposure and human papillomavirus (HPV) infection are among the main risk factors for the development of head and neck squamous cell carcinoma (HNSCC). Interestingly, recent studies show that tumors from HPV positive (HPV+) smokers and non-smokers have similar mutational profiles, which suggests that HPV could prevent mutation induction or accumulation in the intermediate risk group composed of HPV+ smokers. Hence, we tested this observation by analyzing the effects of 4-Nitroquinoline N-oxide (4NQO), a mutagen and smoking mimetic, in NOK (normal oral keratinocytes), NOKE6.E7 (NOK cells transfected with E6.E7 oncogenes of HPV), HPV+ and HPV negative (HPV-) HNSCC cells. Oxidative DNA damage, γH2AX foci formation, DNA repair protein activation, cell cycle phase analysis, apoptotic cell death, cell viability and clonogenic cell survival were analyzed after 4NQO treatment in NOK, NOKE6.E7, HPV+ and HPV- HNSCC cells. 4NQO increased oxidative base damage and γH2AX foci formation in NOKE6.E7, HPV+ and HPV- HNSCC cells. Phosphorylation of homologous recombination (HR) repair proteins was higher in NOKE6.E7 and HPV+ HNSCC cells compared to NOK and HPV- HNSCC cells respectively. HPV+ and HPV- HNSCC cells showed differential activation of cell cycle regulatory proteins, increased apoptosis, and decreased cell viability upon 4NQO-induced DNA damage. Taken together, 4NQO (a smoking mimetic), induced higher activation of HR repair in HPV+ HNSCC cells compared to HPV- HNSCC cells. This may allow for increased mutational resistance and help explain why HPV+ smokers have a worse prognosis than HPV+ non-smokers.

Abstract 1930: 4-nitroquinoline-1-oxide enhances induction of apoptosis preferentially in HPV positive head and neck squamous cell carcinoma cells

Abstract Background: Tobacco use and human papillomavirus (HPV) infection are both strong contributors to the oncogenesis of head and neck squamous cell carcinoma (HNSCC). Despite HPV's causative role in HNSCC, it confers no statistically significant difference in mutational profile between HPV-associated HNSCC smokers and non-smokers. Our earlier preliminary data suggested that HPV positive (+) HNSCC cells have better activation of DNA repair proteins compared to HPV negative (-) HNSCC cells when treated with 4NQO, a mutagen and carcinogen which serves as a surrogate for tobacco exposure. This may partially explain HPV+ HNSCC's higher mutational resistance. In addition to increased DNA repair, HPV+ cells may also eliminate mutations through higher rates of apoptosis compared to HPV- cells. Hence, we wanted to test this hypothesis by analyzing the effects of 4NQO on apoptosis in HPV+ and HPV- HNSCC cells. Methods: NOK, NOK E6.E7, SCC40 (HPV-), SCC104 and UMSCC47 (HPV+) cells were treated with different concentrations of 4NQO (0, 0.1, and 0.2 ug/ml) for 24 and 48 hours. Untreated (control) and 4NQO treated cells were assessed for cell viability using MTT assay. Apoptotic cells were determined using Annexin-V FITC flow cytometry analysis, and Western blot was performed to check the apoptotic proteins for the afore-mentioned control and 4NQO treatments. Results: MTT assay showed decreased cell viability of NOK E6.E7, SCC47 and SCC104 cells in response to 4NQO treatment compared to relatively higher cell viability of SCC40 cells. Flow cytometry confirmed that HPV+ HNSCC cells are relatively more apoptotic compared to HPV- cells following 4NQO treatment at 24 and 48 hours. This is further supported by activation of pro-apoptotic proteins including p53, cleaved caspase 3, cleaved PARP, cleaved caspase 9, and p21 to a greater extent in HPV+ cells at different time points of 4NQO treatment using Western blot analysis. Conclusion: Our in-vitro data suggest HPV may stimulate a protective measure against smoking-induced mutations by increasing apoptosis before cells can contribute to the HNSCC mutational profile. Taken together, these data explain why HPV associated HNSCC have a better prognosis than HPV negative irrespective of smoking history. Citation Format: Gauri Shishodia, Alice Lee, Rhodee Ric Toledo, Emily Zimmerman, Xiaohua Rong, Adam Xiao, Lynn Harrison, Cherie Ann Nathan. 4-nitroquinoline-1-oxide enhances induction of apoptosis preferentially in HPV positive head and neck squamous cell carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1930.

Tumor-Naïve Multimodal Profiling of Circulating Tumor DNA in Head and Neck Squamous Cell Carcinoma.

Circulating tumor DNA (ctDNA) enables personalized treatment strategies in oncology by providing a noninvasive source of clinical biomarkers. In patients with low ctDNA abundance, tumor-naïve methods are needed to facilitate clinical implementation. Here, using locoregionally confined head and neck squamous cell carcinoma (HNSCC) as an example, we demonstrate tumor-naïve detection of ctDNA by simultaneous profiling of mutations and methylation. We conducted CAncer Personalized Profiling by deep Sequencing (CAPP-seq) and cell-free Methylated DNA ImmunoPrecipitation and high-throughput sequencing (cfMeDIP-seq) for detection of ctDNA-derived somatic mutations and aberrant methylation, respectively. We analyzed 77 plasma samples from 30 patients with stage I-IVA human papillomavirus-negative HNSCC as well as plasma samples from 20 risk-matched healthy controls. In addition, we analyzed leukocytes from patients and controls. CAPP-seq identified mutations in 20 of 30 patients at frequencies similar to that of The Tumor Genome Atlas (TCGA). Differential methylation analysis of cfMeDIP-seq profiles identified 941 ctDNA-derived hypermethylated regions enriched for CpG islands and HNSCC-specific methylation patterns. Both methods demonstrated an association between ctDNA abundance and shorter fragment lengths. In addition, mutation- and methylation-based ctDNA abundance was highly correlated (r > 0.85). Patients with detectable pretreatment ctDNA by both methods demonstrated significantly worse overall survival (HR = 7.5; P = 0.025) independent of clinical stage, with lack of ctDNA clearance post-treatment strongly correlating with recurrence. We further leveraged cfMeDIP-seq profiles to validate a prognostic signature identified from TCGA samples. Tumor-naïve detection of ctDNA by multimodal profiling may facilitate biomarker discovery and clinical use in low ctDNA abundance applications.

Effect of 4‐Nitroquinoline‐1‐Oxide on DNA damage and repair in head and neck squamous cell carcinoma cells

Background Tobacco exposure and human papillomavirus (HPV) infection are among the main factors for development of head and neck squamous cell carcinoma (HNSCC). Interestingly, recent studies show that HPV positive (+) smokers and non-smokers have similar mutational profiles, which suggests that mutations induced in smokers could be associated with the protective effects of HPV. Our preliminary data showed that 4NQO, a mutagen and carcinogen which serves as a surrogate for tobacco exposure induces oxidative DNA damage and DSB formation in HNSCC cells at 6 hrs. HPV+ HNSCC cells also showed a relative increase in activation of homologous recombination (HR) DNA repair proteins compared to HPV negative (-) HNSCC cells at 6 hrs. In this study, we wanted to investigate the effect of 4NQO on DNA damage and repair activation at 24 hrs in HPV+ and HPV- HNSCC cells. Methods NOK E6.E7 +/-, UPCI-SCC40 (HPV-), UM-SCC104 and UM-SCC47 (HPV+) cells were utilized to check effects of 4NQO on DNA repair. Oxidative DNA damage, double strand break formation (DSBs) and protein expression were analyzed by FPG-modified alkaline comet assay, γH2AX staining and Western analysis following treatment with 4NQO for 6 hr, replacement with fresh media, and sample collection at 24 hr. Results 4NQO increased oxidative DNA damage, single strand breaks, and yH2AX foci at 6 hrs; however, these lesions were no longer present at 24 hrs. This suggests DNA repair had probably occurred upon removal of 4NQO. We no longer observed an increase in phosphorylation of BRCA1, Rad51, H2AX, and p21 levels in HNSCC cells at 24 hr although it was present at 6 hrs. Conclusion Our in-vitro data suggests that HPV may have a role in the activation of DNA repair proteins by 4NQO at 6 hrs. This may have allowed for successful repair of DNA damage in both HPV+ and HPV- HNSCC cells by 24 hrs. Taken together, these data suggest that HPV may protect against smoking-induced mutations by preferentially increasing HR DNA repair protein activation.

Programmed Death-1/Programmed Death-Ligand 1-Axis Blockade in Recurrent or Metastatic Head and Neck Squamous Cell Carcinoma Stratified by Human Papillomavirus Status: A Systematic Review and Meta-Analysis.

BackgroundProgrammed death-1/programmed death-ligand 1 (PD-1/PD-L1) inhibitors have provided clinical benefit to head and neck squamous cell carcinoma (HNSCC) patients in recent clinical trials. However, it remains unclear as to whether human papillomavirus (HPV) status is associated with improved clinical outcome of anti-PD-1 or anti-PD-L1 immunotherapy in HNSCC.MethodsPubMed, EMBASE, Cochrane Library, and Web of Science were systematically searched up to February 28, 2021. Published clinical trials of HNSCC patients treated with only PD-1 or PD-L1 inhibitors were selected. The primary or secondary outcome of these studies included objective response rate (ORR) stratified by HPV status. The pooled odds ratio (OR) and hazard ratio (HR) were estimated using a fixed-effect model.ResultsA total of seven eligible studies comprising 814 patients were included. The ORR of HPV positive HNSCC patients was significantly higher than that of HPV negative HNSCC patients (OR = 1.77; 95%CI = 1.14-2.74; P = 0.01), and this favorable effect occurred in pooled anti-PD-L1 trials (OR = 2.66; 95%CI = 1.16-6.11; P = 0.02). In comparison, the pooled OR was 1.51 in anti-PD-1 trials (95%CI = 0.90-2.54; P = 0.12). Survival analysis indicated that HPV positive HNSCC patients had a lower risk of overall death as compared to HPV negative HNSCC patients (HR = 0.77; 95%CI = 0.60–0.99; P = 0.04).ConclusionsHPV positive HNSCC patients display improved outcomes with PD-1/PD-L1 axis blockade as compared to HPV negative HNSCC patients. These improved outcomes are likely driven to a greater extent by anti-PD-L1 inhibitors. However, randomized controlled trials with greater numbers of patients are needed for validation of these early findings.

SLC7A11 as a biomarker and therapeutic target in HPV-positive head and neck Squamous Cell Carcinoma

Ferroptosis, a regulated form of cell necrosis was previously reported to be induced upon pharmacological targeting of the cystine transporter SLC7A11 in Head and neck Squamous Cell Carcinoma (HNSCC). Whether tumors arising in a context of chronic infection with Human Papillomavirus (HPV) are sensitive to ferroptosis is unknown. Using RNAseq data (both whole-tumor and single-cell sequencing) we report that HPV positive (HPV+ve) tumors have lower expression levels of SLC7A11 compared to HPV negative (HPV-ve) HNSCC. We examined invitro the effect of erastin, a specific blocker of SLC7A11, applied on two HNSCC cell lines with stable expression of HPV16 E6 and E7 oncoproteins. We report a decrease in total GSH levels and an increased sensitivity to erastin-induced ferroptosis in E6-E7 cells. Cell sensitivity to ferroptosis was specificaly related to a defect in cystine transport since we found no difference in ferroptosis induced by the direct inhibition of GPX4, and N-Acetyl Cystein abolished the difference between WT and E6-E7-expressing cells. Our findings point to SLC7A11 as an HPV-related biomarker of potential therapeutic relevance in HNSCC. Targeting cystine import to promote ferroptosis might be a promising strategy against HPV+ve HNSCC. (188 words).

Senescence Induction by Combined Ionizing Radiation and DNA Damage Response Inhibitors in Head and Neck Squamous Cell Carcinoma Cells.

DNA damage response inhibitors (DDRi) may selectively enhance the inactivation of tumor cells in combination with ionizing radiation (IR). The induction of senescence may be the key mechanism of tumor cell inactivation in this combinatorial treatment. In the current study the effect of combined IR with DDRi on the induction of senescence was studied in head and neck squamous cell carcinoma (HNSCC) cells with different human papilloma virus (HPV) status. The integrity of homologous recombination (HR) was assessed in two HPV positive, two HPV negative HNSCC, and two healthy fibroblast cell cultures. Cells were treated with the DDRi CC-115 (DNA-dependent protein kinase, DNA-pK; dual mammalian target of rapamycin, mTor), VE-822 (ATR; ataxia telangiectasia and Rad3-related kinase), and AZD0156 (ATM; ataxia telangiectasia mutated kinase) combined with IR. Effects on senescence, apoptosis, necrosis, and cell cycle were analyzed by flow cytometry. The fibroblast cell lines generally tolerated IR or combined treatment better than the tumor cell lines. The ATM and ATR inhibitors were effectively inducing senescence when combined with IR. The DNA-PK inhibitor was not an important inductor of senescence. HPV status and HR activity had a limited influence on the efficacy of DDRi. Induction of senescence and necrosis varied individually among the cell lines due to molecular heterogeneity and the involvement of DNA damage response pathways in senescence induction.

Abstract 6185: In vivo screening in HPV positive and HPV negative head and neck squamous cell carcinoma

Abstract Background: The pathogenesis of head and neck squamous cell carcinoma (HNSCC) largely occurs through two pathways, human papillomavirus (HPV) mediated or chemical carcinogenesis secondary to alcohol and tobacco use. HPV status is an important prognostic factor and marker for sensitivity to radiation therapy in HNSCC patients. HPV negative HNSCC has been shown to have increased radioresistance compared to HPV positive HNSCC leading to poorer prognosis. The goal of this work is to utilize in vivo screening technology to identify key differential targets between HPV positive and negative tumors for both therapeutic purposes as well as to identify differential pathways between these two malignancies. Methods: Pooled, barcoded shRNA libraries focused on DNA damage repair (DDR) and currently targetable genes were transduced into three HPV positive (UM-SCC-147, UD-SCC-2, UPCI:SCC152) and three HPV negative (UM-SCC-22A, Cal-27, HN31) cell lines. Each cell line was injected into the mouse flank and allowed to grow to ~100mm3. Tumors were divided into control and radiation groups; the latter being treated with 6-10 Gy at 2 Gy/treatment. For each tumor type radiation was titrated to generate an approximate 20% reduction in tumor size. Tumors were then allowed to grow for an additional 2 weeks and then harvested. Barcodes from each tumor were sequenced and compared to both the reference input cells as well as untreated control tumors. Barcodes observed at decreased frequency in treated tumors indicated a potential target for therapeutic sensitization. Both p-values as well as percentile median fold change for each gene were calculated. These values were averaged for the three HPV negative and three HPV positive cell lines and used for analysis. Results: Multiple genes in the DDR and FDA libraries were identified from the HPV negative and HPV positive groups that may be associated with radioresistance in vivo. Example genes that were identified primarily in the HPV negative group include BTK, TXN, TTK, while those in the HPV positive group include RNF168, RAD50, ESR1. Inhibition of these genes may lead to increased cell death following radiation therapy through diminished DNA repair of radiation-induced damage. Conclusion: Our study identifies targetable genes that may contribute to radioresistance in both HPV negative and HPV positive head and neck squamous cell carcinoma and warrants further analysis. Citation Format: Mohamed K. Abdelhakiem, Liangpeng Yang, Tongxin Xie, Manish Kumar, David Molkentine, Aakash Sheth, Mitchell Frederick, Jeffrey Myers, Curtis R. Pickering, Heath D. Skinner. In vivo screening in HPV positive and HPV negative head and neck squamous cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6185.

Ovarian cancer-derived copy number alterations signatures are prognostic in chemoradiotherapy-treated head and neck squamous cell carcinoma.

DNA copy number alterations (CNAs) are frequent in cancer, and recently developed CNA signatures revealed their value in molecular tumor stratification for patient prognosis and platinum resistance prediction in ovarian cancer. Head and neck squamous cell carcinoma (HNSCC) is also characterized by high CNAs. In this study, we determined CNA in 173 human papilloma virus‐negative HNSCC from a Dutch multicenter cohort by low‐coverage whole genome sequencing and tested the prognostic value of seven cancer‐derived CNA signatures for these cisplatin‐ and radiotherapy‐treated patients. We find that a high CNA signature 1 (s1) score is associated with low values for all other signatures and better patient outcomes in the Dutch cohorts and The Cancer Genome Atlas HNSCC data set. High s5 and s7 scores are associated with increased distant metastasis rates and high s6 scores with poor overall survival. High cumulative cisplatin doses result in improved outcomes in chemoradiotherapy‐treated HNSCC patients. Here we find that tumors high in s1 or low in s6 are most responsive to a change in cisplatin dose. High s5 values, however, significantly increase the risk for metastasis in patients with low cumulative cisplatin doses. Together this suggests that the processes causing these CNA signatures affect cisplatin response in HNSCC. In conclusion, CNA signatures derived from a different cancer type were prognostic and associated with cisplatin response in HNSCC, suggesting they represent underlying molecular processes that define patient outcome.

Single Nucleotide Polymorphism rs6942067 Is a Risk Factor in Young and in Non-Smoking Patients with HPV Negative Head and Neck Squamous Cell Carcinoma.

Genetic factors behind the increasing incidence of human papillomavirus (HPV) negative head and neck squamous cell carcinoma (HNSCC) in young non-smokers are suspected, but have not been identified. Recently, rs6942067, a single nucleotide polymorphism (SNP) located upstream of the DCBLD1 gene, was found associated with non-smoking lung adenocarcinoma. To validate if this SNP is also implicated in HNSCC, participants of The Cancer Genome Atlas HNSCC cohort were investigated for rs6942067 status, associated DCBLD1 expression, and clinical characteristics. Occurrence of the rs6942067 GG genotype is significantly higher in young and in HPV negative non-smoking HNSCC than in other HNSCC. Additionally, rs6942067 GG is associated with higher DCBLD1 expression in HNSCC and patients with high DCBLD1 expression have a worse overall survival at three years, both in univariate and multivariate analysis. Furthermore, high DCBLD1 expression is associated with activation of the integrin signaling pathway and its phosphorylation with EGFR and MET. Collectively, these findings suggest that DCBLD1 plays a critical role in HNSCC and demonstrate an association between rs6942067 and clinical characteristics of young age and HPV negative non-smoking status in HNSCC patients.

Inhibition of CDK4/CDK6 Enhances Radiosensitivity of HPV Negative Head and Neck Squamous Cell Carcinomas

Human papillomavirus negative (HPV-ve) head and neck squamous cell carcinoma (HNSCC) has a poor prognosis compared with HPV+ve HNSCCs. Expression of p16 in HPV+ve HNSCC is thought to mediate radiosensitivity via inhibition of cyclin-dependent kinase (CDK) 4/6. We used a clinically approved CDK4/CDK6 inhibitor, palbociclib, and assessed its effect on radiosensitivity in HNSCC. The effect of palbociclib on radiosensitivity was determined in HPV-ve and HPV+ve HNSCC cell lines using colony survival assays, immunofluorescent staining of repair proteins, homologous recombination assays, cell cycle, and metaphase spread analyses. Only HPV-ve HNSCC cells were radiosensitized by palbociclib, which also occurred at hypoxic levels associated with radioresistance. Palbociclib led to decreased induction of BRCA1 and RAD51 after irradiation. Homologous recombination was diminished and repair of radiation-induced DNA damage was delayed in the presence of palbociclib, leading to increased chromosomal damage. Failure to repair radiation-induced damage led to cell death as a result of mitotic catastrophe. Here, we highlight a therapeutic strategy to improve the radiosensitivity of HPV-ve HNSCC, a patient group that has an unmet and urgent need for improved radiation therapy efficacy.

Association of lnc-IL17RA-11 with increased radiation sensitivity and improved prognosis of HPV-positive HNSCC.

Human papillomavirus (HPV) associated head and neck squamous cell carcinoma (HNSCC) has a far better prognosis than HPV negative HNSCC. Recent studies suggest that long noncoding RNA (lncRNA) moieties may play a role in HPV associated differential HNSCC prognoses. In this study, we examined differential expression of lncRNAs in HPV+ vs HPV- HNSCC using The Cancer Genome Atlas database. LncRNAs were categorized based on expression level and survival analysis. A group of eight lncRNAs was identified in which altered expression was associated with both HPV infection and better prognosis. Subsequently, genes coexpressed with these lncRNAs in HNSCC cells were sorted into corresponding co-expression modules. The lnc-IL17RA-11 coexpression module exhibited the greatest correlation with HPV infection and radiotherapy efficacy. We identified the lnc-IL17RA-11 transcription factor ER-alpha as the most likely HPV infection associated factor promoting increased lnc-IL17RA-11 levels. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis revealed enrichment among lnc-IL17RA-11 co-expressed genes for functions related to DNA replication and cell proliferation. These observations are consistent with a model in which HPV infection upregulates transcription factor ER-alpha, which increases levels of lnc-IL17RA-11 and coexpressed genes that promote HNSCC cell sensitivity to radiotherapy, thereby improving disease prognosis.