HPV-negative HNSCC Research Articles

Tissue microarray analyses of the essential DNA repair factors ATM, DNA-PKcs and Ku80 in head and neck squamous cell carcinoma

BackgroundHead and neck squamous cell carcinoma (HNSCC) negative for Human Papillomavirus (HPV) has remained a difficult to treat entity, whereas tumors positive for HPV are characterized by radiosensitivity and favorable patient outcome. On the cellular level, radiosensitivity is largely governed by the tumor cells` ability to repair radiation-induced DNA double-strand breaks (DSBs), but no biomarker is established that could guide clinical decision making. Therefore, we tested the impact of the expression levels of ATM, the central kinase of the DNA damage response as well as DNA-PKcs and Ku80, two major factors in the main DSB repair pathway non-homologous end joining (NHEJ).MethodsA tissue microarray of a single center HNSCC cohort was stained for ATM, DNA-PKcs and Ku80 and the expression scored based on staining intensity and the percentages of tumor cells stained. Scores were correlated with clinicopathological parameters and survival.ResultsSamples from 427 HNSCC patients yielded interpretable stainings and were scored following an established algorithm. The majority of tumors showed strong expression of both NHEJ factors, whereas the expression of ATM varied more. The expression scores of ATM and DNA-PKcs were not associated with patient survival. For HPV-negative HNSCC, the minority of tumors without strong Ku80 expression trended towards superior survival when treatment included radiotherapy. Focusing stronger on staining intensity to define the subgroup with lowest and therefore potentially insufficient expression levels in the HPV-negative subgroup, we observed significantly better overall survival for patients treated with radiotherapy but not with surgery alone.ConclusionsOur data suggest that HPV-negative HNSCC with particularly low Ku80 expression represent a highly radiosensitive subpopulation. Confirmation in independent cohorts is required.

Exosomal transcript cargo and functional correlation with HNSCC patients’ survival

BackgroundHPV status in a subset of HNSCC is linked with distinct treatment outcomes. Present investigation aims to elucidate the distinct clinicopathological features of HPV-positive and HPV-negative HNSCC and investigate their association with the HNSCC patient survival.Materials and methodsThe total RNA of exosomes from HPV-positive (93VU147T) and HPV-negative (OCT-1) HNSCC cells was isolated, and the transcripts were estimated using Illumina HiSeq X. The expression of altered transcripts and their clinical relevance were further analyzed using publicly available cancer transcriptome data from The Cancer Genome Atlas (TCGA).ResultsTranscriptomic analyses identified 3785 differentially exported transcripts (DETs) in HPV-positive exosomes compared to HPV-negative exosomes. DETs that regulate the protein machinery, cellular redox potential, and various neurological disorder-related pathways were over-represented in HPV-positive exosomes. TCGA database revealed the clinical relevance of altered transcripts. Among commonly exported abundant transcripts, SGK1 and MAD1L1 showed high expression, which has been correlated with poor survival in HNSCC patients. In the top 20 DETs of HPV-negative exosomes, high expression of FADS3, SGK3, and TESK2 correlated with poor survival of the HNSCC patients in the TCGA database.ConclusionOverall, our study demonstrates that HPV-positive and HPV-negative cells’ exosomes carried differential transcripts cargo that may be related to pathways associated with neurological disorders. Additionally, the altered transcripts identified have clinical relevance, correlating with patient survival in HNSCC, thereby highlighting their potential as biomarkers and as therapeutic targets.

Neoadjuvant adebrelimab plus dalpiciclib in HPV-negative locally advanced head and neck squamous cell carcinoma: A phase II clinical trial.

TPS6129 Background: The efficacy and safety of neoadjuvant immunotherapy (NAI) for treating resectable locally advanced head and neck squamous cell carcinoma (LAHNSCC) remain uncertain, and the comprehensive protocol of NAI needs further explored. The tumor suppressor gene CDKN2A plays a pivotal role in cell cycle regulation by modulating cyclin-dependent kinase (CDK) activity. Mutations in CDKN2A lead to continuous activation of CDK4/6, resulting in uncontrolled cell proliferation and tumor growth. Approximately 35.7% of HNSCC patients exhibit CDKN2A mutations, with the incidence rising to 58% among HPV-negative LAHNSCC patients. Studies have shown that combining immunotherapy with a CDK4/6 inhibitor produces a synergistic anti-tumor effect. This study aims to assess the efficacy and safety of combining anti-PD-L1 (adebrelimab) with a CDK4/6 inhibitor (dalpiciclib) as NAI in resectable LAHNSCC patients. Methods: This open-label, single-arm, prospective phase II trial will enroll LAHNSCC patients eligible for surgery. Inclusion criteria include: being 18 to 75 years old at study entry; pathologically confirmed HPV-negative HNSCC (oral, laryngeal, hypopharyngeal, and HPV-negative oropharyngeal carcinoma), determined via p16 immunohistochemistry or HPV DNA tests; with CDKN2A mutation; resectable LAHNSCC, staged III-IVB according to the UICC/AJCC 8th edition TNM system; an ECOG Performance Status score of 0 or 1; no prior relevant anti-tumor treatments; intent for curative treatment; and adequate organ function. Key exclusion criteria are active autoimmune diseases, use of immunosuppressive drugs, or systemic corticosteroids. Eligible patients receive 3 cycles of adebrelimab (1200 mg intravenously every 3 weeks, Day 1, 22 and 43) and 2 cycles of dalpiciclib (150 mg, po, every 4 weeks, day 1-21 and 29-49) before surgery. Dose adjustments are allowed based on toxicity. Surgery will follow 2-4 weeks post-NAI, with subsequent adjuvant radiotherapy or chemoradiotherapy based on risk factors after surgery. The primary endpoints are 1-year disease-free survival (defined as the time from surgical resection to local recurrence) and major pathological response (MPR, defined as ≤10% residual viable tumor cells), with secondary endpoints focusing on safety and predictive biomarkers. Clinical trial information: NCT06199271 .

Abstract 4768: Differential expression of triptolide gene targets in human papillomavirus positive and negative head and neck squamous cell carcinomas

Abstract Head and neck squamous cell carcinoma (HNSCC) is the fifth most common cancer in the US. HPV represents a more recent risk factor for which treatment may be individualized. HPV expresses viral E6 and E7 oncoproteins which downregulate wild type p53 and Rb respectively. Triptolide, a derivative of a traditional Chinese medicinal herb, has been shown to inhibit viral E6, thereby reactivating wild type p53. Therefore, triptolide may target HPV positive HNSCC as a putative treatment. To explore this, we compared mutation, methylation, and expression of triptolide gene targets between HPV positive and HPV negative samples from The Cancer Genome Atlas (TCGA) program. Patient data was queried from the TCGA PanCancer Atlas for HNSCC using cBioPortal. This included a total of 523 patients with 487 meeting inclusion criteria. 415 patients were HPV (-) and 72 were HPV (+).Differences in direct genomic alterations, DNA methylation, mRNA expression, and protein expression of the 34 known triptolide targets were compared between these two groups using the Benjamini-Hochberg Procedure with a significance level of q < 0.05. Significant differences of the studied genes (q < 0.05) are shown in Table 1. There were no significant differences in protein expression. Of the 34 known triptolide targets, HPV positive and negative samples show variable enrichment. Triptolide targets exhibit greater methylation and mRNA expression in HPV positive samples supporting triptolide’s efficacy in these samples. However, other triptolide targets are the differentially expressed and methylated in HPV negative samples, so the compound may have a beneficial effect in this context as well. Further studies are required to test efficacy of triptolide against HPV positive and HPV negative HNSCC in vitro and in vivo models. Table 1. Significant genomic differences between HPV (+) and HPV (-) samples shown. Enriched in HPV (+) Enriched in HPV (-) Gene Log2 ratio q-value Gene Log2 ratio q-value DNA methylation Direct genomic mutations VEGFA 0 5.85E-03 TP53 3.08 4.53E-29 BCL2 0.07 9.78E-18 DNA methylation JUN 0 4.89E-02 RELA 0.07 5.06E-04 CD80 0.06 2.56E-02 STAT3 0.24 3.19E-20 BIRC3 0.01 1.29E-04 TNF 0.07 4.31E-04 CD274 0.07 4.40E-03 XIAP 0.07 8.02E-06 CCR7 0.11 3.56E-08 CXCR4 0.04 3.58E-03 CD1A 0.10 2.36E-05 CD40 0.06 1.00E-05 C3 0.06 6.91E-03 CD14 0.04 8.12E-03 CD86 0.03 2.45E-02 mRNA expression mRNA expression RELA 0.16 1.90E-03 STAT3 0.25 4.22E-03 CDKN1A 0.28 3.00E-02 BCL2 1.95 1.68E-14 PLAU 1.37 3.24E-12 CASP3 0.45 2.55E-07 CXCL8 1.05 1.51E-04 TP53 1.45 3.16E-25 MCL1 0.23 3.59E-04 IL2 0.52 2.13E-06 TGFB1 0.77 3.27E-11 INFG 1.15 2.08E-05 DEFB4A 1.10 1.77E-02 CXCR4 1.18 8.03E-07 BIRC3 1.20 6.14E-06 IL23A 0.77 3.88E-04 CCR7 1.18 2.10E-06 CD40 0.54 2.27E-04 Citation Format: William E. Kamm, Lindsey J. Mortenson, Frank G. Ondrey. Differential expression of triptolide gene targets in human papillomavirus positive and negative head and neck squamous cell carcinomas [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 4768.

EGFR overexpression and macrophage infiltration correlate with poorer prognosis in HPV-negative oropharyngeal cancer via STAT6 signaling.

The prevalence of HPV-negative oropharyngeal cancer (OPC) is higher in Asian countries. Patients with HPV-negative OPC suffer poor outcomes. Multi-omics analysis could provide researchers and clinicians with more treatment targets for this high-risk group. We aimed to explore the prognostic significance of EGFR overexpression and macrophage infiltration in OPC, especially HPV-negative OPC in this study. EGFR alternation was evaluated with TCGA, PanCancer Atlas through cBioProtal. EGFR mRNA expression in HPV-negative head and neck squamous cell carcinoma was analyzed using the Tumor Immune Estimation Resource (TIMER 2.0). We also examined EGFR/STAT6/MRC1 expression in paraffin-embedded tissues from a p16-negative OPC cohort. The correlation between EGFR expression and macrophage activation was explored using Person's correlation coefficient. The impact of biomarkers or macrophage infiltration on 5-year overall survival and recurrence-free survival were analyzed using Kaplan-Meier survival curves. EGFR alteration rate was 15%, 13%, and 0% for HPV-negative HNSCC (excluding OPC), HPV-negative OPC, and HPV-positive OPC. High EGFR expression was associated with increased tumor infiltration of immune cells, such as macrophages. We observed positive correlations between EGFR, STAT6, and MRC1 expression in p16-negative OPC. Higher MRC1 expression was associated with poorer survival rates. There is strong correlation between EGFR overexpression and M2 polarization in patients with p16-negative OPC. Immunotherapy with or without EGFR inhibitor could be considered in these high-risk patients.

Human papillomavirus-associated head and neck squamous cell carcinoma cells rely on glycolysis and display reduced oxidative phosphorylation.

Head and neck squamous cell carcinoma (HNSCC) constitutes a heterogeneous group of cancers. Human papilloma virus (HPV) is associated with a subtype of HNSCC with a better response to treatment and more favorable prognosis. Mitochondrial function and metabolism vary depending on cancer type and can be related to tumor aggressiveness. This study aims to characterize the metabolism of HPV-positive and HPV-negative HNSCC cell lines. Oxidative phosphorylation (OXPHOS) and glycolysis were assessed in intact cells, in four HNSCC cell lines using Seahorse XF Analyzer. OXPHOS was further studied in permeabilized cells using high-resolution respirometry in an Oroboros O2K. Metabolomic analysis was performed using mass spectroscopy. The HPV-negative cell lines were found to display a higher OXPHOS capacity and were also able to upregulate glycolysis when needed. The HPV-positive cell line had a higher basal glycolytic rate but lower spare OXPHOS capacity. These cells were also unable to increase respiration in response to succinate, unlike the HPV-negative cells. In the metabolomic analysis, the HPV-positive cells showed a higher kynurenine/tryptophan ratio. HPV-positive HNSCC preferred glycolysis to compensate for lower OXPHOS reserves, while the HPV-negative HNSCC displayed a more versatile metabolism, which might be related to increased tumor aggressiveness. The higher kynurenine/tryptophan ratio of HPV-positive HNSCC might be related to increased indoleamine 2,3-dioxygenase activity due to the carcinoma's viral origin. This study highlights important metabolic differences between HPV-positive and HPV-negative cancers and suggests that future metabolic targets for cancer treatment should be individualized based on specific tumor metabolism.

Landscape of the Radiosensitizing Properties of Targeted Agents from the NCI CTEP Portfolio across Genomically Diverse 3D Tumor Models

Clinical translation of molecular targeted radiosensitizers remains challenging. To improve the robustness of preclinical data, we sought to broadly examine across a wide spectrum of genomic cancer features the radiosensitizing properties of targeted drugs from the NCI Cancer Therapy Evaluation Program (CTEP). As part of a U01 Consortium, we adapted a robotic high-throughput platform for testing ionizing radiation (IR) plus drug combinations in 3D cultures derived from non-small cell lung carcinoma (NSCLC), head/neck (HNSCC), and pancreatic cancers. All cell lines were annotated, mycoplasma free, and re-authenticated. Signal windows were established for all 3D growth formats. CTEP drugs were added in four concentrations 24 hours prior to IR exposure of 384-well plates. Five days after IR, cell viability was assessed, an endpoint previously benchmarked using colony formation. Each data point was based on 4-6 technical replicates with 2 biological repeats. A multistep statistical method combining Wilcoxon signed-rank and Spearman rank-order correlation tests together with adjusted Benjamini-Hochberg corrections for multiple comparisons was used to identify radiosensitization hits. Select validation experiments of hits were carried out. We screened 73 3D tumor models with 34-42 CTEP drugs each plus IR (n = 10,744 cell line/drug concentration combinations). IR/drug patterns were comparable for the three cancer types. DNA damage response (DDR) inhibitors constituted the most active class of radiosensitizers with DNA-PKcs, PARP, and ATR being the top targets. In contrast, the majority of the other agents radiosensitized only a minority of models. Highlighting NSCLC, out of 1,120 cell line/drug combinations, 328 (29.3%) were associated with statistically significant radiosensitization. PARP inhibitors, of particular interest for clinical translation in this space, demonstrated relatively broad radiosensitization for talazoparib (62%) and olaparib (44%) but less so for veliparib (28%), which correlated with differential PARP trapping potentials. Radiosensitization was evident even for tumor genotypes associated with radioresistance such as mutant KRAS, KEAP1, and STK11. Interestingly, single-agent activity of olaparib was more pronounced in 11/30 3D cultures compared to 2D growth conditions but with only 2/11 having known homologous recombination defects. Lastly, for HPV negative HNSCC (735 combinations), there was radiosensitization in subsets of models for IAP, ATR, and AKT targets, which are under clinical investigation. We present a unique resource for examining radiosensitizing drug properties in 3D cultures. The observed radiosensitization patterns strongly argue for the use of larger panels of clinically relevant tumor models rather than a few non-representative cell lines. Our data highlight the urgent need for predictive biomarkers to guide IR/drug combinations, even for some DDR inhibitors.

Abstract IA09: Characterization of hypoxic tumor microenvironment in HPV-negative HNSCC

Abstract IA09: Characterization of hypoxic tumor microenvironment in HPV-negative HNSCC

Abstract PO-070: New patient-derived head and neck cancer xenograft (PDX) panel for drug development, immuno-oncology and translational research

Abstract Head and neck squamous cell carcinomas (HNSCC) represent a heterogeneous group of epithelial cell malignancies arising from the upper aerodigestive tract. Despite of improved therapies, HNSCC remain a devastating disease. Oropharynx cancers are mainly attributed to infection with human papillomavirus (HPV). Since 2017, HNSCC are classified into HPV-negative and HPV-positive HNSCC. Therapies in the clinic are surgery and/or radiotherapy (RT)/radiochemotherapy (RCT) with a recurrence of about 50%. Therefore, new approaches are needed to improve long-term remission and patient survival. Recent advances in high-throughput molecular profiling have helped to identify genetic dispositions (TP53, FAT1, CDKNA2, NOTCH1 etc.) for HNSCC. We have generated and characterized a diverse panel (n = 85) of patient-derived xenografts (PDX) of HNSCC for preclinical research and immuno-oncological approaches. We successfully established 85 HNSCC PDX from fresh surgery tissue of primary and recurrent carcinomas by direct subcutaneous transplantation into immune-deficient mice. 14 HNSCC PDX were derived from HPV-positive tumors. We treated 28 HNSCC PDX with radiotherapy alone or radiochemotherapy. Heterogeneous individual responses to treatments resemble the clinical situation. For characterization, the established HNSCC PDX were treated with standard of cares (SoC) such as docetaxel, platinum compounds, cetuximab, 5-fluorouracil and investigational drugs. No correlation was observed to mutations and drug response. Molecular subtypes were determined based on gene expression data. Cetuximab response was associated with basal subtype and inflamed/mesenchymal subtype was negative predictive for cetuximab response. Further, 30 HNSCC PDX were analyzed for PD-L1 expression as suitable candidates for the evaluation of checkpoints inhibitors such as nivolumab and pembrolizumab and other novel immunotherapy approaches on humanized mice. To gain a deeper insight into the molecular biology, RNA sequencing was performed for 46 HNSCC models. Based on RNASeq data, an individual Human Leucocyte Antigen (HLA) profile of each PDX was determined and a comprehensive HLA matching analysis of the HNSCC models and 9 peripheral blood mononuclear cell (PBMC) donors was performed to enable personalized, preclinical immuno-oncology studies. Our newly and comprehensively characterized HNSCC PDX panel enables the evaluation of new targeted and immunological therapies in preclinical phase II studies. It provides an exceptional platform for the identification and validation of new targets and enables the preclinical screening of new combinations in translational research. Citation Format: Bernadette Brzezicha, Theresia Conrad, Maria Stecklum, Michael Becker, Konrad Klinghammer, Jens Hoffmann. New patient-derived head and neck cancer xenograft (PDX) panel for drug development, immuno-oncology and translational research [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-070.

Abstract PR04: Deciphering radiotherapy resistance mechanisms in HPV negative and positive head and neck cancers

Abstract Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. The prognosis of HNSCC is poor with a 5-year survival rate for all stages around 55%. HNSCC can be classified into two subtypes, human papilloma virus (HPV)-negative, caused mainly by tobacco use, and HPV-positive subtype. Radiotherapy is one of the cornerstones of the treatment of HNSCC. However, radioresistance is common and is associated with a high risk of recurrence. Radioresistance may be pre-existing, or acquired, and may be direct (tumor-cell autonomous) or indirect, mediated by RT-mediated changes to the TME such as cellular and/or extracellular matrix (ECM) changes to the stroma. These pathways are not fully understood. Better understanding of the intrinsic as well as RT-induced resistance is needed to improve survival of hard-to-treat HNSCC patients. HPV-positive HNSCC have significantly better RT response and prognosis, however currently they are treated with similarly high dose RT which causes significant adverse side-effects. As the incidence of HPV-induced HNSCC is increasing very fast, the current focus is on treatment de-escalation to reduce toxicity without compromising outcome. EGFR upregulation is an established biomarker of treatment resistance and aggressiveness in head HNSCC. EGFR-targeted therapies have shown benefits for HPV-negative HNSCC; surprisingly, inhibiting EGFR in HPV-associated HNSCC led to inferior therapeutic outcomes suggesting opposing roles for EGFR in the two HNSCC subtypes. We have recently shown that EGFR activation regulates the DNA damage response pathway differently in the two HNSCC subtype. Here, we further investigated the link between EGFR with HPV-infected HNSCC particularly the regulation of HPV oncoproteins E6 and E7. We demonstrate that EGFR overexpression suppresses cellular proliferation and increases radiosensitivity of HPV-positive HNSCC cell lines. EGFR overexpression was shown to inhibit protein expression of BRD4, a known cellular transcriptional regulator of HPV E6/E7 expression and DNA damage repair facilitator. Using in vitro 2D and 3D spheroid models of HNSCC we showed that EGFR inhibition by cetuximab restored the expression of BRD4 leading to increased HPV E6 and E7 transcription. Concordantly, pharmacological inhibition of BRD4 led to suppression of HPV E6 and E7 transcription, delayed cellular proliferation and sensitised HPV-positive HNSCC cells to ionising radiation. This effect was shown to be mediated through EGFR-induced upregulation of microRNA-9-5p and consequent silencing of its target BRD4 at protein translational level, repressing HPV E6 and E7 transcription and restoring p53 tumor suppressor functions. These results suggest a novel mechanism for EGFR inhibition of HPV E6/E7 oncoprotein expression through an epigenetic pathway, mediated through microRNA-9-5p/BRD4 regulation. The results may help better future design of radiotherapy combination treatment of HPV-positive and negative HNSCC, such as combination with BRD4 inhibitors to improve HNSCC therapeutic outcome. Citation Format: Mahvash Tavassoli. Deciphering radiotherapy resistance mechanisms in HPV negative and positive head and neck cancers [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 PR04.

Prognostic and clinicopathological significance of MRC2 expression in head and neck squamous cell carcinoma

BackgroundHead and neck squamous cell carcinoma (HNSCC) is one of the most aggressive types of cancers worldwide, with metastasis being the major cause of death. Recent research suggests that changes in the expression of MRC2 (mannose receptor, C-type 2) may play a role in the development and progression of various cancers; however, its expression pattern in HNSCC/ OSCC is unknown. This study aimed to elucidate the clinicopathological significance and prognostic role of MRC2 expression in HNSCC, including OSCC. Materials and methodsIn the present study, we assessed the potential roles of MRC2 in expression, prognostic value, immune infiltration and functional enrichment analysis in HNSCC patients by using different bioinformatics databases. We then validated MRC2 gene expression in 30 OSCC and adjacent normal tissue samples using quantitative reverse transcription PCR (RT-qPCR). ResultsMRC2 mRNA and protein expression were significantly upregulated in OSCC and HNSCC patients compared to that in adjacent normal tissues. Upregulated MRC2 expression was associated with poor overall survival. Increased MRC2 expression has also been linked to an aggressive clinicopathological features including advanced stages, grade, metastasis and HPV status. Interestingly, our in silico results strongly suggest that the MRC2 gene and protein interaction networks are associated with HNSCC development. Moreover, the tumor infiltration level was significantly correlated with HPV-negative HNSCC patients. ConclusionOur results suggest that MRC2 could be used as a novel prognostic marker and therapeutic target for HNSCC.

Deciphering the Tumor-Immune-Microbe Interactions in HPV-Negative Head and Neck Cancer.

Patients with human papillomavirus-negative head and neck squamous cell carcinoma (HPV-negative HNSCC) have worse outcomes than HPV-positive HNSCC. In our study, we used a published dataset and investigated the microbes enriched in molecularly classified tumor groups. We showed that microbial signatures could distinguish Hypoxia/Immune phenotypes similar to the gene expression signatures. Furthermore, we identified three highly-correlated microbes with immune processes that are crucial for immunotherapy response. The survival of patients in a molecularly heterogenous group shows significant differences based on the co-abundance of the three microbes. Overall, we present evidence that tumor-associated microbiota are critical components of the tumor ecosystem that may impact tumor microenvironment and immunotherapy response. The results of our study warrant future investigation to experimentally validate the conclusions, which have significant impacts on clinical decision-making, such as treatment selection.

Impaired DNA double-strand break repair and effective radiosensitization of HPV-negative HNSCC cell lines through combined inhibition of PARP and Wee1.

In head and neck squamous cell carcinoma (HNSCC), tumors negative for Human Papillomavirus (HPV) remain a difficult to treat entity and the morbidity of current multimodal treatment is high. Radiotherapy in combination with molecular targeting could represent suitable, less toxic treatment options especially for cisplatin ineligible patients. Therefore, we tested dual targeting of PARP and the intra-S/G2 checkpoint through Wee1 inhibition for its radiosensitizing capacity in radioresistant HPV-negative HNSCC cells. Three radioresistant HPV-negative cell lines (HSC4, SAS, UT-SCC-60a) were treated with olaparib, adavosertib and ionizing irradiation. The impact on cell cycle, G2 arrest and replication stress was assessed through flow cytometry after DAPI, phospho-histone H3 and γH2AX staining. Long term cell survival after treatment was determined through colony formation assay and DNA double-strand break (DSB) levels were assessed through quantification of nuclear 53BP1 foci in cell lines and patient-derived HPV±tumor slice cultures. Wee1 and dual targeting induced replication stress but failed to effectively inhibit radiation-induced G2 cell cycle arrest. Single as well as combined inhibition increased radiation sensitivity and residual DSB levels, with the largest effects induced through dual targeting. Dual targeting also enhanced residual DSB levels in patient-derived slice cultures from HPV-negative but not HPV+HNSCC (5/7 vs. 1/6). We conclude that the combined inhibition of PARP and Wee1 results in enhanced residual DNA damage levels after irradiation and effectively sensitizes radioresistant HPV-negative HNSCC cells. Ex vivo tumor slice cultures may predict the response of individual patients with HPV-negative HNSCC to this dual targeting approach.

Correlation of CDKN2A genomic alterations with tumor response to palbociclib given before chemoradiation therapy (CRT) to patients with human papillomavirus (HPV)-negative, locally advanced head and neck squamous-cell carcinoma (LA-HNSCC): A single-arm, phase 2 trial.

6015 Background: Cell cycle deregulation is ubiquitous in HNSCC. In HPV-negative disease, the most common genomic alteration of cell-cycling included CDKN2A deletions (57%), mutations (27%) or hypermethylation (12%). Selective CDK4/6 inhibition arrested cell cycling and inhibited tumor growth in cell-line and xenograft models of HPV-negative HNSCC, and CDKN2A alterations were predictive of response. An exploratory analysis of a double-blind, randomized, phase 2 trial of patients with HPV-negative recurrent/metastatic HNSCC showed that CDKN2A alterations were associated with better overall survival (OS) with palbociclib and cetuximab vs placebo and cetuximab (median 9.7 vs 4.6 months, HR 0.38). OS was similar between the two arms in patients without CDKN2A alterations. A phase 2 basket trial of patients with CDKN2A-altered head and neck cancers observed that palbociclib resulted in a target lesion decrease in 25% of patients. Collectively, these data warrant further studies to delineate predictive biomarkers of response to selective CDK4/6 inhibitors in HPV-negative HNSCC. Methods: The primary aims of this single-arm, phase 2 trial were to determine the objective response rate (ORR) of HPV-negative, LA-HNSCC to palbociclib, and to correlate responses to somatic CDKN2A alterations. HPV-negative disease was defined as SCC of the larynx, hypopharynx or oral cavity, or SCC of the oropharynx if negative for p16 by IHC and/or high-risk HPV-RNA by ISH. Genome sequencing (FoundationOne CDx/Tempus xT) was performed on tumor tissue obtained before treatment. Patients received palbociclib 125 mg/d orally on days 1-21 of each 28-day cycle. Tumor response was assessed using RECIST 1.1 with CT scans performed pre and post two cycles of palbociclib. Patients then received CRT. A sample size of 24 patients yielded an 80% power if the ORR was ≥38%, using an exact binomial test of one sample proportion comparison with an upper one-sided nominal significance level of 0.05 and null ORR of ≤17%. Results: 24 patients enrolled and completed two cycles of palbociclib: primary site (larynx-15; hypopharynx-4; oropharynx-4, oral cavity-1), clinical stage (III-7; IV-17), and smoking history (yes-23; no-1). The ORR with palbociclib was 41.7%. Best tumor response included: CR (1), PR (9), SD (13), and PD (1). CDKN2A altered disease was identified in 15 patients (62.5%) [mutation: 8, deletion: 7]. Tumor response to palbociclib occurred in 10 of 15 patients (66.7%) with CDKN2A altered disease versus 0 of 9 patients (0%) without CDKN2A altered disease (p=0.002, Fisher’s Exact Test). Conclusions: The primary hypothesis was met: the ORR with palbociclib in HPV-negative, LA-HNSCC was 41.7%. The ORR with palbociclib was significantly higher in CDKN2A altered disease. Clinical trial information: NCT03389477 .

DURTRERAD: A phase II open-label study evaluating feasibility and efficacy of durvalumab (D) and durvalumab plus tremelimumab (DT) in combination with radiotherapy (RT) in non-resectable locally advanced HPV-negative head and neck squamous cell carcinoma (LA-HNSCC).

2611 Background: Concurrent platinum-based chemo-RT is the standard treatment for LA-HNSCC, despite of limited efficacy in poor-risk patients and long-term toxicity. Targeting PD-L1 with D has activity in recurrent/metastatic (RM)-HNSCC, and the combination with the CTLA4 targeting agent T was considered to augment the immunotherapy efficacy of a chemotherapy-free regimen. Thus DURTRERAD was designed as a randomized two-arm phase II study to investigate, whether D or DT combined with RT was feasible in the patient population and a 1-year progression free survival rate of 50% was reached. Methods: The phase II study planned to enroll 60 patients in each treatment arm. Key selection criteria were patients with advanced HPV negative unresectable HNSCC planned for definitive radiotherapy. Treatments: First dose of immunotherapy 14 days prior to start of RT (70Gy over 7 weeks). D (1500mg q 3 weeks x 13) or DT (D: 1500mg q 3 weeks x 13 + T: 4 x 75 mg in weeks 5,9,13,17). Planned treatment duration was 51 weeks in both arms. Treatment was considered feasible in case of <10% treatment discontinuations. An interim analysis for feasibility was planned after treatment of 6 patients per arm and a futility analysis was performed after treatment of 18 patients, resulting in premature closure of the trial. Results: Patient accrual in 4 centers in Germany from 9/2018 - 9/2020. The interim feasibility analysis of DT revealed that 5/6 patients stopped treatment due to 1 grade 5 and 1 grade 4 immune related AE and 3 non-immune related AEs, respectively. Thus, the DT arm was prematurely terminated, and only the D monotherapy arm continued. Futility analysis of D monotherapy: One of 12 patients stopped treatment due to immune related toxicity, and 7 patients experienced disease progression on treatment or died within the first year. The median PFS was 39 weeks, the 1 year PFS rate was 42%. Conclusions: Even though D was previously proven as an active agent in RM-HNSCC, and there DT was not associated with increased toxicity, DT in combination with RT was not feasible in our poor prognostic, vulnerable patient cohort of HPV negative LA-HNSCC. D monotherapy in combination with RT was well tolerated, but revealed a low level of efficacy, when administered in the trial schedule, starting 2 weeks prior to RT. Similar to results in lung cancer, start of immunotherapy after completion of radiotherapy may be of interest. Clinical trial information: NCT03624231 .

Discovery of Human Papilloma Virus and patient-specific neoantigens targeted by CD8+ T cells in HNSCC

Abstract Head and neck squamous cell carcinoma (HNSCC) is amongst the rapidly growing form of cancer associated with high mutation burden and Human Papilloma virus (HPV) infection presenting approximately 800,000 new cases and 400,000 deaths annually. HPV-positive (HPV) HNSCC has been strongly associated with better prognosis than HPV-negative HNSCC due to the immune activation in the tumor microenvironment including cytotoxic CD8+ T cells. One of the key missing links is the relative contribution of HPV-reactive and neoantigen-reactive CD8+ T cells in anti-tumor response. To address this, we utilized epitope libraries presented in Signaling and Antigen-presenting Bifunctional Receptors (SABRs), which were previously described by our lab. As a proof-of-concept, we generated SABR libraries presenting 8392 epitopes derived from HPV or from personalized neoantigens from five HNSCC patients. In parallel, we used single cell RNA sequencing to identify 25 clonally expanded TCRs from tumor-infiltrating lymphocytes of the patients. Using SABR screens, we have identified several putative HPV-derived and neoantigen-derived epitopes targeted by CD8+ T cells in HNSCC patients. We have established a robust pipeline for identification of patient-specific epitope specificities from tumor-infiltrating CD8+ T cells. The goal is to determine whether and how effective anti-HPV or anti-neoantigen T cell responses may contribute to therapeutic efficacy using human samples from clinical trials and murine HNSCC models. Our study will aid in the discovery of novel tumor-associated antigens in HPV+ HNSCC patients, which can be successfully employed for precise adoptive T cell transfer therapies.

Suppression of head and neck cancer cell survival and cisplatin resistance by GRP78 small molecule inhibitor YUM70.

Head and neck squamous cell carcinoma (HNSCC) is one of the leading causes of cancer-related death worldwide. Surgical resection, radiation and chemotherapy are the mainstay of HNSCC treatment but are often unsatisfactory. Cisplatin is a commonly used chemotherapy in HNSCC; however, cisplatin resistance is a major cause of relapse and death. The 78-kD glucose-regulated protein (GRP78) is the master regulator of the unfolded protein response (UPR) and is implicated in therapeutic resistance in cancer. The role of GRP78 in cisplatin resistance in HNSCC remains unclear. YUM70 is a newly discovered hydroxyquinoline analogue and found to be an inhibitor of GRP78. The effect of YUM70 in HNSCC cell lines is unknown. Knockdown of GRP78 by siRNAs was performed to investigate the effect of GRP78 reduction in endoplasmic reticulum (ER)-stress induced and general apoptosis. Western blots examining apoptotic markers were performed on three HPV-negative HNSCC cell lines. WST-1 assay was performed to determine cell viability. In reverse, we utilized AA147, an ER proteostasis regulator to upregulate GRP78, and apoptotic markers and cell viability were determined. To test the ability of YUM70 to reverse cisplatin resistance, cisplatin-resistant HNSCC cell lines were generated by prolonged, repeated exposure to increasing concentrations of cisplatin. Colony formation assay using the cisplatin-resistant HNSCC cell line was performed to assess the in vitro reproductive cell survival. Furthermore, to test the ability of YUM70 to reverse cisplatin resistance in a physiologically relevant system, we subjected the 3D spheroids of the cisplatin-resistant HNSCC cell line to cisplatin treatment with or without YUM70 and monitored the onset of apoptosis. Reduction of GRP78 level induced HNSCC cell death while GRP78 upregulation conferred higher resistance to cisplatin. Combined cisplatin and YUM70 treatment increased apoptotic markers in the cisplatin-resistant HNSCC cell line, associating with reduced cell viability and clonogenicity. The combination treatment also increased apoptotic markers in the 3D spheroid model. The GRP78 inhibitor YUM70 reduced HNSCC cell viability and re-sensitized cisplatin-resistant HNSCC cell line in both 2D and 3D spheroid models, suggesting the potential use of YUM70 in the treatment of HNSCC, including cisplatin-resistant HNSCC.

Genomic signature of Fanconi anaemia DNA repair pathway deficiency in cancer.

Fanconi anaemia (FA), a model syndrome of genome instability, is caused by a deficiency in DNA interstrand crosslink repair resulting in chromosome breakage1-3. The FA repair pathway protects against endogenous and exogenous carcinogenic aldehydes4-7. Individuals with FA are hundreds to thousands fold more likely to develop head and neck (HNSCC), oesophageal and anogenital squamous cell carcinomas8 (SCCs). Molecular studies of SCCs from individuals with FA (FA SCCs) are limited, and it is unclear how FA SCCs relate to sporadic HNSCCs primarily driven by tobacco and alcohol exposure or infection with human papillomavirus9 (HPV). Here, by sequencing genomes and exomes of FA SCCs, we demonstrate that the primary genomic signature of FA repair deficiency is the presence of high numbers of structural variants. Structural variants are enriched for small deletions, unbalanced translocations and fold-back inversions, and are often connected, thereby forming complex rearrangements. They arise in the context of TP53 loss, but not in the context of HPV infection, and lead to somatic copy-number alterations of HNSCC driver genes. We further show that FA pathway deficiency may lead to epithelial-to-mesenchymal transition and enhanced keratinocyte-intrinsic inflammatory signalling, which would contribute to the aggressive nature of FA SCCs. We propose that the genomic instability in sporadic HPV-negative HNSCC may arise as a result of the FA repair pathway being overwhelmed by DNA interstrand crosslink damage caused by alcohol and tobacco-derived aldehydes, making FA SCC a powerful model to study tumorigenesis resulting from DNA-crosslinking damage.

EGFR-induced suppression of HPV E6/E7 is mediated by microRNA-9-5p silencing of BRD4 protein in HPV-positive head and neck squamous cell carcinoma

EGFR upregulation is an established biomarker of treatment resistance and aggressiveness in head and neck cancers (HNSCC). EGFR-targeted therapies have shown benefits for HPV-negative HNSCC; surprisingly, inhibiting EGFR in HPV-associated HNSCC led to inferior therapeutic outcomes suggesting opposing roles for EGFR in the two HNSCC subtypes. The current study aimed to understand the link between EGFR and HPV-infected HNSCC particularly the regulation of HPV oncoproteins E6 and E7. We demonstrate that EGFR overexpression suppresses cellular proliferation and increases radiosensitivity of HPV-positive HNSCC cell lines. EGFR overexpression inhibited protein expression of BRD4, a known cellular transcriptional regulator of HPV E6/E7 expression and DNA damage repair facilitator. Inhibition of EGFR by cetuximab restored the expression of BRD4 leading to increased HPV E6 and E7 transcription. Concordantly, pharmacological inhibition of BRD4 led to suppression of HPV E6 and E7 transcription, delayed cellular proliferation and sensitised HPV-positive HNSCC cells to ionising radiation. This effect was shown to be mediated through EGFR-induced upregulation of microRNA-9-5p and consequent silencing of its target BRD4 at protein translational level, repressing HPV E6 and E7 transcription and restoring p53 tumour suppressor functions. These results suggest a novel mechanism for EGFR inhibition of HPV E6/E7 oncoprotein expression through an epigenetic pathway, independent of MAPK, but mediated through microRNA-9-5p/BRD4 regulation. Therefore, targeting EGFR may not be the best course of therapy for certain cancer types including HPV-positive HNSCC, while targeting specific signalling pathways such as BRD4 could provide a better and potentially new treatment to improve HNSCC therapeutic outcome.

Abstract 3209: Everolimus inhibits angiogenesis and lymphangiogenesis to affect tumor growth in TP53 mutant HNSCC

Abstract Head and neck squamous cell carcinoma (HNSCC) constitutes the eighth most common cancer globally, with the eighth highest mortality rate amongst all cancer types. TP53 is the most frequently mutated gene in HPV -negative HNSCC. HPV-negative HNSCC harboring p53 mutations have the worst clinical outcomes with 50-60% local regional and distant recurrences causing increased morbidity and mortality. TP53 mutations are associated with shorter recurrence-free and overall survival. Angiogenesis and lymphangiogenesis play a causal role in tumor recurrence and lymph node metastasis. Therefore, inhibiting tumor angiogenesis and lymphangiogenesis is important in to preventing tumor recurrence and metastasis of TP53 mutant HNSCC. Previous studies demonstrated that mTOR activates STAT3 to upregulate HIF-1α and its target proteins, VEGF-A and VEGF-C, key molecules involved in angiogenesis and lymphangiogenesis, respectively. Further, mTOR inhibitors suppress autocrine and paracrine growth stimulation of tumor and lymphatic endothelial cells by impairing the VEGF-C/VEGF-3 axis and releasing soluble VEGFR-2. Accordingly, our objective was to investigate the effect of an mTOR inhibitor, everolimus, on angiogenesis and lymphangiogenesis of HPV-negative TP53 mutant HNSCC. Everolimus treatment of HPV-negative HNSCC cell lines harboring a variety of TP53 mutations, significantly downregulated protein and mRNA levels of VEGF-A and VEGF-C. Moreover, everolimus downregulated HIF-1α protein levels suggesting a HIF-1alpha-dependent VEGF modulation. Interestingly, treatment of human microvascular and lymphatic endothelial cells (HMEC-1 and HMEC-1A, respectively) with everolimus was also associated with a significant reduction in cell proliferation, in vitro tube formation, and migration. Protein and mRNA levels of HIF-1α, VEGF-A, and VEGF-C were downregulated by everolimus treatment in both HMEC-1 and HMEC-1A cell lines. Taken together, our data suggest that everolimus prevents angiogenesis and lymphangiogenesis through the inhibition of HIF-1α, indicating a promising role for mTOR inhibitors in treating HPV-negative TP53 mutant HNSCC patients at high risk for recurrence. Citation Format: Md Maksudul Alam, Janmaris Marin Fermin, Mark Knackstedt, Mackenzie J. Noonan, Emily K. Daniel, Tara Moore-Medlin, Xiaohua Rong, Alok R. Khandelwal, Cherie-Ann O. Nathan. Everolimus inhibits angiogenesis and lymphangiogenesis to affect tumor growth in TP53 mutant HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3209.