Head And Neck Squamous Cell Carcinoma Primary Tumors Research Articles

Abstract 2466: tRNA-derived fragments as immunotherapy response targets in head and neck squamous cell carcinoma

Abstract Background: Despite significant advances in immunotherapy-based treatment options for Head and Neck Squamous Cell Carcinoma (HNSCC), patient prognosis and survival remains poor. To enhance response to immune-based therapies, specific targeting of immunomodulatory pathways has been investigated through co-stimulatory gene targets, the microbiome, and more. An emerging biomolecule is Transfer RNAs (tRNAs), which are a class of RNAs that transport amino acids from the cytoplasm to ribosomes, and synthesize new proteins encoded by mRNA transcripts. tRNA-derived fragments (tRFs) are a category of cleaved, mature tRNA that participate in post-transcriptional regulation and have been implicated in the modulation of cancer and other immune-associated diseases. While tRFs are primarily implicated in diagnostic signatures for various cancers, few studies have investigated their role in modulating the immunotherapy-based response in HNSCC. In this study, we characterized the role of tRFs in modulating immunotherapy gene targets pertinent to HNSCC. Methods: We extracted tRF read counts from MINTbase 2.0 for 455 primary HNSCC tumor samples and 44 adjacent normals. Differential expression analysis was first performed to identify tRFs implicated in HNSCC tumors, with respect to adjacent normal tissue. Next, these tRFs were associated with immunotherapy target genes and response markers with Spearman’s correlation. Further characterization of these tRFs was performed by correlating their abundance to infiltrating immune cells, pathway enrichment of immunotherapy targets, and clinical variables. Results: Significant differential expression of tRFs were found between HNSCC primary tumor samples and adjacent normal tissue, including 7 tRFs (FDR < 0.05, |log fold change| > 0.5). All were enriched in normal samples. AlaCGC5tRF appeared to be an important regulator of immunotherapy target genes through increased co-expression of PTPN11, STAT3, and PTEN (p-value < 0.05, R > 0) and decreased co-expression of CD58 and TNFRSF14 (p-value < 0.05, R < 0). Immune Checkpoint Inhibitor (ICI) genes were significantly correlated with several tRFs. Among several pertinent tRF markers, AlaCGC5tRF was strongly associated with negatively correlated with infiltrating activated NK cells naïve CD4 T cells (p-value < 0.05, R < 0). Conclusion: Our study is the most comprehensive to date to characterize the role of tRF elements in modulating HNSCC immunotherapy targets. We found several potential tRF biomarkers that may be indicative of enhanced immunotherapy treatment susceptibility, including AlaCGC5tRF and ArgTCT3tRf. Further studies are needed to investigate how these tRFs may be targeted alongside immunotherapies to enhance response to treatment. Citation Format: Rishabh Yalamarty, Daniel John, Annie Do, Tianyi Chen, Megan Ngo, Pranava Gande, Matthew Uzelac, Wei Tse Li, Weg M. Ongkeko. tRNA-derived fragments as immunotherapy response targets in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2466.

Memory-like Differentiation, Tumor-Targeting mAbs, and Chimeric Antigen Receptors Enhance Natural Killer Cell Responses to Head and Neck Cancer.

Head and neck squamous cell carcinoma (HNSCC) is an aggressive tumor with low response rates to frontline PD-1 blockade. Natural killer (NK) cells are a promising cellular therapy for T cell therapy-refractory cancers, but are frequently dysfunctional in patients with HNSCC. Strategies are needed to enhance NK cell responses against HNSCC. We hypothesized that memory-like (ML) NK cell differentiation, tumor targeting with cetuximab, and engineering with an anti-EphA2 (Erythropoietin-producing hepatocellular receptor A2) chimeric antigen receptor (CAR) enhance NK cell responses against HNSCC. We generated ML NK and conventional (c)NK cells from healthy donors, then evaluated their ability to produce IFNγ, TNF, degranulate, and kill HNSCC cell lines and primary HNSCC cells, alone or in combination with cetuximab, in vitro and in vivo using xenograft models. ML and cNK cells were engineered to express anti-EphA2 CAR-CD8A-41BB-CD3z, and functional responses were assessed in vitro against HNSCC cell lines and primary HNSCC tumor cells. Human ML NK cells displayed enhanced IFNγ and TNF production and both short- and long-term killing of HNSCC cell lines and primary targets, compared with cNK cells. These enhanced responses were further improved by cetuximab. Compared with controls, ML NK cells expressing anti-EphA2 CAR had increased IFNγ and cytotoxicity in response to EphA2+ cell lines and primary HNSCC targets. These preclinical findings demonstrate that ML differentiation alone or coupled with either cetuximab-directed targeting or EphA2 CAR engineering were effective against HNSCCs and provide the rationale for investigating these combination approaches in early phase clinical trials for patients with HNSCC.

The tumor microenvironment-relay station for prognosis and therapy response

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide with adismal prognosis. Besides tobacco and alcohol abuse, human papilloma virus (HPV) infection is an independent risk factor, particularly in oropharyngeal squamous cell carcinomas (OPSCC). One key determinant of therapy response and prognosis is the tumor immune microenvironment (TIME). Astrong anti-tumor immune response represents an important mechanism of tumor cell killing. Another major determinant is the tumor metabolism. One feature of tumor cells is their reliance on glycolysis instead of oxidative phosphorylation (OXPHOS) for energy production despite sufficient oxygen supply.The presented studies were based on several different clinically and pathologically well annotated HNSCC cohorts. Immunohistochemical stainings were performed on tissue microarrays and whole slides against p16, different immune cell markers, and metabolic markers. DNA and mRNA were extracted to detect HPV and assess immune-related and metabolic genes via RT-PCR and Nanostring.An initial assessment of HPV infection frequencies in different HNSCC cohorts showed that socioeconomic and regional factors strongly influence HPV prevalence. An analysis of the immunological and metabolic differences of HPV-positive and HPV-negative OPSCC demonstrated an enhanced anti-tumor immune response together with increased levels of OXPHOS metabolism in HPV-positive OPSCC. In asubsequent study, astrong anti-tumor immune response together with an OXPHOS metabolism was associated with improved short-term survival in HNSCC. Finally, TIME differences of HNSCC primary tumors and recurrent tumors were analyzed to understand the poor therapeutic response of recurrences and discovered an overall decrease of tumor infiltrating lymphocytes with significant loss of CD8-positive Tcells and Blymphocytes as well as asignificant decrease of tertiary lymphoid structures in recurrences after chemoradiation.These results demonstrate the clinical and molecular differences of HPV-positive and HPV-negative HNSCC with afocus on OPSCC. Furthermore, they delineate the interdependencies of TIME and tumor metabolism in HNSCC in general and stress their impact on therapy response. Additionally, they show an impairment of anti-tumor immune response in recurrences after chemoradiation, which indicates immune evasion as one recurrence driver.

Interplay between Partial EMT and Cisplatin Resistance as the Drivers for Recurrence in HNSCC.

This study aims to investigate the role of partial epithelial to mesenchymal transition (pEMT)-related proteins in modulating Cisplatin resistance in head and neck squamous cell carcinoma (HNSCC). SCC-25 cells were pre-treated with TGF-beta1 followed by transient Krüppel-like Factor 4 (KLF4)-overexpression and Cisplatin treatment. Cell growth, cell morphological changes and cell migration were assessed using Juli BR live cell video-microscopy. In addition, Ki-67 and Slug immunostaining and follow-up image cytometric analysis of primary and recurrent HNSCC tumors were performed to evaluate the proliferation index (PI) and the EMT-like phenotype. We observed that proliferating and Slug-positive tumor cells expand after therapy in HNSCC. Subsequently, protein analysis revealed the stabilization of Slug, upregulation of Vimentin and phospho-p38 (p-p38) in Cisplatin-resistant SCC-25 cells. Moreover, KLF4-overexpression contributed to Cisplatin sensitivity by reduction of Slug at the protein level. This work strongly suggests that an pEMT-like pathway is activated in recurrent and Cisplatin-resistant HNSCC. Finally, stable KLF4-overexpression might sensitize HNSCC tumor cells for Cisplatin treatment.

CD8+ and FoxP3+ T-Cell Cellular Density and Spatial Distribution After Programmed Death-Ligand 1 Check Point Inhibition.

To analyze CD8+ and FoxP3+ T-cell cellular density (CD) and intercellular distances (ID) in head and neck squamous cell carcinoma (HNSCC) samples from a neoadjuvant trial of durvalumab +/- metformin. Paired pre- and post-treatment primary HNSCC tumor samples were stained for CD8+ and FoxP3+. Digital image analysis was used to determine estimated mean CD8+ and FoxP3+ CDs and CD8+-FoxP3+ IDs in the leading tumor edge (LTE) and tumor adjacent stroma (TAS) stratified by treatment arm, human papillomavirus (HPV) status, and pathologic treatment response. A subset of samples was characterized for T-cell related signatures using digital spatial genomic profiling. Post-treatment analysis revealed a significant decrease in FoxP3+ CD and an increase in CD8+ CDs in the TAS between patients receiving durvalumab and metformin versus durvlaumab alone. Both treatment arms demonstrated significant post-treatment increases in ID. Although HPV+ and HPV- had similar immune cell CDs in the tumor microenvironment, HPV+ pre-treatment samples had 1.60 times greater ID compared with HPV- samples, trending toward significance (p= 0.05). At baseline, pathologic responders demonstrated a 1.16-fold greater CD8+ CDs in the LTE (p= 0.045) and 2.28-fold greater ID (p= 0.001) than non-responders. Digital spatial profiling revealed upregulation of FoxP3+ and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) in the TAS (p= 0.006, p= 0.026) in samples from pathologic responders. Analysis of CD8+ and FoxP3+ detected population differences according to HPV status, pathologic response, and treatment. Greater CD8+-FoxP3+ ID was associated with pathologic response. CD8+ and FoxP3+ T-cell distributions may be predictive of response to immune checkpoint inhibition. gov (Identifier NCT03618654). 3 Laryngoscope, 133:1875-1884, 2023.

Mutational profiles of head and neck squamous cell carcinomas based upon human papillomavirus status in the Veterans Affairs National Precision Oncology Program.

Patients with advanced head and neck squamous cell carcinoma (HNSCC) associated with human papillomavirus (HPV) demonstrate favorable clinical outcomes compared to patients bearing HPV-negative HNSCC. We sought to characterize the association between HPV status and mutational profiles among patients served by the Veterans Health Administration (VHA). We performed a retrospective analysis of all Veterans with primary HNSCC tumors who underwent next-generation sequencing (NGS) through the VHA's National Precision Oncology Program between July 2016 and February 2019. HPV status was determined by clinical pathology reports of p16 immunohistochemical staining; gene variant pathogenicity was classified using OncoKB, an online precision oncology knowledge database, and mutation frequencies were compared using Fisher's exact test. A total of 79 patients met inclusion criteria, of which 48 (60.8%) had p16-positive tumors. Patients with p16-negative HNSCC were more likely to have mutations in TP53 (p < 0.0001), and a trend towards increased mutation frequency was observed within NOTCH1 (p = 0.032) and within the composite CDK/Rb pathway (p = 0.065). Mutations in KRAS, NRAS, HRAS, and FBXW7 were exclusively identified within p16-positive tumors, and a trend towards increased frequency was observed within the PI3K pathway (p = 0.051). No difference in overall mutational burden was observed between the two groups. In accordance with the previous studies, no clear molecular basis for improved prognosis among patients harboring HPV-positive disease has been elucidated. Though no targeted therapies are approved based upon HPV-status, current efforts to trial PI3K inhibitors and mTOR inhibitors across patients with HPV-positive disease bear genomic rationale based upon the current findings.

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

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

Impact of HPV status on immune responses in head and neck squamous cell carcinoma

The main objective of our study was to understand the impact of immune cell composition and the tumor-reactivity of tumor infiltrating lymphocytes (TIL) in HPV-positive (HPV+) and HPV-negative (HPV-) head and neck squamous cell carcinoma (HNSCC). TIL cultures were established from primary HNSCC tumors, the T cell subsets were phenotypically characterized using flow cytometry, and Interferon (IFN)-γ ELISA assay was used to determine TIL function. NanoString Immune Profiler was used to determine an immune signature by HPV-status, and multiplex immunohistochemistry (MIHC) was used to quantify immune cell distributions and their spatial relationships. Results showed that HPV+ and HPV- HNSCC had similar capacity to expand IFN-γ reactive TIL populations, and these TIL populations had similar characteristics. NanoString analysis revealed increased differential expression of genes related to B cell functions in HPV+ HNSCC, which were significant at a Benjamini-Yekutieli adjusted p-value of < 0.001. MIHC also displayed increased CD8+ T cell and CD19/CD20+ B cell densities in the tumor region of HPV+ HNSCC as opposed to HPV- HNSCC (p < 0.01). Increases in a combined metric of tumor B cell content and stromal plasma cell content was associated with increased progression-free survival in HPV- HNSCC patients treated with immune checkpoint inhibitor therapy (p = 0.03). In summary, TIL populations expanded from HPV+ and HPV- HNSCC displayed similar IFN-γ reactivity. However, we identified a strong B-cell signature present within HPV+ HNSCC, and higher B and plasma cell content associated with improved PFS in HPV- HNSCC patients treated with immune checkpoint inhibitors.

Self-assembling protein nanocarrier for selective delivery of cytotoxic polypeptides to CXCR4+ head and neck squamous cell carcinoma tumors

Loco-regional recurrences and distant metastases represent the main cause of head and neck squamous cell carcinoma (HNSCC) mortality. The overexpression of chemokine receptor 4 (CXCR4) in HNSCC primary tumors associates with higher risk of developing loco-regional recurrences and distant metastases, thus making CXCR4 an ideal entry pathway for targeted drug delivery. In this context, our group has generated the self-assembling protein nanocarrier T22-GFP-H6, displaying multiple T22 peptidic ligands that specifically target CXCR4. This study aimed to validate T22-GFP-H6 as a suitable nanocarrier to selectively deliver cytotoxic agents to CXCR4+ tumors in a HNSCC model. Here we demonstrate that T22-GFP-H6 selectively internalizes in CXCR4+ HNSCC cells, achieving a high accumulation in CXCR4+ tumors in vivo, while showing negligible nanocarrier distribution in non-tumor bearing organs. Moreover, this T22-empowered nanocarrier can incorporate bacterial toxin domains to generate therapeutic nanotoxins that induce cell death in CXCR4-overexpressing tumors in the absence of histological alterations in normal organs. Altogether, these results show the potential use of this T22-empowered nanocarrier platform to incorporate polypeptidic domains of choice to selectively eliminate CXCR4+ cells in HNSCC. Remarkably, to our knowledge, this is the first study testing targeted protein-only nanoparticles in this cancer type, which may represent a novel treatment approach for HNSCC patients.

Preliminary Computed Tomography Radiomics Model for Predicting Pretreatment CD8+ T-Cell Infiltration Status for Primary Head and Neck Squamous Cell Carcinoma.

Immunotherapy has emerged as a treatment option for head and neck squamous cell carcinoma (HNSCC), with tumor response being linked to the CD8+ T-cell inflammation. The purpose of this study is to assess whether computed tomography (CT) radiomic analysis can predict CD8+ T-cell enrichment in HNSCC primary tumors. This retrospective study included 71 patients from a head and neck cancer genomics cohort with CD8+ T-cell enrichment status. Pretreatment contrast-enhanced neck CT scans were retrospectively reviewed using 3D Slicer for primary lesion segmentation.The SlicerRadiomics extension was used to extract 107 radiomic features. Ridge regression and lasso regression were applied for feature selection and model construction. Lasso regression defined Coarseness as the most important variable, followed by SmallDependenceEmphasis, SmallAreaLowGrayLevelEmphasis, Contrast.1, and Correlation.Ridge regression defined Coarseness as the most important variable, followed by SmallDependenceLowGrayLevelEmphasis, Contrast.1, DependenceNonUniformityNormalized, and Idmn. These variables identified by lasso and ridge regressions were used to create a combined logistic regression model. The area under the curve (AUC) for the lasso-generated model was 0.786 (95% confidence interval [CI], 0.532-1.000), and the AUC for the ridge-generated model was 0.786 (95% CI, 0.544-1.000). Combining the radiomic variables identified by lasso and ridge regressions with clinical characteristics including alcohol use, tobacco use, anatomic site, and initial T stage produced a model with an AUC of 0.898 (95% CI, 0.731-1.000). T-cell inflammation status of HNSCC primary tumors can be predicted using radiomic analysis of CT imaging and thereby help identify patients who would respond well to immunotherapy.

Abstract 1794: Human cancer associated fibroblast subsets can differentially regulate immunotherapy response in head and neck cancer patients

Abstract The stromal cancer associated fibroblasts (CAF) have been postulated to modulate immune responses, but their role in clinical immunotherapy has not been rigorously explored in detail. To determine potential biomarkers of response and resistance for αPD-1 therapy in head &amp; neck cancer patients, we performed high dimensional immunophenotyping to include single cell RNA sequencing (scRNA-SEQ) from a neoadjuvant trial of 50 advanced-stage head and neck squamous cell carcinoma (HNSCC) patients that were treated with nivolumab. Sufficient suspension cells from the tumors harvested pre and post nivolumab treatment allowed single-cell RNA sequencing on 4 patients as well as bulk RNA sequencing on 40 patients. Using the Algorithm for the Reconstruction of Accurate Cellular Networks (ARACNe) and Virtual Inference of Protein-activity by Enriched Regulon (VIPER) bioinformatic analysis platform on the matched scRNA-Seq dataset to capture low expressing genes such as transcription factors, we identified 14 distinct cell types present in HNSCC patients. While CD8 T cells did not show increased number among the responder cohort, T cell cluster analysis showed enhanced IFNγ, granzyme, and perforin. Of these 14 cell types, the CAF cluster showed significant changes among the nivolumab responder cohort. Upon deeper analysis of this fibroblast population, we identified 5 distinct clusters of cancer associated fibroblast subsets (CAF-A, B, C, D, and E) of which, one cluster, CAF-A correlated with patient response to αPD-1 therapy. In order to determine the significance of this CAF subset's function, we isolated CD45-EpCAM-CD31-CD29+FAP+ CAF-A's from primary HNSCC tumor specimens and co-cultured with primary human T cells. Analysis by flow cytometry showed that CAF-A suppresses both CD4+ and CD8+ T cell proliferation as well as activation of these T cells. When we queried the cancer genome atlas, CAF-A gene signature correlated with increased survival, corroborating the important regulatory role of these CAF subsets in the context of endogenous immune responses. Overall, our findings demonstrate the functional importance of CAF subsets in modulating the immunoregulatory milieu of the human HNSCC for the first time, and justifies a more in depth characterization of these stromal cells in human squamous carcinoma in the current era of cancer immunotherapy. Citation Format: Diana Graves, Michael J. Korrer, Aleksandar Z. Obradovic, Sohini Roy, Andrea Califano, Charles G. Drake, Young J. Kim. Human cancer associated fibroblast subsets can differentially regulate immunotherapy response in head and neck cancer patients [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 1794.

SEC61G identified as a prognostic biomarker of head and neck squamous cell carcinoma

PurposeIt is of obvious interest to identify clinical prognosis-related oncogenes in HNSCC (head and neck squamous cell carcinoma).MethodsBased on the available datasets within the TCGA (The Cancer Genome Atlas) and the GEO (Gene Expression Omnibus) databases, the potential mechanism of action of the SEC61G (SEC61 translocon subunit gamma) gene in HNSCC tumorigenesis was explored by several bioinformatics approaches.ResultsThere was a higher expression level of SEC61G in primary HNSCC tumor tissues than in normal tissues. Moreover, highly expressed SEC61G was statistically associated with the poor survival prognosis of HNSCC patients. When HPV (human papilloma virus) was considered, we also observed a relatively lower proportion of “arm-level gain” and “high amplification” types of CNA (copy-number alteration) in the HNSCC-HPV (+) group than in the HNSCC-HPV (−) group. Additionally, we identified SEC61G CAN-correlated genes, such as CCT6A (chaperonin-containing TCP1 subunit 6A) and HUS1 (HUS1 checkpoint clamp component), and found a correlation between SEC61G copy-number segments and prognosis related to overall and progression-free survival intervals of HNSCC patients. Moreover, the molecular regulation mechanisms of the spliceosome, ribosome, proteasome degradation, cell adhesion, and immune infiltration of B and CD8+ T cells may contribute to the involvement of SEC61G in the pathogenesis of HNSCC.ConclusionsThe SEC61G gene was identified for the first time as a prognostic biomarker of HNSCC. The detailed underlying mechanism merits further research.

A novel diphtheria toxin-based bivalent human EGF fusion toxin for treatment of head and neck squamous cell carcinoma.

Epidermal growth factor receptor (EGFR) is often overexpressed in head and neck squamous cell carcinoma (HNSCC) and represents a top candidate for targeted HNSCC therapy. However, the clinical effectiveness of current Food and Drug Administration (FDA)‐approved drugs targeting EGFR is moderate, and the overall survival rate for HNSCC patients remains low. Therefore, more effective treatments are urgently needed. In this study, we generated a novel diphtheria toxin‐based bivalent human epidermal growth factor fusion toxin (bi‐EGF‐IT) to treat EGFR‐expressing HNSCC. Bi‐EGF‐IT was tested for in vitro binding affinity, cytotoxicity, and specificity using 14 human EGFR‐expressing HNSCC cell lines and three human EGFR‐negative cancer cell lines. Bi‐EGF‐IT had increased binding affinity for EGFR‐expressing HNSCC compared with the monovalent version (mono‐EGF‐IT), and both versions specifically depleted EGFR‐positive HNSCC, but not EGFR‐negative cell lines, in vitro. Bi‐EGF‐IT exhibited a comparable potency to that of the FDA‐approved EGFR inhibitor, erlotinib, for inhibiting HNSCC tumor growth in vivo using both subcutaneous and orthotopic HNSCC xenograft mouse models. When tested in an experimental metastasis model, survival was significantly longer in the bi‐EGF‐IT treatment group than the erlotinib treatment group, with a significantly reduced number of metastases compared with mono‐EGF‐IT. In addition, in vivo off‐target toxicities were significantly reduced in the bi‐EGF‐IT treatment group compared with the mono‐EGF‐IT group. These results demonstrate that bi‐EGF‐IT is more effective and markedly less toxic at inhibiting primary HNSCC tumor growth and metastasis than mono‐EGF‐IT and erlotinib. Thus, the novel bi‐EGF‐IT is a promising drug candidate for further development.

Validation of Gene Profiles for Analysis of Regional Lymphatic Metastases in Head and Neck Squamous Cell Carcinoma.

The progress of Head and Neck Squamous Cell Carcinoma (HNSCC) is dependent on both cancer stem cells (CSCs) and immune suppression. This study was designed to evaluate the distribution of CSCs and the characteristic immune suppression status in HNSCC primary tumors and lymph nodes. A total of 303 lymph nodes from 25 patients, as well as tumor and adjacent normal tissue samples, were evaluated by a quantitative PCR assay of the markers of CSCs and the characteristic immune suppression. Expressions of selected genes in The Cancer Genome Atlas (TCGA) datasets were also analyzed. In the primary tumors, we found that expressions of CSCs markers (ALDH1L1, PECAM1, PROM1) were down-regulated, while immune suppression markers FOXP3, CD47, EGFR, SOX2, and TGFB1 were up-regulated significantly when compared to that in adjacent normal tissues. In the lymph nodes, expressions of both CSCs, and immune suppression markers were upregulated significantly compared with that in primary tumors. The mRNA expression of selected CSCs and immune suppression markers exhibited the highest expression in the level II of metastasis, then declined in the level III and remained constant at a reduced value in levels IV and V of metastases. These results reveal a comprehensive understanding of the unique genetic characteristics associated with metastatic loci and potential routes of lymphatic dissemination of HNSCC, which helps to explain why the level II has a high incidence of lymph node metastasis, and why skip metastasis straight to the level IV or level V is rarely found in the clinic.

Molecular dissection of the oncogenic role of ETS1 in the mesenchymal subtypes of head and neck squamous cell carcinoma.

Head and Neck Squamous Cell Carcinoma (HNSCC) is a heterogeneous disease of significant mortality and with limited treatment options. Recent genomic analysis of HNSCC tumors has identified several distinct molecular classes, of which the mesenchymal subtype is associated with Epithelial to Mesenchymal Transition (EMT) and shown to correlate with poor survival and drug resistance. Here, we utilize an integrated approach to characterize the molecular function of ETS1, an oncogenic transcription factor specifically enriched in Mesenchymal tumors. To identify the global ETS1 cistrome, we have performed integrated analysis of RNA-Seq, ChIP-Seq and epigenomic datasets in SCC25, a representative ETS1high mesenchymal HNSCC cell line. Our studies implicate ETS1 as a crucial regulator of broader oncogenic processes and specifically Mesenchymal phenotypes, such as EMT and cellular invasion. We found that ETS1 preferentially binds cancer specific regulator elements, in particular Super-Enhancers of key EMT genes, highlighting its role as a master regulator. Finally, we show evidence that ETS1 plays a crucial role in regulating the TGF-β pathway in Mesenchymal cell lines and in leading-edge cells in primary HNSCC tumors that are endowed with partial-EMT features. Collectively our study highlights ETS1 as a key regulator of TGF-β associated EMT and reveals new avenues for sub-type specific therapeutic intervention.

Abstract 3399: Uncovering hidden sources of transcriptional dysregulation arising from inter- and intra-tumor heterogeneity

Abstract Introduction: This study develops an innovative computational framework, Expression Variation Analysis (EVA), to model transcriptional dysregulation in cancer. Heterogeneity poses a major challenge in translational research. For example, inter-tumor heterogeneity limits the biomarker discovery and intra-tumor heterogeneity enables therapeutic resistance. Moreover, in some cancers driver mutations are insufficient to account for the widespread transcriptional variation responsible for these outcomes. Thus, new computational tools to model transcriptional variation are essential. Methods: EVA is a unified computational framework to model transcriptional variation in cancer. Briefly, EVA quantifies transcriptional heterogeneity for one set of samples or cells from one phenotype using the expected dissimilarity between pairs of expression profiles. U-statistics theory can then quantify the statistical significance of the difference in transcriptional heterogeneity between phenotypes. Results: We apply EVA to perform a comprehensive characterization of transcriptional variation in head and neck squamous cell carcinoma (HNSCC). At a pathway level, transcriptional variation in HNSCC tumors is higher than normal controls. Applying EVA to integrate ChIP-seq data with RNA-seq reveals that these pervasive transcriptional differences occur in enhancers. Similarly, applying EVA at a gene level to model splicing reveals more heterogeneity in transcript usage in tumor samples than normals. HPV- HNSCC tumors are unique in having mutations in genes that regulate the splicing machinery, and the HPV- tumors with these alterations have a greater number of dysregulated splice variants than those without. Nonetheless, the EVA analysis identifies a similar number of alternative splice variants in HPV+ as HPV- tumors suggesting an alternative mechanism of transcriptional heterogeneity in HPV+ disease. Adapting EVA to single cell data demonstrates that increased fibroblast composition is associated with greater variation in immune pathway activity in HNSCC. Moreover, we observe greater transcriptional heterogeneity in HNSCC primary tumors than lymph node metastasis consistent with a clonal outgrowth. Conclusions: We demonstrate that the statistical framework from EVA enables differential heterogeneity analysis in HNSCC ranging from pathway dysregulation, splice variation, epigenetic regulation, and single cell analysis. This algorithm provides a critical framework to model the hidden multi-molecular mechanisms underlying the complex patient outcomes that are pervasive in cancer. Citation Format: Bahman Afsari, Leslie Cope, Daria A. Gaykalova, Donald Geman, Sidharth Puram, Loyal A. Goff, Alexander Favorov, Elana Judith Fertig. Uncovering hidden sources of transcriptional dysregulation arising from inter- and intra-tumor heterogeneity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3399.

Abstract 4697: Expression variation analysis for tumor heterogeneity in single-cell RNA-sequencing data

Abstract Introduction: Tumor heterogeneity provides a complex challenge to cancer treatment and is a critical component of therapeutic response, disease recurrence, and patient survival. Single-cell RNA-sequencing (scRNA-seq) technologies reveal the prevalence of intra- and inter-tumor heterogeneity. Computational techniques are essential to quantify the differences in variation of these profiles between distinct cell types, tumor subtypes, and patients to fully characterize intra- and inter-tumor molecular heterogeneity. To address this, we devised a new algorithm, Expression Variation Analysis in Single Cells (EVAsc), to perform multivariate statistical analyses of differential variation of expression in gene sets for scRNA-seq. Methods: EVAsc provides a robust statistical test to compare the heterogeneity of transcriptional profiles of genes in a pathway between groups of cells from two phenotypes. Using simulated data, we demonstrated that this method is robust for imputed scRNA-seq data with high sensitivity and specificity to detect pathways with true differential heterogeneity. We then applied EVAsc to public domain scRNA-seq tumor datasets in breast cancer and head and neck squamous cell carcinoma (HNSCC) to quantify the landscape of tumor heterogeneity in several key applications in cancer genomics, i.e. immunogenicity, cancer subtypes, and metastasis. Results: We demonstrated that immune pathway heterogeneity in hematopoietic cell populations in breast tumors corresponded to the amount diversity present in the T-cell repertoire of each individual. In HNSCC patients, we found dramatic differences in pathway dysregulation across basal primary tumors, indicative of inter-tumor heterogeneity within a single subtype. Within the basal primary tumors we also identified increased immune dysregulation in individuals with a high proportion of fibroblasts present in the tumor microenvironment. Moreover, cells in HNSCC primary tumors had significantly more heterogeneity across pathways than their matched metastatic cells, consistent with a model of clonal outgrowth. Conclusions: The results of these analyses demonstrate the broad utility of EVAsc to quantify inter- and intra-tumor heterogeneity from scRNA-seq data without reliance on low dimensional visualization. EVAsc is a robust multivariate statistical method to quantify differential variation of pathway gene expression and provides the ability to explore transcriptional variation in numerous disease and normal contexts at a single cell resolution. Accurate characterization of inter-sample variation from scRNA-seq data of tumors is critical to quantify the cellular heterogeneity that drives tumor progression through dysregulation of key cancer pathways. Thus, identifying dysregulated pathways in individual tumors may be an important biomarker for clinical response to immunotherapy. Citation Format: Emily F. Davis-Marcisak, Pranay Orugunta, Genevieve Stein-O'Brien, Sidharth V. Puram, Evanthia Roussos Torres, Alexander Hopkins, Elizabeth M. Jaffee, Alexander V. Favorov, Bahman Afsari, Loyal A. Goff, Elana J. Fertig. Expression variation analysis for tumor heterogeneity in single-cell RNA-sequencing data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4697.

Abstract 98: Stromal-targeting with quercetin in patient-derived models of head and neck squamous cell carcinoma (HNSCC)

Abstract Head and neck squamous cell carcinoma (HNSCC) is an aggressive cancer with a high recurrence rate. Recurrences occur in ~25% cases of early -stage patients (Stage I-II) and 50-60% cases of advanced stage (Stage II-IV) patients. Cumulative evidences suggest that tumor stroma, especially cancer-associated fibroblast (CAF) plays a protective role and contributes to drug-resistance in HNSCC, leading to recurrences after treatment. Quercetin is a flavonoid purified from natural plants, such as green tea, onions and berries. It has been reported to suppress CAFs and reduce collagen accumulation surrounding tumors, potentially facilitating drug delivery to tumors. Here, we hypothesize that quercetin targeting of CAFs may enhance the anti-tumor efficacy of cisplatin, and potentially other antitumor therapies in HNSCC. Using a primary co-culture system with primary HNSCC cells (70%) and the paired CAFs (30%) from HNSCC patients, we investigated the effect of quercetin and cisplatin combination treatment in vitro. We found that combination of quercetin and cisplatin elicited a significantly greater growth inhibition (70% of inhibition) as compared to 37% of inhibition with cisplatin and 15% of inhibition with quercetin alone. This result appeared to demonstrate some anti-CAF effects and antitumor effects exerted by this combination strategy.Using patient-derived HNSCC primary tumor cells, stromal-rich xenografts were successfully developed in vivo. The stromal-suppressive effect of quercetin was then examined upon treatment with increasing doses of quercetin (DMF, 2.5, 5 or 10 μM in 5 microliter volume, 2 times injection). Potential alteration of the tumor architecture by quercetin (vs. vehicle treatment) was examined by H&amp;E staining. A marked reduction of stromal component was found in quercetin -treated tumors vs. vehicle-treated tumors. Importantly, we also found that these quercetin-treated tumors have 80.4% reduction in α-SMA protein expression (6.0% in vehicle vs.1.2% in treatment group), strongly indicating a significant stromal suppressive action of quercetin in an HNSCC primary culture-derived xenograft model. Furthermore, quercetin (10 μM) treatment reduced collagen expression by ~80% (vs. vehicle treatment) in the HNSCC primary culture-derived xenograft model by Masson’s trichrome staining. In conclusion, quercetin can effectively suppress the activated status of CAFs and may potentially enhance anti-tumor effect of cisplatin in an HNSCC patient-relevant manner. The antitumor efficacy of quercetin combination strategies should warrant further investigations. Citation Format: Hui Li, Wenying Piao, Pik Yuk Lau, Chin Wang Lau, Jason Ying Kuen Chan, Yu Xiong Su, Leaf Huang, Kai Li Hu, Vivian Wai Yan Lui. Stromal-targeting with quercetin in patient-derived models of head and neck squamous cell carcinoma (HNSCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 98.

Abstract 4033: RAC1 genomic aberrations as predictive biomarkers for head and neck squamous cell carcinoma (HNSCC)

Abstract RAC1 is a small GTPase with known oncogenicity. Mutations of RAC1 are highly relevant to melanoma. Yet, little is known about RAC1 aberrations in other human cancers, including HNSCC. Pan-cancer analysis of 32 cancer types from the TCGA reveals that RAC1 mutations only affect 12/32 cancer types and HNSCC is the 2nd most frequently affected (2.64%; 14/510 cases), after melanoma (4.59%, 22/479 cases). 28/34 cancer types harbor RAC1 copy number alterations, including HNSCC (2.08% cases, all are amplifications). Among all 14 HNSCC-associated RAC1 mutations found in the TCGA cohort, RAC1 p.A159V mutation (residing in the highly conserved G5 Box of RAC1) appears to be particularly prevalent in HNSCC (6/14 cases), suggesting a likely mutant-specific role in HNSCC. HNSCC patients with somatic RAC1 aberrations [gene amplification/copy number gain/hotspot mutations (p.P29, p.K116 and p.A159)] have poorer overall survival (OS)(P=4.555e-5; median survival of 30.91 months vs 68.43 months) and disease-free survival (DFS) (P=5.49e-5; 27.89 months vs unreached median) vs. RAC1-unaltered HNSCC patients. RAC1-mutated tumors are also associated with a higher rate of TP53 mutation (P=2.07e-6), and a lower rate of HPV infection (P=7.397e-6; 7.7% vs. 87.2%; Fisher’s Exact test). Further, RAC1-altered patients are of more advanced T clinical staging (T3/4 vs T1/2; P=0.0312) and with more gross/microscopic extension (P=0.0111; 59/119 vs 54/201). These evidences strongly suggest that HNSCC with RAC1 aberrations are more aggressive and may serve as a prognostic biomarker for HNSCC. To further elucidate the biological function of RAC1 mutations in HNSCC, we analyzed the mutational profile of RAC1-mutated HNSCC tumors which showed enriched mutations of 3 tumor suppressor genes: FAT1, FAT4 and CSMD3 (P=0.0001, P=0.0038 and P=0.0467 respectively). Gene set enrichment analysis (GSEA) of RAC1-mutated HNSCC also demonstrates significant dysregulation of immune-related gene sets (vs. RAC1-WT tumors) including increased interleukin-6 (IL-6) production (P&amp;lt;0.0001), a pro-tumorigenic inflammatory cytokine known to be involved in HNSCC tumorigenesis and progression. A previous study showed that overexpression of RAC1 activating mutation (p.V12) could upregulate IL-6 production in a cervical cancer model, HeLa, which is supportive of a RAC1/IL-6 axis in HNSCC. This is further supported by our RNAseq finding that RAC1-mutated HNSCC tumors do have significant upregulation of MYD88 (P=0.0436), a gene that has been shown to induce IL-6 secretion in HNSCC cells. Subsequent Tumor Immune Estimation Resource (TIMER) analysis shows that RAC1-mutated HNSCC primary tumors have higher infiltrating neutrophil levels than RAC1-WT tumors (P=0.019). Our findings may uncover a novel tumorigenic mechanism by RAC1 mutations in HNSCC, first linking IL-6 dysregulation to an oncogene mutation in HNSCC. Citation Format: Hoi Lam Ngan, Yuchen Liu, Peony Hiu Yan Poon, Vivian Wai Yan Lui. RAC1 genomic aberrations as predictive biomarkers for head and neck squamous cell carcinoma (HNSCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4033.

Overexpression of PIK3CA in head and neck squamous cell carcinoma is associated with poor outcome and activation of the YAP pathway

ObjectivesPhosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) is commonly altered in many human tumors, leading to the activation of p110α enzymatic activity that stimulates growth factor-independent cell growth. PIK3CA alterations such as mutation, gene amplification and overexpression are common in head and neck squamous cell carcinoma (HNSCC) and. We aim to explore how these alterations and clinical outcome are associated, as well as the molecular mechanisms involved. Material and methodsMutation and copy-number variation in PIK3CA, and whole-genome expression profiles, were analyzed in primary HNSCC tumors from The Cancer Genome Atlas (TCGA) cohort (n = 243). The results were validated in an independent cohort form the University Hospital of A Coruña (UHAC, n = 62). Expression of the PIK3CA gene protein product (PI3K p110α) and nuclear YAP were assessed in tissue microarrays in a cohort from the University Hospital 12 de Octubre (UH12O, n = 91). ResultsOnly high expression of the PIK3CA gene was associated with poor clinical outcome. The study of gene expression, transcription factor and protein signatures suggested that the activation of the Hippo-YAP pathway, involved in organ size, stem cell maintenance and tumorigenesis, could underlie tumor progression in PI3KCA overexpressing tumors. Tissue arrays showed that PI3K p110α levels correlated with YAP nuclear localization in HNSCC tumors. ConclusionsHigh expression of PIK3CA in HNSCC primary tumors identifies patients at high risk for recurrence. In these tumors, progression could rely on the Hippo-YAP pathway instead of the canonical Akt/mTOR pathway. This observation could have important implications in the therapeutic options for patients.