Biomarkers For Head And Neck Squamous Cell Carcinoma Research Articles

Elemental Zn and its Binding Protein Zinc-\u03b12-Glycoprotein are Elevated in HPV-Positive Oropharyngeal Squamous Cell Carcinoma

Human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSCC) is biologically distinct from HPV-negative HNSCC. Outside of HPV-status, few tumor-intrinsic variables have been identified that correlate to improved survival. As part of exploratory analysis into the trace elemental composition of oropharyngeal squamous cell carcinoma (OPSCC), we performed elemental quanitification by X-ray fluorescence microscopy (XFM) on a small cohort (n = 32) of patients with HPV-positive and -negative OPSCC and identified in HPV-positive cases increased zinc (Zn) concentrations in tumor tissue relative to normal tissue. Subsequent immunohistochemistry of six Zn-binding proteins—zinc-α2-glycoprotein (AZGP1), Lipocalin-1, Albumin, S100A7, S100A8 and S100A9—revealed that only AZGP1 expression significantly correlated to HPV-status (p < 0.001) and was also increased in tumor relative to normal tissue from HPV-positive OPSCC tumor samples. AZGP1 protein expression in our cohort significantly correlated to a prolonged recurrence-free survival (p = 0.029), similar to HNSCC cases from the TCGA (n = 499), where highest AZGP1 mRNA levels correlated to improved overall survival (p = 0.023). By showing for the first time that HPV-positive OPSCC patients have increased intratumoral Zn levels and AZGP1 expression, we identify possible positive prognostic biomarkers in HNSCC as well as possible mechanisms of increased sensitivity to chemoradiation in HPV-positive OPSCC.

Review of emerging biomarkers in head and neck squamous cell carcinoma in the era of immunotherapy and targeted therapy.

Biomarkers in head and neck squamous cell carcinoma (HNSCC) emerge rapidly in recent years, especially for new targeted therapies and immunotherapies. Recent, relevant peer-reviewed evidence were critically reviewed and summarized. This review article briefly introduces essential biomarker concepts, including purposes and classifications (predictive, prognostic, and diagnostic markers), and the phases of biomarker development. We summarize current biomarkers in order of clinical utility; p16 and human papillomavirus status remain the most important and validated biomarkers in HNSCC. The rationale for biomarker study design continues to evolve with technological advances, especially whole-exome or whole-genomic sequencing. Noninvasive body fluid and liquid biopsy biomarkers appear to hold strong potential for development as tools for early cancer detection, cancer diagnosis, monitoring of disease recurrence, and outcome prediction. In light of discrepancies among different technologies, standardized approaches are needed. Biomarkers from cancer tissue or blood in HNSCC could direct new anticancer therapies.

Pretreatment Serum Levels of Soluble Cytokeratin Fragments (Cyfra 21-1, TPS, MonoTotal) in Relation to Clinical and Pathobiological Aspects of Head and Neck Squamous Cell Carcinomas.

The aim of this study was to analyze the pretreatment cytokeratin serum levels in head and neck squamous cell carcinoma (HNSCC) by three assays in relation to selected clinicopathological characteristics in an effort to find diagnostic/prognostic biomarkers for HNSCC and determine the best assay. In total, 46 patients with HNSCC with different subsite (oropharyngeal-21 cases, hypopharyngeal-4 and laryngeal-21) were included in this prospective study. MonoTotal, Cyfra 21-1, and TPS radioimmunoassay kits were used to analyze cytokeratin fragments serum levels. Statistically significant differences in serum levels of TPS and Cyfra 21-1 were found between low (stage I-II)- and high-stage (stage III-IV) tumors (p=0.0057; p=0.0138 respectively). Cyfra21-1 assay showed significant differences between tumors of different sites with prominent elevation being found in oropharyngeal carcinomas and between patients with p16 positive and p16 negative HNSCC (p=0.0242), being elevated in p16 positive tumors. The present study is the first to compare cytokeratin serum levels between various subgroups of HNSCC using three different assays. Cyfra 21-1 seems to be the most useful for clinical practice. The relation between elevated Cyfra 21-1 serum levels and p16 expression requires further investigation.

Biological Rationale for Radiation-induced Release of Circulating Tumor DNA

Rapid release of circulating tumor DNA (ctDNA) during cytotoxic therapy could provide an early indication of treatment efficacy. However, the biological basis for treatment-induced ctDNA release is poorly understood. To study this phenomenon, we selected head and neck squamous cell carcinoma (HNSCC) as an archetypal epithelial cancer treated with ionizing radiation (IR). We hypothesized that IR-induced ctDNA release would be correlated with intrinsic radiosensitivity, human papillomavirus (HPV) association, and propensity to undergo apoptosis. Nine HNSCC cell lines (3 HPV+, 6 HPV-) were grown in tissue culture for in vitro ctDNA kinetic analysis. Intrinsic radiosensitivity was determined by clonogenic assays. Apoptosis was measured by luminescent DEVD cleavage assay, triggered by staurosporine, and blocked by z-vad-fmk. Senescence was measured by SA-β-galactosidase activity, triggered in the fibroblast cell lines IMR90 and WI38 by transduction with oncogenic HrasG12V, and blocked by navitoclax. Three HNSCC cell lines (1 HPV+, 2 HPV-) were grown as subcutaneous xenografts in Nod-Rad-Gamma (NRG) mice for in vivo ctDNA kinetic analysis. Endpoint xenograft tumors were assessed for necrotic area (H&E) and for markers of apoptosis (cleaved-caspase 3), senescence (p21), vessel density (CD31), and hypoxia (CAIX) by immunohistochemistry. ctDNA was quantified from media or plasma by quantitative PCR using human-specific LINE-1 primers. Following IR, HNSCC cell lines exhibited variable magnitudes and kinetics of ctDNA release in vitro and in vivo with peak release observed after 72-96 hours. IR-induced ctDNA release was dose-dependent but not associated with intrinsic radiosensitivity, HPV status, or propensity to undergo apoptosis. Although caspase inhibition resulted in a near complete reduction in IR-induced caspase activity (-84%±8%), a comparatively minor reduction in ctDNA release (-29%±9%) was observed. There was an inverse association between IR-induced senescence and ctDNA release. HrasG12V-triggered senescence reduced ctDNA release, while navitoclax treatment reversed this effect. HNSCC models with greater IR-induced ctDNA release displayed more necrosis and less senescence in endpoint xenograft tumors; in contrast, neither vessel density nor hypoxia could account for differences in IR-induced ctDNA release between models. IR-induced ctDNA spikes are commonly observed within 2-3 days of treatment in preclinical HNSCC models. Our striking findings reveal the complex interplay between distinct mechanisms of cell death following IR exposure and their influence on ctDNA release. Necrosis and senescence are the major determinants of IR-induced ctDNA release, while apoptosis has a comparatively minor impact. This study will inform future work on implementing ctDNA as a response biomarker in HNSCC and other cancer types.

Evaluation of quantitative imaging parameters in head and neck squamous cell carcinoma.

Functional imaging such as 18F-fluoro-deoxy-glucose positron emission tomography/computed tomography (FDG-PET/CT), 18F-fluoro-misonidazole (F-MISO)-PET/CT, and diffusion-weighted magnetic resonance imaging (DW-MRI) can assess complex biological phenomena in tumors reflecting underlying disease biology. The aim of this prospective observational study was to correlate quantitative imaging parameters derived from pretreatment biological imaging such as FDG-PET/CT, F-MISO-PET/CT, and DW-MRI with each other and with clinical outcomes in patients with head and neck squamous cell carcinoma (HNSCC) treated with definitive radio(chemo)therapy. Twenty patients with pharyngo-laryngeal cancers underwent pretreatment biological imaging. Gross tumor volume (GTV) was delineated on axial planning CT (GTV<inf>CT</inf>). Quantitative FDG-PET/CT parameters included maximum, mean, minimum standardized uptake values (SUV<inf>max-FDG</inf>, SUV<inf>mean-FDG</inf>, SUV<inf>min-FDG</inf>); metabolic tumor volume (MTV); and total lesion glycolysis (TLG). F-MISO-PET/CT parameters included hypoxic tumor volume (HTV); maximum, mean, minimum SUV; and fractional hypoxic volume (FHV). Mean apparent diffusion coefficient (ADC<inf>mean</inf>) was derived from DW-MRI. There was moderately strong positive correlation (r=0.616, P=0.005) between GTV<inf>CT</inf> and MTV. HTV derived from F-MISO-PET/CT at 3-hours (HTV<inf>3hrs-F-MISO</inf>) showed strong positive correlation with GTV<inf>CT</inf> (r=0.753, P<0.0001) and MTV (r=0.796, P<0.0001) respectively. ADC<inf>mean</inf> showed strong positive correlations with SUV<inf>mean-5hrs-F-MISO</inf> (r=0.713, P=0.021) and SUV<inf>min-5hrs-F-MISO</inf> (r=0.731, P=0.016) respectively. A moderate negative correlation (r=-0.500, P=0.049) was observed between ADC<inf>mean</inf> and MTV. At a median follow up of 44 months, the 5-year Kaplan-Meier estimates of loco-regional control, disease-free survival, and overall survival were 53%, 43%, and 40% respectively. Larger volume of primary tumor (GTV<inf>CT</inf>>22cc and MTV>7.9cc) and increasing hypoxia (HTV<inf>3hr-F-MSO</inf>>4.9cc) were associated with worse outcomes. Functional imaging represents an attractive and non-invasive modality to assess complex biological phenomena in solid tumors. Larger tumor volume and increasing hypoxia emerged as putative prognostic imaging biomarkers in HNSCC.

Diagnostic Tumor Markers in Head and Neck Squamous Cell Carcinoma (HNSCC) in the Clinical Setting.

Head and neck squamous cell carcinoma (HNSCC) represents a group of tumors arising in the oral cavity, oropharynx, and larynx. Although HNSCC is traditionally associated with tobacco and alcohol consumption, a growing proportion of head and neck tumors, mainly of the oropharynx, are associated with Human Papilloma Virus (HPV). Recurrent/metastatic disease is characterized by dismal prognosis and there is an unmet need for the development of biomarkers for detection of early disease, accurate prediction of prognosis, and appropriate selection of therapy. Based on the REMARK guidelines, a variety of diagnostic and prognostic biomarkers are being evaluated in clinical trials but their clinical significance is doubtful. Herein, we will focus on biomarkers in HNSCC used in the clinical setting and we will illustrate their clinical relevance.

Morphomolecular analysis of the immune tumor microenvironment in human head and neck cancer.

Immunotherapy is effective in head and neck squamous cell carcinoma (HNSCC), but only a minority of patients responds to immune checkpoint blockade (ICB). To contribute to a better understanding of the underlying immune biology, we combined histomorphological evaluation and molecular analysis of the HNSCC immune microenvironment in the TCGA cohort. Analyzing digital HE-stained slides, a method for classification of tumor infiltrating lymphocytes (TILs) in the intra-epithelial compartment (ieTILs, present vs. absent) and the stromal compartment (strTILs, high vs. low) was established. We also analyzed the abundance of eight immune cell populations (estimated from RNAseq data) and PD-L1 mRNA expression. Status of ieTILs and status of strTILs were concordant for 61%, but discordant for 39% of tumors. In univariate survival analysis, ieTILs were a positive prognostic marker for DFS in the study cohort (HR = 0.66, p = 0.015) and in the HPV- subcohort (HR = 0.68, p = 0.04), but not in the HPV + subcohort. T cells were a positive prognostic marker for DFS in the study cohort (HR = 0.80, p = 0.03) and in the HPV + subcohort (HR = 0.20, p = 0.001), but not in the HPV- subcohort. In univariate survival analysis, PD-L1 mRNA expression was neither associated with DFS nor with OS. However, in bivariate and multivariate analyses including both PD-L1 mRNA levels and T cells, PD-L1 was a negative prognostic marker of DFS and OS, while T cells remained a positive prognostic marker. In conclusion, ieTILs and strTILs were non-redundant biomarkers in HNSCC and should be evaluated separately. The identified prognostic markers should be evaluated for predictivity in ICB-treated patients.

Upregulation of the long non-coding RNA CASC9 as a biomarker for squamous cell carcinoma

BackgroundFew diagnostic and prognostic biomarkers are available for head-and-neck squamous cell carcinoma (HNSCC). Long non-coding RNAs (lncRNAs) have shown promise as biomarkers in other cancer types and in some cases functionally contribute to tumor development and progression. Here, we searched for lncRNAs useful as biomarkers in HNSCC.MethodsPublic datasets were mined for lncRNA candidates. Two independent HNSCC tissue sets and a bladder cancer tissue set were analyzed by RT-qPCR. Effects of lncRNA overexpression or downregulation on cell proliferation, clonogenicity, migration and chemosensitivity were studied in HNSCC cell lines.ResultsData mining revealed prominently CASC9, a lncRNA significantly overexpressed in HNSCC tumor tissues according to the TCGA RNAseq data. Overexpression was confirmed by RT-qPCR analyses of patient tissues from two independent cohorts. CASC9 expression discriminated tumors from normal tissues with even higher specificity than HOTAIR, a lncRNA previously suggested as an HNSCC biomarker. Specificity of HNSCC detection by CASC9 was further improved by combination with HOTAIR. Analysis of TCGA pan-cancer data revealed significant overexpression of CASC9 across different other entities including bladder, liver, lung and stomach cancers and especially in squamous cell carcinoma (SCC) of the lung. By RT-qPCR analysis we furthermore detected stronger CASC9 overexpression in pure SCC of the urinary bladder and mixed urothelial carcinoma with squamous differentiation than in pure urothelial carcinomas. Thus, CASC9 might represent a general diagnostic biomarker and particularly for SCCs. Unexpectedly, up- or downregulation of CASC9 expression in HNSCC cell lines with low or high CASC9 expression, respectively, did not result in significant changes of cell viability, clonogenicity, migration or chemosensitivity.ConclusionsCASC9 is a promising biomarker for HNSCC detection. While regularly overexpressed, however, this lncRNA does not seem to act as a major driver of development or progression in this tumor.

NOVA1 induction by inflammation and NOVA1 suppression by epigenetic regulation in head and neck squamous cell carcinoma

Neuro-oncological ventral antigen 1 (NOVA1) is known as a neuron-specific pre-mRNA binding splicing factor. Previously, it was shown to be highly upregulated in T lymphocytes, as well as fibroblasts/stromal spindle cells, in tertiary lymphoid tissues formed by the benign immune-inflammatory process, while it was frequently downregulated in tumor cells and other cells within the tumor microenvironment. Here, we sought to identify the mechanisms of NOVA1 modulation in head and neck squamous cell carcinoma (HNSCC). NOVA1 was induced by inflammatory-immune signals within the tumor microenvironment and was suppressed by epigenetic dysregulation, such as that with miR-146. We found attenuated expression of NOVA1 to be associated with non-oropharynx sites such as oral cavity, hypopharynx, and larynx, human papilloma virus (HPV)-negative SCC defined by immunohistochemistry for p16INK4a expression, fewer tumor infiltrating lymphocytes, and poor patient outcomes. Moreover, changes were discovered in epithelial mesenchymal transition-associated markers according to NOVA1 status. This study provides some insights to the underlying mechanism of NOVA1 regulation and suggests that NOVA1 may serve as a prognostic biomarker and potential therapeutic target for HNSCC in the future.

Prognostic 4-lncRNA-based risk model predicts survival time of patients with head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a common malignant disease with high mortality rates. Recently, long non-coding RNAs (lncRNAs) have been demonstrated to participate in a number of important biological functions and could serve as prognostic biomarkers in the field of oncology. Therefore, the present study aimed to identify an lncRNA-based model that was associated with prognosis. RNA-sequencing data was downloaded from The Cancer Genome Atlas and R software was used to analyze the data. Univariate analyses, robust likelihood analyses and multivariate analyses were performed to screen out key lncRNA candidates associated with prognosis and construct a risk model. A Kaplan-Meier plot was constructed for survival analysis. LncBase and Starbase were used to identify the miRNA and protein targets. Gene set enrichment analysis was used for functional analysis. As a result, a 4-lncRNA (ALMS1-IT1, RP11-359J14.2, CTB-178M22.2 and RP11-347C18.5) based risk model was identified and patients in the high-risk group were revealed to have a lower survival rate than patients in the low-risk group. A nomogram that could predict the survival of patients was plotted. A total of 79 target miRNAs and 61 target proteins were identified. The gene set enrichment analysis results revealed that nutrient metabolism pathways were enriched in the high-risk group and immune regulation pathways were enriched in the low-risk group. In summary, a 4-lncRNA based risk model was identified that was associated with prognosis, which may serve as a prognosis prediction biomarker for HNSCC.

Antitumour effects of metformin and curcumin in human papillomavirus positive and negative head and neck cancer cells.

The incidence of oropharyngeal squamous cell carcinoma (OPSCC) has significantly increased in recent decades due to human papillomavirus (HPV)-mediated oncogenesis. Unfortunately, a growing number of HPV-positive (+) OPSCC survivors are living with the irreversible side effects of treatment. The novel, well-tolerated chemotherapeutics with improved side effect profiles are, therefore, in high demand. Metformin is one such drug, widely used as a first-line oral agent in the treatment of type 2 diabetes mellitus. Curcumin is another well-tolerated agent quickly gaining attention for its medicinal properties. Both metformin and curcumin have been shown to display anticancer properties. This study aimed to determine the antitumor effects of these agents, individually and combined, in HPV+​​​​ ​​​and HPV-negative (-) head and neck squamous cell carcinoma (HNSCC) cell lines. This was achieved by assessing the efficacy of varying drug concentrations on the overall cell viability, proliferation, and expression of common HNSCC biomarkers. The results from protein and RNA expression data are highly variable, as expected, with multiple pathways being affected in cancer. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays and immunofluorescence microscopy suggest that both agents are capable of slowing proliferation and inducing apoptosis. We conclude that curcumin and metformin display effective antitumor effects in both HPV+ and HPV- HNSCC cell lines. The curcumin effects appear more pronounced in the HPV- cell lines. Metformin appears to be more effective at reducing the overall cell numbers in HPV+ cell lines. Metformin and curcumin combined did not appear to have synergistic effects on the proliferation or apoptosis of the treated cell lines.

Combined Identification of Three miRNAs in Serum As Effective Diagnostic Biomarkers for HNSCC

Background: Head and neck squamous cell carcinoma (HNSCC) is a disastrous disease with substantial morbidity and mortality. This study aims to explore the effective diagnostic and prognostic biomarker for HNSCC. Methods: MiRNA expression data and corresponding clinical information of HNSCC from The Cancer Genome Atlas (TCGA) database were analyzed comprehensively to identify the differentially expressed miRNAs. The predictive ability of different classification was analyzed of the three-miRNA combinations. Then, diagnostic and prognostic value were evaluated and verified in clinical patients. Results: A total of 486 HNSCC samples and 44 normal samples were obtained from the TCGA database. After analysis, 128 differentially expressed miRNAs in HNSCC tissues were identified, and 10 miRNAs were finally selected for further study. Classification analysis developed a threemiRNA signature of hsa-mir-383, hsa-mir-615 and hsa-mir-877 with an average AUC (Area Under the Curve) of 99.4% in differentiating tumor tissues from normal tissues. The combination of the three miRNAs in serum yielded an AUC value of 0.986 (95%CI, 0.974-0.998; P＜0.0001) with 97.5% sensitivity and 94.6% specificity, which was superior to that of a single or a two-miRNA signature. One-way ANOVA test results suggested the expression levels of hsa-mir-615 and hsa-mir-877 were significantly changed with different tumor grades, with the p-values of 0.001 and 0.056, respectively, while hsa-mir-383 showed no significant correlation with tumor grade (P=0.18). And no significant correlations were found between hsa-mir-383, hsa-mir-615 or hsa-mir-877 and tumor stage with the p-values of 0.14, 0.95 and 0.81, respectively. Survival analysis indicated different expression levels of hsa-mir-383 led to significantly different survival rates (P<0.022), while no significant survival differences were observed between the high and low expression set of hsamir-615 (P<0.401) or hsa-mir-877 (P<0.403). This result was verified in 81 validation patients. The low expression level of hsa-mir-383 was significantly correlated with poor prognosis (P=0.0404), while the expression level of hsa-mir-615 or hsa-mir-877 showed no significant relationship with the prognosis (hsa-mir-615, P=0.6247; hsa-mir-877, P=0.5199). Conclusion: A three-miRNA signature of hsa-mir-383, hsa-mir-615, and hsamir-877 may serve as an excellent diagnostic biomarker for HNSCC, and therein hsa-mir-383 shows potential prognostic significance for HNSCC patients. Funding Statement: Shandong Provincial Health Commission Foundation (No. 2016WS0410), China postdoctoral Scinence Foundation Grant (No. 2019M652380), Fundamental Research Funds of Shandong University (No.2018CJ047). This work is also supported by Qilu Young Scholar Program of Shandong University. Declaration of Interests: The authors declare that they do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted. Ethics Approval Statement: All patients and normal controls gave informed consent. This study was approved by the Medical Institutional Ethical Committee of the Provincial Hospital Affiliated to Shandong University.

Abstract 1812: LncRNAs as prognostic biomarkers in head and neck squamous cell carcinoma (HNSCC)

Abstract Long noncoding RNAs (lncRNAs) are transcripts that are greater than 200 nucleotides in length and do not encode proteins in contrast to mRNAs. They regulate a variety of cellular processes including cell proliferation and metastasis, and they have a role in cancer development. To identify lncRNAs that may have biological significance in head and neck squamous cell carcinoma (HNSCC), we globally profiled dysregulated lncRNAs in HNSCC. We analyzed RNA-seq data from HNSCC and paired adjacent normal tissue obtained from The Atlas of Noncoding RNAs in Cancer (TANRIC) database. Of 3576 lncRNAs, we identified 927 lncRNAs to be significantly (FDR &amp;lt;0.05) and differentially regulated by 2-fold among the HNSCC samples (upregulated-688, downregulated-239). Next, to evaluate the potential function of the lncRNAs, we performed lncRNA-mRNAs co-expression analysis for the top 10 upregulated lncRNAs and found that three of the lncRNAs (LINC01614, LINC02081, and LINC00460) are associated with cancer-related biological pathways including extracellular matrix organization, cell adhesion, and collagen catabolism. TP53 mutation status and presence of human papillomavirus (HPV) have been frequently associated with the survival in HNSCC. Therefore, we investigated differential expression of lncRNAs in 426 HNSCC based on TP53 mutation and HPV status. We identified 135 lncRNAs (upregulated-105, downregulated-30) to be significantly (FDR &amp;lt;0.05) and differentially regulated by 2-fold among the mutant vs wild-type TP53 samples. Among HPV-negative vs positive samples, we identified 714 differentially regulated lncRNAs (upregulated-203, downregulated-511). Interestingly, we found that LINC01614, LINC02081, and LINC00460 were also upregulated in mutant TP53 and HPV-negative HNSCC compared to wild-type TP53 and HPV-positive HNSCC. Survival analysis of the HNSCC patient data revealed that higher expression of LINC01614, LINC02081, and LINC00460 were significantly associated with poor overall survival. In addition, we were able to validate that higher expression of LINC02081 and LINC00460 were associated with poor overall survival in an independent data of 130 HNSCC generated at Moffitt Cancer Center. In summary, we identified lncRNAs that are differentially expressed in HNSCC compared to the normal tissue and may have a role in cancer development and metastasis pathways. LINC02081 and LINC00460 lncRNAs are associated with poor prognosis. Further evaluation of these lncRNAs is warranted to understand the mechanism and to potentially develop prognostic biomarkers in HNSCC. Citation Format: Ritu Chaudhary, Xuefeng Wang, Biwei Cao, Nicholas T. Gimbrone, Robbert JC Slebos, Christine H. Chung. LncRNAs as prognostic biomarkers in 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 1812.

Abstract 1809: LncRNA-SOX2-OT contributes to head and neck cancer cell proliferation by regulating DDIT4

Abstract Background: Long non-coding RNAs (lncRNAs) have been shown to serve as an oncogenic role in human cancers. However, the role of lncRNAs in head and neck squamous cell carcinoma (HNSCC) is still unclear. Methods and Results: Our previous microarray analysis showed that lncRNA SOX2-OT was upregulated in HNSCC tumor tissues and was correlated with DDIT4. To determine whether SOX2-OT served as a poor prognosis factor and targeted DDIT4 to facilitate cell proliferation and tumorigenesis in human HNSCC, qRT-PCR tested the relative expression of SOX2-OT and DDIT4 in 60 pairs of LSCC cancer tissues and the corresponding adjacent non-cancer tissues. Cell functional assay was conducted in vitro. We validated the up-regulation of SOX2-OT and DDIT4 in HNSCC tissues. Furthermore, we found that high SOX2-OT expression was significantly associated with advanced tumor differentiation, and worse prognosis. In vitro experiments demonstrated that SOX2-OT functioned as an oncogene by promoting cell proliferation and inhibiting cell apoptosis, and SOX2-OT targeted DDIT4 as its competing endogenous RNA in HNSCC cells. Conclusions: Our findings for the first time identify the oncogenic role of SOX2-OT in HNSCC, demonstrating that SOX2-OT is a potential prognostic biomarker and promising therapeutic target in HNSCC. Note: This abstract was not presented at the meeting. Citation Format: Ru Wang, Yifan Yang, Chen Tan, Ling Feng, Jugao Fang. LncRNA-SOX2-OT contributes to head and neck cancer cell proliferation by regulating DDIT4 [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 1809.

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.

Abstract 440: Use of longitudinal samples from the DOD serum repository to identify candidate biomarkers for HNSCC

Abstract The Department of Defense Serum Repository (DODSR) represents a unique resource enabling longitudinal studies of cancer risk, progression, and response to therapy. The DODSR was initiated in 1989, initially as a means for HIV surveillance, and is comprised of serum samples from active and reserve military personnel drawn at enlistment and annually or biennially throughout the service members' participation in the Military Health System, accompanied by the service member’s electronic health records. This enables the identification of serum samples for all service members with a particular diagnosis, such as Head and Neck Squamous Cell Carcinoma (HNSCC), a cancer significantly represented in the military population with substantial impact on military operations. For HNSCC, 175 cases diagnosed between 2003 and 2013 were identified, as well as 175 corresponding healthy controls matched for age at the time of diagnosis, gender, and ethnicity. Serum samples drawn at the time of diagnosis, 2 and 4 years prior to diagnosis, and 2 years after diagnosis were retrieved and analyzed by targeted mass spectrometry analysis using a panel of 148 qualified selected reaction monitoring (SRM) assays selected from the curated literature and discussions with military oncologists at the Murtha Cancer Center. Initial analyses of 212 samples (38 cases and 38 matched controls, each with 2-4 longitudinal samples) from the total 978 samples available focused on comparisons of pre-diagnosis and at diagnosis serum proteins. Preliminary results identified proteins that were differentially abundant between serum samples taken from HNSCC cases at the time of diagnosis, compared to age-matched controls. Proteins such as A2GL displayed a trend of gradually increasing abundance as a function of time to diagnosis, in HNSC cases, suggesting behavior that may be proven useful for early detection of HNSCC, prior to overt symptoms. These preliminary results suggest that the DODSR can be used to provide serum samples of sufficiently high quality for extensive proteomic analysis, and form cohorts sufficiently large to enable meaningful statistics. Access to longitudinal samples representing years prior to diagnosis enables the identification of serum factors that predict the risk of cancer and may also enable early diagnosis. Access to post-treatment serum samples accompanied with data indicating the clinical response to treatment enables the development of algorithms predicting response to treatment and may eventually contribute to the selection of appropriate therapies. Citation Format: Ju Yeon Lee, Tujin Shi, Vladislav Petyuk, Athena Schepmoes, Thomas Fillmore, Wayne Cardoni, George Coppit, Joseph Goodman, Shiv Srivastava, Craig Shriver, Tao Liu, Karin Rodland. Use of longitudinal samples from the DOD serum repository to identify candidate biomarkers for 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 440.

LCL161, a SMAC-mimetic, Preferentially Radiosensitizes Human Papillomavirus-negative Head and Neck Squamous Cell Carcinoma.

Targeting inhibitor of apoptosis proteins (IAP) with second mitochondria-derived activator of caspase (SMAC) mimetics may promote cancer cell death. We tested whether cIAP1 predicts poor prognosis in head and neck squamous cell carcinoma (HNSCC) and whether a novel Smac-mimetic, LCL161, could radiosensitize human papillomavirus-positive (HPV+) and -negative (HPV-) HNSCC. The association of BIRC2 (encoding cIAP1) mRNA level with HPV status in HNSCC was analyzed using The Cancer Genome Atlas (TCGA) database. cIAP1 was assessed by IHC on an HNSCC tissue microarray (TMA, n = 84) followed by correlation analysis with HPV status and patient outcomes. Human cell culture and animal models of HNSCC were used to analyze the outcome and molecular characteristics following radiotherapy in combination with LCL161. cIAP1 expression is increased in HPV- compared with HPV+HNSCC tumors in the TCGA database. In our TMA, cIAP1 was overexpressed in HNSCC compared with normal tissues (P = 0.0003) and associated with a poor overall survival (P = 0.0402). cIAP1 levels were higher in HPV- than that in HPV+HNSCC tumors (P = 0.004) and patients with cIAP1+/HPV- HNSCC had the worst survival. LCL161 effectively radiosensitized HPV- HNSCC cells, which was accompanied with enhanced apoptosis, but not HPV+ HNSCC cells. Importantly, LCL161 in combination with radiotherapy led to dramatic tumor regression of HPV- HNSCC tumor xenografts, accompanied by cIAP1 degradation and apoptosis activation. These results reveal that cIAP1 is a prognostic and a potential therapeutic biomarker for HNSCC, and targeting cIAP1 with LCL161 preferentially radiosensitizes HPV- HNSCC, providing justification for clinical testing of LCL161 in combination with radiation for patients with HPV- HNSCC.

Widespread Expression of Sonic Hedgehog (Shh) and Nrf2 in Patient Treated with Cisplatin Predicts Outcome in Resected Tumors and Potential Therapeutic Target for Head and Neck Cancer

Background: Sonic hedgehog (Shh) and Nrf2 play an immense role in chemotherapeutic resistance. These genes have been found to be deregulated in head and neck squamous cell carcinoma (HNSCC). The purpose of this study is to analyze the expression, function and clinical prognostic relationship of Shh and Nrf2 in HNSCC in the context of therapeutic resistance and cancer stem cells (CSCs). Methods: We analyzed a cohort of HNSCC patients to identify potential candidate therapeutic biomarkers correlating with overall survival (OS) and disease-free survival (DFS) from our own data and validated the results using data from TCGA. Expression of Shh and Nrf2 was silenced by siRNA and cell growth, sphere growth and chemotherapeutic resistance were evaluated. Findings: Widespread abundant expression of Shh and Nrf2 protein was associated with shorter OS and DFS. The combination of Shh and Nrf2 expression levels was found to be a mostly significant predictor of patient DFS. The tumor stromal index (SI) was correlated with Shh expression and inversely associated with shorter DFS. Inhibition of Shh by siRNA or cyclopamine resulted in the attenuation of resistant CSC self-renewal, invasion, clonogenic growth and re-sensitization to chemotherapeutic agents. Interpretation: Concomitant high expression of Shh and Nrf2 proved to be an independent predictor of poor OS and DFS in HNSCC patients. These findings suggest that Shh and Nrf2 might serve as therapeutic targets as well as promising dual prognostic therapeutic biomarkers for HNSCC. Funding Statement: Grant-in-Aid from Bangladesh Medical Research Council (BMRC), and University Grant Commission (UGC), Ministry of Education of Bangladesh with the assistance of the World Bank under Higher Education Quality Enhancement Project (HEQEP)– Window -4 (Grant Id. CP4023). Declaration of Interests: The authors declare that they have no conflict of interests. Ethics Approval Statement: This study was approved by the ethics committee of the University of Chittagong and Chittagong Medical Hospital, Chittagong, Bangladesh, and was performed in accordance with the ethical standards laid down in the 1964 declaration of Helsinki and all subsequent revisions. Written informed consent was obtained from all patients used in this study.

Oncogenic Role of ZFAS1 lncRNA in Head and Neck Squamous Cell Carcinomas.

Background: Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous disease with high mortality. The identification of specific HNSCC biomarkers will increase treatment efficacy and limit the toxicity of current therapeutic strategies. Long non-coding RNAs (lncRNAs) are promising biomarkers. Accordingly, here we investigate the biological role of ZFAS1 and its potential as a biomarker in HNSCC. Methods: The expression level of ZFAS1 in HNSCC cell lines was analyzed using qRT-PCR. Based on the HNSCC TCGA data, the ZFAS1 expression profile, clinicopathological features, and expression of correlated genes were analyzed in patient tissue samples. The selected genes were classified according to their biological function using the PANTHER tool. The interaction between lncRNA:miRNA and miRNA:mRNA was tested using available online tools. All statistical analyses were accomplished using GraphPad Prism 5. Results: The expression of ZFAS1 was up-regulated in the metastatic FaDu cell line relative to the less aggressive SCC-25 and SCC-040 and dysplastic DOK cell lines. The TCGA data indicated an up-regulation of ZFAS1 in HNSCCs compared to normal tissue samples. The ZFAS1 levels typically differed depending on the cancer stage and T-stage. Patients with a lower expression of ZFAS1 presented a slightly longer disease-free survival and overall survival. The analysis of genes associated with ZFAS1, as well its targets, indicate that they are linked with crucial cellular processes. In the group of patients with low expression of ZFAS1, we detected the up-regulation of suppressors and down-regulation of genes associated with epithelial-to-mesenchymal transition (EMT) process, metastases, and cancer-initiating cells. Moreover, the negative correlation between ZFAS1 and its host gene, ZNFX1, was observed. The analysis of interactions indicated that ZFAS1 has a binding sequence for miR-150-5p. The expression of ZFAS1 and miR-150-5p is negatively correlated in HNSCC patients. miR-150-5p can regulate the 3′UTR of EIF4E mRNA. In the group of patients with high expression of ZFAS1 and low expression of miR-150-5p, we detected an up-regulation of EIF4E. Conclusions: In HNSCC, ZFAS1 displays oncogenic properties, regulates important processes associated with EMT, cancer-initiating cells, and metastases, and might affect patients’ clinical outcomes. ZFAS1 likely regulates the cell phenotype through miR-150-5p and its downstream targets. Following further validation, ZFAS1 might prove a new and valuable biomarker.

A longitudinal study on circulating miR-21 as a therapeutic effect marker in head and neck squamous cell carcinoma.

The aim of the study is to investigate plasma miR-21 for a possible therapeutic effect determination marker in head and neck squamous cell carcinoma (HNSCC). Plasma samples are obtained from 86 HNSCC patients and 29 non-cancer volunteers who had been treated at Mie University Hospital between May 2015 and December 2016, and plasma miR-21 expression was measured using real-time quantitative reverse transcription polymerase chain reaction. In addition, plasma miR-21 level of advanced HNSCC patients including 22 non-recurrent cases and 11 recurrent cases before and after treatment was analyzed using a longitudinal design. Plasma miR-21 expression in 86 HNSCC patients was obviously higher than in 29 control patients (P < 0.0001). The area under the curve (AUC) for plasma miR-21 was 0.756 (95% confidence interval: 0.661-0.851). Furthermore, our longitudinal study of plasma miR-21 showed that the expression level of plasma miR-21 was significantly reduced at the time point of 2 months after treatment in case of no recurrence. On the other hand, plasma miR-21 was not decreased after treatment in case of 10 patients who had developed recurrences during the follow-up period. This study may provide new insights into the role of plasma miR-21 as a biomarker for HNSCC, and plasma miR-21 would be useful for early detection of tumor recurrence after operation or chemoradiotherapy.