Incidence and Outcomes of Patients With Oral Cavity Squamous Cell Carcinoma and Fourth Primary Tumors

6562 Background: Oral cavity squamous cell carcinoma (OCSCC) frequently presents as clinically advanced disease with poor prognosis. When diagnosed at early stages, survival rates approach 80%, underscoring the need for validated, cost-effective detection methods. OCSCC is driven by the serial acquisition of genetic alterations. Tumor-defining somatic mutations are attractive biomarkers and hence their presence in saliva may be associated with malignancy as shown in a few proof-of-concept studies, including our previous work. Based on this premise, we present a low-cost, accurate, next generation sequencing (NGS) test with high clinical utility aimed at detecting mutations in the saliva for early diagnosis and potential screening of OCSCC. Methods: We have designed a custom NGS panel that covers exons of 7 most frequently mutated genes in OSCC. This minimal gene set derived from the analysis from 3 public datasets, predicted incidence of at least one somatic aberration in 89% of patients. We recruited 91 treatment-naïve OCSCC patients and profiled DNA from tissue and matched pre-operative saliva using this test. We also tested DNA from 12 subjects with premalignant lesions with high-grade oral dysplasia and matched saliva. Results: Using stringent variant calling criteria, at least one somatic variant was detected in 88 (96%) of the 91 primary tumors. 90.9% of the matched saliva were concordant, with only a minor decrease in early stage disease. Tumor-specific mutations (≥5% AF) in driver genes were detected in 10 (83.3%) dysplastic lesions, suggesting that driving clonal events may occur early in disease development. Interestingly, in 3 matched saliva of the dysplastic samples, the same mutations were detected. To ensure a variant is not a false positive call, we performed a vigorous multistep analytical validation of this saliva-based test: (i) independent re-sequencing of 24 saliva confirmed 94% reproducibility; (ii) no functionally relevant variants were detected in saliva from 12 of 13 healthy subjects without history of tobacco and alcohol usage; (iii) reproducibility, sensitivity, and specificity were confirmed using a positive control with 7 loci at 0.25% AF across 8 independent saliva sequencing runs and a certified negative control and was found to be on par with droplet digital PCR. Conclusions: These data highlight the feasibility of saliva-based testing for early diagnosis of OCSCC and premalignant lesions.

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Malignant cell-specific CXCL14 promotes tumor lymphocyte infiltration in oral cavity squamous cell carcinoma

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-001048 ◽

2020 ◽

Vol 8 (2) ◽

pp. e001048

Author(s):

Anuraag Parikh ◽

JuneHo Shin ◽

William Faquin ◽

Derrick T Lin ◽

Itay Tirosh ◽

...

Keyword(s):

Squamous Cell Carcinoma ◽

T Cell ◽

Oral Cavity ◽

Tumor Growth ◽

Cell Carcinoma ◽

Squamous Cell ◽

Malignant Cell ◽

Malignant Cells ◽

Lymphocyte Infiltration ◽

Primary Tumors

ObjectivesTo explore lymphocyte infiltration as a potential mechanism behind CXCL14-mediated tumor growth suppression in oral cavity squamous cell carcinoma (OSCC).MethodsWe analyzed single cell RNA-sequencing (scRNA-seq) data from OSCC to identify expression changes among malignant cells in lymph nodes (LN) versus primary tumors. CXCL14 expression in murine OSCC cell lines was quantified using qRT-PCR. Short hairpin RNA knockdown of CXCL14 was performed in mouse oral cavity (MOC)1 cells, and CXCL14 overexpression was performed in MOC2 cells. Cells in each condition were injected into C57BL/6 mice with and without T cell depletion, and tumor volume was measured. At 30 days, tumors were dissociated and analyzed by flow cytometry for CD45+CD3+ T cells. CXCL14 expression was correlated with gene expression signatures of tumor infiltrating lymphocytes (TIL) in scRNA-seq data, as well as TCGA tumors.ResultsscRNA-seq revealed CXCL14 as the most significantly downregulated gene among malignant cells in LNs relative to primary tumor, supporting a role in preventing invasion and/or metastasis. In a murine immunocompetent model, CXCL14 expression was higher in indolent MOC1 cells than in more aggressive MOC2 cells. Tumor growth in vivo was significantly increased by CXCL14 knockdown in MOC1 cells relative to control, with a corresponding decrease in TIL. In MOC2 cells, tumor growth was significantly reduced by CXCL14 overexpression relative to control and TIL were increased. Both effects were lost with T cell depletion. In a human tumor scRNA-seq cohort, we found that only malignant cell CXCL14, but not non-malignant cell or fibroblast CXCL14, was associated with TIL. Bulk CXCL14 from the TCGA cohort had no association with TIL.ConclusionsHigher CXCL14 expression by tumor cells is associated with reduced tumor growth and increased TIL, supporting immune-mediated suppression of tumor growth in OSCC. Given that CXCL14 is downregulated in LN metastases compared with primary tumors, our data raise the possibility that CXCL14-mediated immune infiltration may discourage invasion and metastasis. In human scRNA-seq data, only malignant cell-specific CXCL14 was associated with TIL, suggesting a critical context-dependent effect of CXCL14 expression.

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