Evidence of a role for oropharyngeal cancer cells with low HPV gene expression in treatment failure.

6058 Background: A subset of tumor cells with reduced HPV gene expression, here termed HPV-lo cells, are proposed contribute to therapy resistance in HPV+ oropharyngeal cancers (OPCs). However, the biologic and clinical significance of this cell state is unclear because it has been analyzed in relatively few HPV+ OPCs and total tumor cells and remains to be directly linked to therapy resistance and recurrence. We aimed to evaluate HPV-lo cells for their biologic traits, cytotoxic drug responses, and association with recurrence. Methods: Single cell mRNA sequencing (scRNAseq) was performed on 64,822 tumor cells from five HPV+ OPC PDXs, and HPV-lo cells were defined by presence of <1.0 normalized HPV mRNA reads. Expression profiles distinguishing HPV-lo cells were assessed by gene set enrichment analysis. Changes in HPV-lo cell frequency and gene expression were examined by scRNAseq after two weeks of in vivo cisplatin treatment in NSG mice. To test for association of HPV-lo cells with recurrence, a single institution cohort of 851 therapy-naïve p16+ OPC patients receiving primary surgery was used to curate 50 pT1/2 tumors that later recurred (cases) and match 50 tumors that were cured (controls) for pathologic stage, smoking history, and adjuvant therapy. A clinical in situ hybridization (ISH) assay was used to probe for HPV E6/E7 in the case-control cohort. Digital image analysis segmented the ISH(+) vs. ISH(-) cells in tumor regions plus the subset of ISH(-) cells comprised of CD45-IHC(+) tumor infiltrating leukocytes (TILs). The ISH(-) tumor cell fraction was estimated by calculating total %ISH(-) cells - %TILs. Results: Content of HPV-lo cells in PDXs ranged from 32% to 65%. These cells showed significant downregulation of E2F target genes and upregulation of p53 target genes, supporting presence of reduced HPV E6/E7 activity. Their relative quiescence was further supported by transcriptional inference of fewer cells in the S/G 2 /M cell cycle fractions. HPV-lo cell content in the PDXs negatively correlated with cisplatin response measured by T/C ratio (r=-0.96, p=0.007), and the size and gene expression profile of this fraction were largely unaltered by cisplatin. In the case-control cohort, the HPV ISH(-) tumor cell fraction was larger in cases (p<0.001), which often contained large tumor regions devoid of ISH(+) cells. The %ISH(-) tumor cells provided favorable discrimination of cases vs. controls based on an area under the ROC curve of 0.77 (p<0.001, OR=66, 95% CI=11-547). Conclusions: HPV+ OPCs contain a subset of tumor cells with reduced HPV gene expression and a relatively quiescent phenotype, and increased size of this cell fraction appears predictive of recurrence. Cell state dynamics maintaining this fraction during cytotoxic therapy may contribute treatment failure. Thus, HPV-lo cells merit evaluation for generalizability as a biomarker and mechanistic interrogation as an etiology for tumor recurrence.