Higher expression of HPV16 derived E7_LI Transcript Observed in Men with HIV and Recurrent Anal Cancer
Squamous cell carcinoma of the anus (SCCA) or anal cancer (AC) is an understudied cancer with a high occurrence rate in people with HIV (PWH), especially men having sex with men (MSM). Furthermore, AC recurs in approximately one fourth of patients who undergo standard care with chemoradiation therapy (CRT). Using bulk RNA sequencing data of AC obtained from 12 patients with non-recurrent (NR, N=9) or recurrent (R, N=3) cancer, we previously showed upregulated expression of key immune genes in the NR compared to the R group. Although the main causative agent of anal cancer is high-risk human papillomavirus (HPV), association of host and viral RNA transcript expression contributing to AC recurrence has not been extensively studied. The objective of the current study was to determine whether enrichment of specific HPV genotypes and/or HPV gene expression patterns differentiate the two groups and if any specific viral (HPV) and host (human) immune mediators correlate with each other. Using bulk RNA sequencing data and VIRTUS 2, we detected viral RNA reads mapping to 7 high-risk and 6 low-risk HPV types of which the high-risk HPV16 observed in 83% (10/12) AC tumors (7/9 non-recurrent and 3/3 recurrent). Rate of all HPV genomes trended towards a decrease in non-recurrent anal cancer isolates and correlation between HPV types was more commonly observed in low-risk ones. Analysis of HPV 16 gene expression profile showed a significantly lower positivity rate for a polycistronic transcript encoding for E7^L1 in the non-recurrent group (1/9, NR versus 3/3, R, p value <0.05). An unbiased correlation analysis of HPV-human transcript expression showed a direct correlation between HPV transcripts and human genes involved in cell growth. The data also identified human transcripts showing an inverse correlation with HPV gene expression. These included genes involved in negative regulation of growth, proliferation and immune response.  Taken together, these data indicate that concurrent analyses of viral and host factors in the same tumor can identify potential new therapeutic targets to ameliorate cancer recurrence post treatment. Overall design: 12 FFPE primary tissues from patients with confirmed anal cancer (AC) diagnosis prior to chemoradiation therapy (CRT), 3 who went on to have recurrent AC post CRT and 9 who did not recur (non-recurrent). RNA was purified with Quick-DNA/RNA FFPE kit (R1009, Zymo) and developed into cDNA libraries using the SMARTer Stranded Total RNA-Seq Kit v2 (Takara). Pooled libraries were then sequenced on a NovaSeq 6000 (Illumna) to a depth of > 40 million paired-end 150 bp reads per sample. Phred quality was > 30 for all samples.