Clonal Expansion of Antigen Specific T-Cells during Radiation Therapy for HPV Associated Cervical Cancers Is Regulated By the Vaginal Microbiome

Abstract

Cervical cancers are known to be highly radiation sensitive; however, the rate of response to treatment is variable and poor responders are more likely to recur. The microbiota of colon cancers has been reported to play a role in response to chemotherapy and immune activation. We hypothesized that the cervical microbiome may impact radiation response and T-cell expansion. To address this, we completed prospective serial T-cell receptor (TCR) and microbiome sequencing of 30 patients with CCa throughout chemoradiation (CRT). Patients with newly diagnosed CCa were enrolled on a prospective study to characterize changes in the TCR profile and vaginal microbiome during CRT. Cervical samples were obtained from 30 patients before and during (weeks 1, 3, and 5) radiation. TCR profiling was performed through ImmunoSEQ to amplify and sequence the TCR CDR3b regions. Total unique templates, clonality, maximum productive frequency (MPF) and MPF of the top 10 clones (MPF10) were quantified. Disease response was categorized as exceptional response (ER), standard response (SR) or poor response (PR) based on week 5 MRI and 3 month PET/CT. Microbiome was characterized via 16S rDNA sequencing. Shannon diversity index (SDI) was used to compare microbiome diversity. Impact of specific microbial species were assessed using Linear Effect Size (LEFSe) analysis. Among all patients, the total templates (p=0.22), sample clonality (p=0.3) and MPF (p=0.07) were similar among SR, ER and PR at baseline. At week 5, total templates was similar among all groups (p=0.13); however, ER samples demonstrated higher clonality (ER median 0.05 [0.04-0.06] vs SR median (0.01 [0.009-0.02]; p=0.02). MPF10 decreased over time for PR/SR and increased over time for ER, with a higher fold change in MPF10 from week 3 to week 5 for ER (1.51 [1.10-2.61]) vs PR (0.60 [0.30-0.94]; p=0.02) or SR (0.80 [0.54-1.10]; p=0.03). Alpha diversity of the cervical microbiome was similar among responders (SR/ER) and non-responders (PR); however, distinct genus-level differences existed between SR/ER and non-responders PR, with enrichment of Porphyromonas in SR/ER and enrichment of Lactobacilli in PR. A Lactobacillus:Porphyromonas ratio (LPR) >= 1 (N=7) vs. LPR <1 (N=23). A Lactobacillus:Porphyromonas ratio (LPR) >= 1 was associated with decreased RFS (12.3 months vs NR; p=0.004) with failure rate of 43% (3/7) versus 4% (1/23) noted in the low LPR (<1; N=23). High LPR at baseline was also associated with decreased fold change in MPF10 by week 5 (p=0.015). Exceptional responders to radiation treatment are more likely to have clonal expansion of antigen specific T-cells within CCa. Predominance of vaginal Lactobacillus is associated with poor radiation response and lack of clonal T-cell expansion. Loss of E6/7 expression driven by lactobacillus may provide a mechanism to account for this observation. Further validation will be needed prior to development of interventions targeting the local tumor microbiome.