Saturday, June 18, 20222:00 PM - 3:00 PM PL01 Presentation Time: 2:00 PM

Purpose

Chemoradiation (CRT) may modulate the intratumoral and peripheral immune milieu by serving as an in situ vaccine. The impact of the rapid dose delivery of brachytherapy (BT) on the T-cell repertoire has not been examined. HPV-related cancers express viral oncoproteins E6/E7, which could function as tumor antigens (Ag) and are thus ideal for investigating the impact of CRT in generating tumor-specific immunity.

Methods

11 pts with cervical cancer were enrolled on a multi-institutional prospective tissue banking protocol from 1/2020—2022. Pts underwent standard external beam RT (EBRT) spanning 5-7 wks with concurrent weekly 40mg/m² cisplatin, then BT boost with either high dose rate (HDR) in 5 fx or pulse dose rate (PDR) in 2 fx. Cervical tumor swabs were taken at 5 timepoints: baseline (T1), wk 1 (T2, after 5 fx), wk 3 (T3, after 10-15 fx), wk 5 (T4, at conclusion of EBRT and prior to 1st BT), prior to 2nd BT (T4B), and at 1st follow-up (T5, 12 wks post-treatment). Samples underwent functional expansion of HPV Ag-specific T cells before DNA extraction and multiplex deep sequencing of the CDR3 region of TCR-β using immunoSEQ PCR (Adaptive Biotech). TCR productive frequency (freq), templates, clonality, and rearrangements were compared across timepoints. Separately, HPV-responsive T-cell clones underwent ex vivo expansion by incubation with HPV long peptide sequences of E6/E7 overnight (ON). TCR sequences were compared between HPV-stimulated T cells and evaluated against incubations with CEF (positive control peptides) or media (negative control). Statistical analysis was via Mann-Whitney U.

Results

Median age was 47 yrs (range 27-57). 6 pts (54.5%) had SCC; 5 (45.5%) had adenocarcinoma. 4 pts (36.4%) had FIGO 2018 ≤Stage IIB disease, 45.5% were Stage III, and 18.2% were Stage IVA/B. 7 pts (63.6%) received PDR BT and 4 pts (36.4%) underwent HDR BT. At median follow-up of 7.5 mo, all pts are alive and with NED. In serially sampled peritumoral microenvironment (TME) isolates, TCR productive clonality increased after first BT, culminating in a significant increase at T5 during the first post-treatment follow-up (Fig 1A). There was a trend (ns) toward increased productive templates throughout CRT and BT (Fig 1B), but this decreased by T5. Productive freq was significantly higher at T5 compared to prior to BT (T1—T4), Fig 1C. Productive rearrangements trended toward abundance throughout CRT and BT (ns), but decreased at T5 (Fig 1D). Functional HPV Ag stimulation assays of peritumoral T-cells in the full cohort did not show significant changes in the TCR repertoire after ON incubation with HPV E6/7 peptides. However, 4 pts had notable E6/E7-responsive increases in these metrics at either T4 or T4B—namely, abundance of productive templates (ranges: 1.9—30.3 fold-increase from baseline), freq (1.25—5.5), rearrangements (1.5—13.8), and clonality (1.31—3.8).

Conclusions

We characterized TCR remodeling during BT in this first translational analysis, demonstrating (i) increased peritumoral TCR productive freq and clonality post-BT at T5, and (ii) transient rise in productive templates and rearrangements through CRT and BT, with a numeric return to baseline post-BT at T5. Some pts had functional Ag-specific activation upon HPV stimulation during and after BT. These findings suggest that despite the cytotoxic effects of radiation, BT alters the TME to facilitate expansion of specific T-cell populations, which may contribute to treatment efficacy.