Immune checkpoint molecule expression measured using circulating cell-free RNA isolated from the blood of metastatic prostate cancer patients.

380 Background: Recent studies have identified panels of immune-related genes linked to progression and survival. Expressions usually are measured using RNA from tissue samples. The aim of the present study was to utilize circulating RNA (cfRNA) isolated from hormone-naïve (HN) and castrate-resistant prostate cancer (CRPC) patients to measure gene expressions of PD-L1, cytotoxic T-lymphocyte associated protein 4 (CTLA-4), T-cell immunoglobulin and mucin domain 3 (TIM-3) and lymphocyte activated gene 3 (LAG-3). Androgen receptor (AR) and AR-variant 7 (AR-v7) expressions were also measured in these samples to test for any possible relationships with the immune checkpoint genes. Methods: Blood samples were collected from 53 metastatic prostate cancer patients. cfRNA was extracted from patients’ plasma and reverse transcribed into complementary DNA. The gene expressions (mRNA levels) of the above genes were determined by quantitative reverse transcription-PCR. Beta-actin was used to normalize gene expressions to total RNA content. Results: Each of the immune signatures tested (PD-L1, TIM-3, LAG-3, CTLA-4) was expressed in over 50% (29/53) of the patients’ blood samples. AR was expressed in 25/53 (47%) samples. Four of these patients (8%) expressed AR-v7, indicating resistance to anti-AR drugs. Patient disease status included: 25% (13/53) CRPC, 74% (39/53) HN, and 1 unknown. Within the CRPC group, a significant negative correlation was found between TIM-3 and CTLA-4 expression (-0.833), and CTLA-4 also correlated with disease progression (0.719). Within the HN group, a significant negative correlation was measured between TIM-3 and PD-L1 (0.589). AR expression did not correlate with other targets measured among all patients or within the CRPC or HN groups. Conclusions: This study demonstrates successful quantitation of immune checkpoint gene expressions as well the AR and AR-v7 genes in plasma of prostate cancer patients. This result opens the possibility of the use of a noninvasive method to measure, monitor and track the dynamic interplay of these genes with changing disease status. Clinical trial information: NCT02853097.