Clonotypic Diversification of Intratumoral T Cells Following Sipuleucel-T Treatment in Prostate Cancer Subjects

Abstract

Sipuleucel-T is an autologous cellular therapy for asymptomatic, or minimally symptomatic, metastatic castrate-resistant prostate cancer, designed to stimulate an immune response against prostate cancer. In a recent clinical trial (NCT00715104), we found that neoadjuvant sipuleucel-T increased the number of activated T cells within the tumor microenvironment. The current analysis examined whether sipuleucel-T altered adaptive T-cell responses by expanding pre-existing T cells or by recruiting new T cells to prostate tissue. Next-generation sequencing of the T-cell receptor (TCR) genes from blood or prostate tissue was used to quantitate and track T-cell clonotypes in these treated subjects with prostate cancer. At baseline, there was a significantly greater diversity of circulating TCR sequences in subjects with prostate cancer compared with healthy donors. Among healthy donors, circulating TCR sequence diversity remained unchanged over the same time interval. In contrast, sipuleucel-T treatment reduced circulating TCR sequence diversity versus baseline as measured by the Shannon index. Interestingly, sipuleucel-T treatment resulted in greater TCR sequence diversity in resected prostate tissue in sipuleucel-T-treated subjects versus tissue of nonsipuleucel-T-treated subjects with prostate cancer. Furthermore, sipuleucel-T increased TCR sequence commonality between blood and resected prostate tissue in treated versus untreated subjects with prostate cancer. The broadening of the TCR repertoire within the prostate tissue supports the hypothesis that sipuleucel-T treatment facilitates the recruitment of T cells into the prostate. Our results highlight the importance of assessing T-cell response to immunotherapy both in the periphery and in tumor tissue. Cancer Res; 76(13); 3711-8. ©2016 AACR.

Figure 1.

Example pairwise sequence scatter matrix of healthy donors and sipuleucel-T–treated subjects. A representative scatter matrix of sequence frequencies between baseline (week 0) and week 2 blood samples in healthy donors and sipuleucel-T–treated subjects. In general, more total productive TCR sequences were observed in the blood of sipuleucel-T–treated subjects compared with healthy donors at all three time points, with a greater flux in overall TCR sequences between week 0 and week 2 in sipuleucel-T–treated subjects.

Figure 2.

Assessments of unique TCR sequences in the blood for healthy donors and for sipuleucel-T–treated subjects were performed on PBMCs taken before the preparation of each sipuleucel-T treatment. A, TCR sequence profiles in healthy donors and sipuleucel-T–treated subjects with prostate cancer over time. Sipuleucel-T–treated subjects had significantly higher mean number of productive TCR sequences at baseline (week 0) compared with week 2 and week 4 (P < 0.01, paired t test on the log scale). No differences in mean productive sequences in healthy donors over time were detected. B, Shannon diversity index for TCR repertoire in blood from healthy donors and sipuleucel-T–treated subjects with prostate cancer. The median Shannon diversity index of healthy donors (left) and a sipuleucel-T–treated subject at baseline (week 0) and weeks 2 and 4. TCR sequence diversity was higher in sipuleucel-T–treated subjects compared with healthy donors. Shannon diversity index decreased at weeks 2 and 4 compared with baseline in sipuleucel-T–treated subjects (P < 0.001), whereas Shannon diversity index remained stable over time in healthy donors.

Figure 3.

Alterations in TCR sequence frequency over time in healthy donors and sipuleucel-T–treated subjects. The FC in sequence frequency versus baseline was categorized as increased (FC ≥ 2), unchanged (0.5 < FC < 2), or decreased (FC ≤ 0.5). A, analysis of TCR sequences present at any time point (all clones). The percentage of sequences was calculated and categorized as increased, unchanged, or decreased on the basis of the sequence frequency count FC for week 2 relative to week 0 (baseline) and week 4 relative to week 0. Data presented are the percentages in each category averaged over all subjects (healthy donors and sipuleucel-T–treated subjects) and included all sequences with a measurable frequency count at weeks 0, 2, or 4. B, analysis of TCR sequences present at all timepoints (common sequences). The percentage of sequences was calculated and categorized as increased, unchanged, or decreased based on the sequence FC for week 2 relative to week 0 (baseline) and week 4 relative to week 0. Data presented are the percentages in each change category averaged over all subjects and included only sequences with measureable frequency counts at both week 0 and week 2 (week 2 analysis) and both week 0 and week 4 (week 4 analysis).

Figure 4.

Change in abundance of TCR sequences during sipuleucel-T treatment. A, a ladder plot of the change in rank of the top 100 sequences at each time point during the course of treatment for representative examples of one healthy donor and one sipuleucel-T–treated subject. B, the SDs of the selected top 100 sequences were averaged for each sample across the three time points. For sequences with missing data points, the lower value observed in the same sample was imputed. The extent of rank shuffling of nucleotides is much greater in the sipuleucel-T–treated subjects compared with the healthy donors (P < 0.02, Wilcoxon one-sided test).

Figure 5.

Assessment of TCR sequences in blood and prostate tissue. A, BUB overlap index between blood and resected prostate tissue. Each point represents the BUB overlap index between the T-cell sequences in the blood samples at weeks 0, 2, and 4 with the radical prostatectomy tissue of the same subject. The horizontal lines represent the median value of the BUB index across the available samples at the corresponding time point. The BUB index is significantly greater at week 2 compared with week 0 (P = 0.01, Wilcoxon test). B, the Shannon diversity index of the TCR repertoire in resected prostate tissue samples from untreated and sipuleucel-T–treated subjects. TCR results were quantified globally across the biopsy/postsurgical tissue. The horizontal lines represent the median value of the Shannon diversity index across the available samples for each group of subjects. Resected prostate tissues from sipuleucel-T–treated subjects with prostate cancer had a significantly greater Shannon diversity index compared with resected prostate tissues from subjects with prostate cancer who did not receive sipuleucel-T (P ¼ 0.01, Wilcoxon test). This represents an increase in T-cell sequence diversity in the prostate among sipuleucel-T–treated subjects.