TCR Sequencing Can Identify and Track Glioma-Infiltrating T Cells after DC Vaccination

Abstract

Although immunotherapeutic strategies are emerging as adjunctive treatments for cancer, sensitive methods of monitoring the immune response after treatment remain to be established. We used a novel next-generation sequencing approach to determine whether quantitative assessments of tumor-infiltrating lymphocyte (TIL) content and the degree of overlap of T-cell receptor (TCR) sequences in brain tumors and peripheral blood were predictors of immune response and overall survival in glioblastoma patients treated with autologous tumor lysate-pulsed dendritic cell immunotherapy. A statistically significant correlation was found between a higher estimated TIL content and increased time to progression and overall survival. In addition, we were able to assess the proportion of shared TCR sequences between tumor and peripheral blood at time points before and after therapy, and found the level of TCR overlap to correlate with survival outcomes. Higher degrees of overlap, or the development of an increased overlap following immunotherapy, was correlated with improved clinical outcome, and may provide insights into the successful, antigen-specific immune response. Cancer Immunol Res; 4(5); 412-8. ©2016 AACR.

Figure 1. Elevated estimated TIL content in pretreatment tumor effectively segregates the survival of glioblastoma patients after DC vaccination

The TTP and OS was compared between patients whose estimated TIL content was dichotomized into the top quartile (Red) or the lower 3 quartiles (orange). The estimated TIL content was dichotomized at 0.787. A log-rank test was performed to compare the TTP (A) and the OS (B). TTP; **p<0.0087. OS; **p<0.0038. (C) Representative immunofluorescent staining of CD8 TIL from patients in the top quartile (-232, -708) and lower 3 quartiles (-828, -815). Quantitation of CD8 was performed using inForm image analysis software.

Figure 2: TCR Overlap between pretreatment and post-treatment peripheral blood and/or tumors are associated with survival. /B> Correlation between pretreatment peripheral blood TCR sequences (x-axis) and post-treatment peripheral blood TCR sequences (y-axis) overlaid with presence of T-cells within the TIL populations

: TCRs present only in peripheral blood samples; Blue: TCRs present only in pretreatment TILs and peripheral blood; Green: TCRs present only in post-treatment TILs; Orange: persistent TCRs present in both sets of TILs and peripheral blood. (A-B, High-High TCR Overlap Group) (C, Low-High TCR Overlap Group) (D-E, Low-Low TCR Overlap Group). A) Patient 1-708 (overall survival= 28.7 months); B) Patient 17-232 (overall survival= 71.3 months); C) Patient 32-204 (overall survival= 12.6 months); D) Patient 6-815 (overall survival= 6.3 months); E) Patient 21-828 (overall survival= 4.5 months).

Figure 3. Dynamic monitoring of tumor-infiltrating TCR sequences in glioblastoma patients treated with autologous tumor lysate-pulsed DC vaccination

/B> Assessment of the TCR overlap between the initial tumor (prior to DC vaccination) and PBMC taken at time points pre-vaccine, post-vaccine, and at relapse (B) Assessment of the TCR overlap between the recurrent tumor (after DC vaccination) and PBMC taken at time points pre-vaccine, post-vaccine, and at relapse.

Figure 4. Development of a TCR overlap model that demonstates how to track the evolution of tumor-infiltrating TCRs after DC vaccination in glioblastoma patients

The quantity and diversity of TCR transcripts are determined from glioblastoma tumor samples and peripheral blood. The TCR overlap was defined as the percentage of unique TCR beta chain sequences identified in each tumor that were also identified in the peripheral blood (before treatment, after treatment, and at relapse). We formulated a model depicted in the Venn diagrams. (Top) Patients who have a high TCR overlap with the blood from the initial tumor and relapsed tumor are predicted to have long survival outcomes. (Middle) Patients whose initial tumor sample have low TCR overlap with the blood, but develop a high TCR overlap in the relapsed tumor after DC vaccination, are predicted to have intermediate survival. (Bottom) Patients who have a low TCR overlap with both the initial and relapsed tumor and blood are predicted to have short survival outcomes.