Background
T-cell receptor (TCR) repertoire profiling is essential to understanding the cancer microenvironment. Assays that can profile TCRs from formalin fixed paraffin embedded (FFPE) samples have been lacking. We collaborated with Cellecta Inc., who have developed the novel multiplex RT-PCR-based DriverMap Adaptive Immune Receptor (AIR) profiling assay that can profile T- or B-cell CDR3 or full-length receptor regions and is optimized for use with small amounts of starting material. In our current study, we used the AIR assay to explore the TCR repertoire of T cells infiltrating chondrosarcoma tumors, a sarcoma subtype that weakly responds to classical immunotherapy in the advanced setting. Our findings in this study highlight the enrichment of chondrosarcomas with conserved T cells known as Mucosa-associated invariant T cells (MAITs).
Methods
24 chondrosarcoma FFPE cases were selected. RNA and DNA TCR-seq on 11 cases was performed by Cellecta using DriverMap AIR Profiling Service. TCR-seq was performed on tumoral and peritumoral regions in selected cases. MAIT cells were identified based on the presence of specific CDR3 regions and a combination of TRAV 1-2 and TRAJ33 or TRAJ20 or TRAJ12; findings were then confirmed using the MAIT MATCH search tool with a cut-off value of 0.9. To explore the presence of MAITs ligand MR-1, immunohistochemistry (IHC) was performed.
Results
In a comparative analysis, TCR sequencing was seen to yield better results with RNA in some samples, while others were more effective with DNA. Though DNA samples were better in quality, the AIR RNA assay showed higher sensitivity, detecting more clonotypes. Clonotype analysis revealed that 76% of the shared CDR3 regions were identified as MAIT cells, and some were highly shared (CAVRDSNYQLIW and CAVMDSNYQLIW were shared among 81%). Subsequent TCR sequencing of the tumor and peritumoral punches revealed a relative enrichment of MAIT cells in the tumor compared with the peritumor despite being less diverse. Lastly, we found strong consistent expression of MR1 in all but one chondrosarcoma sample (mean MR1 expression, 58%) in both the peritumoral compartment and within the tumor.
Conclusions
Our results demonstrate that the assay offers sensitivity and cost-effectiveness for high-throughput immune biomarker discovery applications with FFPE samples. Our analysis of the TCR repertoire revealed the presence of a subset of conserved immune cells (MAIT) that were found to be enriched in the tumor. To maximize the detection of antigen-activated clonotypes, we recommend profiling through both DNA (higher quality) and RNA assays (higher sensitivity).