B- and T-cell clonality using peripheral blood cell-free RNA (cfRNA) in liquid biopsy Free

Introduction: Using cell-free RNA (cfRNA) in liquid biopsy (LBx) is emerging as an added approach for refining and expanding the utilization of LBx. Although cfDNA had been used in determining B- and T-cell clonality in lymphoid neoplasm, this approach is not sensitive in cases with low circulating cfDNA. Since lymphoid cells express large quantities of heavy and light immunoglobulins (Igs) and T-cell receptors (TCR), we explored using cfRNA for detecting B-cell and T-cell clonality.

Methods: Using hybrid capture and next generation sequencing (NGS) of cfRNA, we clonotyped and evaluated the expression levels of various Igs and alpha, beta, and gamma TCRs in 925 LBx samples. These samples included 671 normal or with low level clonal hematopoiesis of indeterminate potential (CHIP), 449 samples confirmed B-cell neoplasm or plasma cell neoplasm based on mutation profile, 51 confirmed T-cell neoplasms, 327 confirmed myeloid neoplasms, and 98 confirmed solid tumors. To establish expression level of a clonal population, we sequenced RNA extracted from 550 tissue samples with clonal lymphoid neoplasms and 188 tissue samples without clonality. B- and T-cell clonality in tissue samples were confirmed by either flow cytometry or PCR-based assay. cfRNA was extracted and sequenced as previously described. The RNA panel is targeted 1600 genes including all TCRs and heavy and light chain Igs. For B- and T-cell repertoire analysis, sequences were mapped and assembled into clonotypes using MiXCR software.

Results: Based on known clonality in tissue samples, we established 10-fold expression level of the highest clonotype as compared to the second highest clone as a stringent cut-off point for confirming B- and T-cell clonality. Clonotype-naïve testing demonstrated B-cell clonality in 36% of the tested 449 cfRNA samples with B-cell neoplasms, in 7% of the 671 normal/CHIP cases, 8% of the 51 T-cell lymphoma cases, 13% of the 327 myeloid neoplasms and 14% of the 98 solid tumor. T-cell clonality using T-cell receptor beta or gamma demonstrated T-cell clonality in 22% of T-cell neoplasms, 3% of normal/CHIP case and B-cell cases, 5% of myeloid neoplasms, and 7% of solid tumors. Due to limited diversity in TCR-alpha, high non-specific clonality was observed in all types of tissue. However, if we exclude the clonotype TRAV1-1, TCR-alpha is more specific and increases detected clonality in T-cell lymphoma to 25%. Conclusions: Our data suggests that B- and T-cell clonality testing using cfRNA in LBx is a reliable approach even in clonotype-naïve testing. However, for routine comprehensive liquid biopsy evaluation of lymphoid neoplasms, clonality should be combined with mutation profiling. Testing for clonality using cfRNA in clonotype-informed setting is highly more sensitive and might be ideal for minimal residual disease (MRD) evaluation. Our approach of sequencing 1600 genes allows us to combine clonality with mutations in determining MRD.