PDCT-11. QUANTITATIVE TRACKING OF T-CELL CLONES AFTER ADOPTIVE CELLULAR THERAPY TARGETING RECURRENT MEDULLOBLASTOMA USING MASSIVELY PARALLEL RNA SEQUENCING TECHNOLOGY

Abstract

Adoptive cellular therapy (ACT) using transfer of tumor-specific lymphocytes has emerged as a potent strategy for treatment of advanced and refractory malignancies. We have established a novel next-generation sequencing approach based on comprehensive characterization of the T-cell receptor (TCR) repertoire changes in patients with recurrent medulloblastoma and PNETs undergoing adoptive cellular therapy at our center (Re-MATCH protocol, FDA IND BB-14058). Total RNA was isolated from patient PBMC samples collected prior to adoptive cellular therapy and weekly for one month and then 5 months following immunotherapy treatment. cDNA was generated with addition of a common adapter at 5’ end of cDNA using SMART technology. PCR was performed to amplify TCR alpha and beta chains and included adapter and index sequences compatible with the Illumina sequencing platform. Libraries were sequenced on Illumina MiSeq using 600 cycle reagent kit with paired end, 300 + 300 base pair reads. T-cell sequences were analyzed using MiXCR software package, hosted on BaseSpace, Illumina’s cloud computing environment. The observed sequence reads in the peripheral blood allowed us to identify 54 TCRAV and 64 TCRBV exons which indicated a good coverage of TCR genes by our cDNA sequencing approach. The data revealed that certain T-cells were clonally expanded after ACT, with an increasing number of “hyper-expanded” TCR clones (comprising >1% of all TCR beta or alpha sequences) after adoptive cellular therapy. These TCR clones established a new balance in the peripheral blood that was stable over the next 5 months following the completion of cellular immunotherapy. This new method is useful for routine monitoring of clonotypic TCR expansion in the peripheral blood of patients undergoing immunotherapy, requires low blood sample volume, and may significantly enhance capacity to monitor the response to immunotherapy in patients with brain tumors.