Reliability of Liquid Biopsy and Next Generation Sequencing in Monitoring Residual Disease Post-Hematopoietic Stem Cell Transplant

Introduction: Using next generation sequencing (NGS) in monitoring residual disease in patients with myeloid neoplasms is complicated by the significant heterogeneity in these diseases and the frequent presence of CHIP (clonal hematopoiesis of indeterminate potential) in patients with hematologic neoplasms on which these neoplasms arise. This is particularly relevant post hematopoietic stem cell transplant (HSCT). We explored the ability of using plasma cell-free DNA (cfDNA) in monitoring patients after HSCT and evaluated the potential of using liquid biopsy as a replacement for bone marrow biopsy. Method: cfDNA was isolated from 204 peripheral blood samples obtained from 75 patients, collected at various time points ranging from 27 days to 650 days (median 178 days) post-transplant. DNA from 102 bone marrow (BM) samples was extracted and sequenced using the same panel and approach as cfDNA. Diagnoses included 30 acute myeloid leukemia (AML), 2 chronic myelogenous leukemia (CML), 5 chronic myelomonocytic leukemia (CMML), 4 lymphoma, 10 myelodysplastic syndrome (MDS), 2 multiple myeloma (MM), 9 myeloproliferative neoplasm (MPN), 1 aplastic anemia, and 11 acute lymphoblastic leukemia. cfDNA was sequenced by NGS using 177 gene panel on Illumina platform. Single primer extension (SPE) approach with UMI was used. Sequencing depth was increased to more than 2000X after removing duplicates. Low-level mutations were confirmed by inspecting BAM file. Results: 156 cfDNA samples (76%) tested negative and 48 samples from 30 different patients were positive. The negative samples were collected from 28 days to 650 days post-transplant (median 277 days). The positive samples were collected from 27 days to 650 days post-transplant (median 188 days). One of these positive patients was in full clinical relapse at the time of testing. No negative patient who remained negative had clinical relapse. Five patients converted from negative to positive and 12 from positive to negative with subsequent testing. Three from the converted to positive patients developed clinical relapse. Patients who were positive without clinical relapse had median variant allele frequency (VAF) of 0.85% (range: 0.01-13.25) and typically one mutated gene. The mutated genes in this group were: JAK2, IDH2, ASXL1, TET2, DNMT3A, ASXL1, PTPN11, SF3B1, MPL, CEBPA1. Patients who had clinical relapse (#4) had median VAF of 16.33% (0.4%-57.63%) with multiple mutated genes. The mutated genes in this group were: