Reliability of Liquid Biopsy and Next Generation Sequencing in Monitoring Residual Disease Post-Hematopoietic Stem Cell Transplant
Authors: Albitar Maher , Zhang Hong , Pecora Andrew L. , Ip Andrew , Goy Andre H. , Estella Jeffrey Justin , De Dios Ivan , Ma Wanlong , Kaur Sukhdeep , Suh Hyung C. , Donato Michele L. , Rowley Scott D. 
Published: November 2021

Abstract
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: TP53, FLT3, ASXL1, CEBPA, EZH1, NRAS, SETBP1, TET2. To evaluate relevance to BM testing, we compared BM samples with cfDNA samples collected within 120 days of each other. This showed 17 pairs with concordant negative results, 10 with concordant positive results, 5 pairs with positive by cfDNA but negative by BM cells, and one pair with positive by BM but negative by cfDNA. This BM positive sample was performed at 78 days after the cfDNA sample and showed mutation in DNMT3A gene at VAF of 0.63%. Four of the 5 pairs with positive cfDNA but negative BM were collected approximately 3 months after bone marrow and the 5th case was one month prior to BM sample.
Conclusion: These data suggest that monitoring residual disease after HSCT using cfDNA and NGS is a reliable approach and may replace the need of bone marrow biopsy. However, low-level mutations should not be used as the sole criterion for determining relapse. Variant allele frequency and the mutated gene should be considered in evaluating actionable findings.

Disclosures
Pecora: Genetic testing cooperative: Membership on an entity's Board of Directors or advisory committees; Genetic testing cooperative: Other: equity investor. Rowley: ReAlta Life Sciences: Consultancy.