Whole Genome Sequencing On Donor Cell Leukemia in a Patient with Multiple Myeloma Identified Gene Mutations That May Provide Insights to Leukemogenesis.

Abstract 2414

Donor cell leukemia (DCL) is a rare occurrence and refers to leukemia of donor origin in patients who have received allogeneic hematopoietic stem cell transplantation (HSCT). We have previously described a male patient with IgG-κ myeloma who received non-myeloablative allogeneic HSCT from a HLA-matched brother and developed complex karyotype acute myeloid leukemia (AML) of donor origin 10 years after transplantation. He achieved complete remission (CR) with standard induction and consolidation chemotherapy but relapsed one year afterwards. We hypothesized that a comparison of the donor HSC before transplantation (pre-leukemic) and the subsequent AML at whole genome level will provide a unique dataset that may shed light on the pathogenesis of leukemia.

DNA was extracted from an aliquot of donor mobilized peripheral blood mononuclear cells (mPBMNC) frozen before transplantation as well as unfractionated and CD34⁺ myeloblasts of the patient's bone marrow at diagnosis and subsequent relapse of AML. The complete donor origin of the AML was confirmed by PCR based on polymorphic STRs. Whole-genome sequencing (WGS) was performed to sequence paired-end reads generated by Illumina HiSeq 2000. Reads were aligned to the human referecne genome (hg19, NCBI37) by SOAP3 and analysed to detect single nucleotide variants (SNVs), small insertion and deletion (indels) and copy number variations (CNVs). Selected genes after filtering were independently validated by Sanger sequencing.

There were 835M and 810M 100bp paried-end reads with insert distance of 500bp generated from donor mPBMNC and CD34⁺ myeloblasts of the relapsed DCL with respective mean depths of 43.2X and 42.6X after alignment. The digital karytoyping based on the read depth was consistent with that by conventional cytogenetic study. 3,979,582 and 1,020,717 SNVs and indels were detectable from both samples. Based on the Catalog of Somatic Mutations in Cancer (COSMIC) and excluding those asian specific wildtypes annotated in 1000 genome project, 11 SNVs and 15 indels within coding sequence with potential roles as tumor suppressors or oncogenes were identified. On the other hand, there were 128,752 and 56,330 SNVs and indels detected exclusively in DCL. Those putative non-pathogenic SNP and those changes locating outside the gene regions were filtered. Within the gene region, SNVs in introns and synonymous mutations were also filtered. 142 non-synonymous SNVs (139 missense and 3 nonsense mutations) were identified of which 25 were considered as statistically highly confident and 17 of them could be confirmed by Sanger Sequencing. Twelve of these were also identified from the whole BM sample of DCL at diagnosis. These candidates include transcription factor (SALL1), metabolic enzymes (UGT1A5, SPEG), membrane protein (TMC6, SCN3A), cytoskeleton protein (MYH10), ribonucleoprotein (RAVER1), secreted protein (WNT7A), protein involved in DNA damage repair (APLF) and others (PRPF8, ZNF518B and MKRN3). 26 indels were indentified in the coding region of which 5 were considered as statistically highly confident, however, only one indel could be confirmed by Sanger Sequencing in the relapse sample and was not present in the diagnostic sample.

The WGS performed in paired pre-leukemic (donor HSC) and leukemic (DCL) human samples has provided us with unique opportunities to dissect the genetic changes in HSC that may contribute to the initiation of AML with complex karyotype. The potential impacts of bone marrow microenvironment in this patient with myeloma in inducing DCL are also being evaluated.