Clonal Evolution Revealed by Whole Genome Sequencing in a Case of Primary Myelofibrosis Transformed to Secondary Acute Myeloid Leukemia

Abstract 706

While the JAK2 V617F mutation and several other genetic alterations have been identified in myeloproliferative neoplasms (MPNs), a comprehensive delineation of the genomic changes underlying these diseases has been lacking. Chronic MPNs such as primary myelofibrosis (PMF) exhibit a propensity for transformation to secondary acute myeloid leukemia (sAML), for which the prognosis is poor. In this era of targeted therapies, a deeper understanding of the genetic complexity and clonal architecture of these diseases is essential.

We describe a woman who first presented with splenomegaly, pancytopenia, and leukoerythroblastosis at the age of 51. A bone marrow biopsy demonstrated severe fibrosis, consistent with a diagnosis of PMF. Cytogenetics were normal. Bone marrow samples were banked at that time. The patient had an excellent response to treatment with thalidomide, ultimately achieving a complete hematologic remission, but was eventually switched to lenalidomide due to neuropathy. Seven years after initial PMF diagnosis, the patient transformed to sAML. A bone marrow biopsy revealed 49% blasts, and cytogenetics were normal. Testing for JAK2 V617F was positive. Bone marrow samples were again banked. The patient received induction chemotherapy with IDA-FLAG and attained a complete remission, followed by consolidation chemotherapy with four cycles of high-dose cytarabine. Subsequently, the patient declined bone marrow transplantation. Approximately 1.5 years after sAML diagnosis, the patient again developed pancytopenia with leukoerythroblastosis, consistent with relapsed/residual PMF, but with no evidence of sAML relapse. Lenalidomide was restarted at that time. Approximately 2.5 years after sAML diagnosis, the patient remains alive with transfusion-dependent anemia and thrombocytopenia.

Whole genome sequencing (WGS) was performed on bone marrow samples banked at PMF and sAML diagnosis, with skin included as a germline surrogate. Haploid coverage of 63.9x (PMF), 60.2x (sAML), and 37.5x (skin) was obtained. A total of 38 high confidence (HC) tier 1 (coding and splice site) single nucleotide variants (SNVs) were identified in the PMF and/or sAML samples but not in the skin. Six of these somatic SNVs were in genes previously known to be involved in MPNs and/or sAML. Both the PMF and sAML samples were predominantly homozygous for JAK2 V617F. Copy-neutral loss of heterozygosity in the first 8.7 Mb of chromosome 9 was identified, confirming uniparental disomy involving JAK2 V617F. U2AF1 was mutated in both the PMF and sAML phases. A mutation in MYB was detected in the PMF but not sAML sample, suggesting the presence of a clone that may have contributed to PMF development, but that was dispensable for transformation. A minor subclone at the PMF stage containing a nonsense mutation in ASXL became substantially enriched upon transformation, illustrating the clonal complexity within a predominantly JAK2 V617F-positive bone marrow. Mutations in IDH1 and RUNX1 were observed in sAML but not PMF, indicating they were likely acquired subsequent to the ASXL1 mutation and also contributed specifically to transformation. These findings suggest that transformation to sAML in this patient was largely driven by the combination of mutations in ASXL1, IDH1, and RUNX1, with possible contribution from a small number of additional mutations.

To fully define the clonal hierarchy of PMF transformed to sAML in this patient, deep sequencing validation of all tier 1 SNVs, all tier 2–3 HC SNVs, and all putative indels and structural variants identified by WGS is currently in progress. This includes putative mutations in several novel genes that may be pathogenic. A sample banked during sAML remission (two years after sAML diagnosis) has also been included in the validation sequencing. This will enable us to determine whether any residual mutations/clones may be identifiable in this particular patient, which has implications for potential future relapse. This study illustrates the capacity of WGS to identify the critical genetic drivers of MPN pathogenesis, and to define the basis for clonal evolution in MPNs and sAML.