Clonal Evolution Revealed By Exome Sequencing in a Case of Primary Myelofibrosis Associated with Subsequent Development of Aggressive Systemic Mastocytosis/Mast Cell Leukemia
Authors: Oh Stephen T. , Miller Christopher A. , Gindin Yevgeniy , Brost Taylor M. , Chan Jason , Fulbright Mary C. , Fisher Daniel A.C. , Duncavage Eric J. , O'Laughlin Michelle , Griffith Malachi , Griffith Obi L. , Wartman Lukas D. 
Published: October 2019

Abstract
A 77 year-old man initially presented in July 2013 with anemia, splenomegaly, and constitutional symptoms. A bone marrow biopsy revealed a hypercellular marrow with megakaryocytic hyperplasia and atypia and mild reticulin fibrosis, consistent with a diagnosis of primary myelofibrosis (PMF). Cytogenetics revealed a normal karyotype. JAK2 V617F testing was negative. Initiation of treatment with the JAK inhibitor ruxolitinib led to marked symptomatic improvement. The patient was then enrolled in a Phase 2 study with the anti-lysyl oxidase-like-2 (LOXL2) monoclonal antibody simtuzumab, administered via IV infusion every two weeks (while continuing ruxolitinib). He tolerated the simtuzumab infusions well initially, but with the 11th and 12th infusions experienced rigors, hypotension, and hypoxia. This occurred ~8 months after his initial PMF diagnosis. A repeat bone marrow biopsy revealed large aggregates of mast cells comprising 30-40% of the marrow cellularity (with 16% mast cells enumerated on the aspirate). A subset of the mast cells exhibited spindled morphology, and CD25 co-expression was demonstrated by flow cytometry in a subset of CD117-positive cells. Testing for the KITD816V mutation was positive. Tryptase levels were significantly elevated (375 ng/mL). These findings were consistent with a diagnosis of aggressive systemic mastocytosis with an associated hematologic non-mast cell lineage disorder (ASM-AHNMD).
Compassionate-use approval for the KIT inhibitor midostaurin was obtained, and treatment with midostaurin (in addition to continuation of ruxolitinib) was initiated. The patient initially reported symptomatic improvement with midostaurin treatment, but after several months his symptoms began to worsen, with a corresponding increase in tryptase (875 ng/mL). A repeat bone marrow biopsy revealed overt evolution to mast cell leukemia (MCL) with > 90% mast cell involvement. Based on the presence of an IDH2mutation identified on a clinical next-generation sequencing assay, the patient was evaluated for a clinical trial with the mutant IDH2 inhibitor AG-221. Unfortunately, the patient decompensated and expired before he could enroll in the study.
To identify contributing driver mutations and to delineate clonal hierarchy associated with disease initiation and progression in this unique case of PMF with concomitant ASM, exome sequencing was performed on serial samples obtained at the following disease stages:PMF diagnosis (pre-ruxolitinib) (Day 0)PMF on ruxolitinib (before ASM diagnosis) (Day 181)ASM diagnosis (post-anti-LOXL2 antibody, pre-midostaurin) (Day 394)Progression to MCL (on midostaurin) (Day 519)Matched skin (normal) sample
Likely driver mutations in IDH2 and SRSF2 were identified at ~40-50% variant allele frequency (VAF) in all samples and were therefore likely present in the founding clone. The KIT D816V mutation was found at 23% VAF at Day 0, then ~40% VAF in all other samples, suggesting it was present in a daughter subclone of the IDH2/SRSF2-containing clone that became dominant over time with disease progression. These findings also suggest that targeting KIT with midostaurin would be unlikely to eradicate the founding clone. Rather, selective targeting of IDH2 and/or SRSF2 could potentially ameliorate both diseases (PMF and ASM/MCL).
To provide pre-clinical evidence of the potential utility of targeting IDH2, peripheral blood mononuclear cells obtained at the time of ASM diagnosis were plated in liquid culture in the presence or absence of the mutant IDH2 inhibitor AGI-6780. After 14 days in culture, the differentiation status of the cultured cells was examined by mass cytometry (CyTOF). Treatment with AGI-6780 resulted in a marked enhancement of myeloid differentiation (denoted by CD15 and CD66b expression) along with a corresponding decrease in CD34+ progenitor cells. These effects were not seen in cells cultured in the absence of AGI-6780. These results are consistent with prior studies in acute myeloid leukemia indicating that the beneficial effects of mutant IDH2 inhibition are likely related to inducing differentiation of primitive cells. In summary, this study highlights the capacity of serial genomic analysis to define the clonal architecture that drives disease initiation and evolution, and to distinguish founding vs subclonal mutations to identify the most promising targets for therapeutic intervention.

Disclosures
Oh: Janssen: Research Funding; CTI: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding.