Mutational Profile of Children with Myeloproliferative Neoplasms

Introduction Myeloproliferative neoplasms (MPNs) are clonal bone marrow disorders of over proliferation driven largely by mutations in the JAK2, CALR, and MPL genes. Mutational profiles including driver and secondary mutations in adult subjects with MPNs have been well studied and recent work has determined that driver mutations occur as early as in utero. There is little described about mutational profiles in pediatric patients with MPNs. Given the importance of clonal evolution and the relevance of MPN-associated mutations for risk assessment, clinical features, and outcomes, it is important to develop an understanding of the mutational spectrum in young subjects. Therefore, we sought to evaluate a pediatric MPN cohort for potential MPN-associated mutations.

Methods We isolated mononuclear cells from the blood and/or bone marrow of 29 pediatric patients (median age 10 years, range 2-21) with MPNs. DNA was extracted and sequenced on the HEMEPACT platform, a gene panel including almost 600 genes focused on myeloid malignancies. Variants were called using a combination of variant callers. For single-nucleotide variants (SNV), we used CaVEMan, Mutect, and Strelka, and for small insertions and deletions also used Pindel. VAF was uniformly reported across all called variants using a realignment procedure. Likely artifact variants were filtered out based on: (i) The number of callers calling a given variant and the combination of filters from the triple callers. (ii) Variants with VAF <2% in all samples or less than 3 mutant supporting reads were excluded.

Results In this cohort, 18 subjects had essential thrombocytosis (ET), 8 subjects had polycythemia vera (PV), 2 had pre-fibrotic myelofibrosis, and 1 had myelofibrosis. Clinical sequencing identified 9 subjects with ET with a JAK2V617F mutation, 3 ET subjects with CALR mutations, and 6 ET subjects who were "triple negative" (TN) for driver mutations. Six subjects with PV were found to have a JAK2V617F mutation, while 2 had JAK2 exon 12 mutations. Both subjects with pre-fibrotic myelofibrosis had CALR mutations, and the subject with myelofibrosis had a MPL mutation.

HEMEPACT identified mutations in 10 additional cancer-associated genes (Figure 1A). After JAK2 and CALR, the most frequently mutated genes were TET2 and SETBP1. Two subjects had TET2 mutations that co-occurred with JAK2V617F (Figure 1B), and both had very low TET2 variant allele frequencies (VAFs). Two subjects had SETBP1 mutations, one co-occurring with JAK2V617F, and 1 with CALR mutation (Figure 1B), both of whom had high SETBP1 VAFs, possibly implying a germline mutation. No mutations in ASXL1, EZH2, or DNMT3A were identified. Of the 6 TN subjects, 3 had mutations detected on HEMEPACT (in CHEK2, CUX1, and RB1). Eight of the 29 subjects (28%) had more than 1 mutation detected, and the average number of mutations was 1.2 per subject.

In contrast, in a small cohort of adult (median age 41 years, range 21-82) subjects with ET (n=2) and PV (n=7), 6 of 9 subjects (67%) had more than 1 mutation identified, and the average number of mutations per subject was 2. JAK2V617F variant allele frequency of the adult cohort was 4-times higher than in the pediatric cohort (median=0.16 vs. median=0.04).

Conclusion This is one of the largest analyses to date of the mutational spectrum of pediatric MPNs. TET2 and SETBP1 mutations co-occurred with driver mutations, and no DNMT3A or ASXL1 mutations were seen in these young patients. While fewer pediatric than adult subjects had multiple mutations, there were some young patients who had already developed multiple mutations in childhood and it is important to better understand the role clonal hematopoiesis may play in pediatric MPNs. Buccal samples in our pediatric patients showed evidence of contamination and identifying the best way to sample the germline from children with these disorders is crucial for the study of MPNs in young patients. There is much to learn about the mutational spectrum of pediatric MPNs, including the role of germline mutations in disease pathogenesis, drivers of MPN development in triple-negative MPNs, mutation acquisition over time, and role of various mutations in MPN progression and outcomes. With this initial evaluation we have begun the process of answering some of these questions.

Rampal:Galecto: Consultancy, Membership on an entity's Board of Directors or advisory committees; Stemline: Consultancy, Research Funding; Blueprint: Consultancy; CTI: Consultancy; Sunimoto Dainippon: Consultancy; Sierra Oncology: Consultancy; Disc Medicines: Consultancy; Stemline: Consultancy, Research Funding; Gilead: Consultancy; Jazz Pharmaceuticals: Consultancy; PharmaEssentia: Consultancy; Kartos: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Zentalis: Consultancy, Research Funding; Promedior: Consultancy; Novartis: Consultancy; Celgene/BMS: Consultancy; Incyte: Consultancy, Research Funding; Constellation Pharmaceuticals, Inc., a MorphoSys Company: Consultancy, Research Funding. Levine:Astra Zeneca and Kura: Other: honoraria for invited lectures ; Imago, Mission Bio, Bakx, Zentalis, Ajax, Auron, Prelude, C4 Therapeutics and Isoplexis: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Syndax, Incyte, Janssen, Astellas, Morphosys and Novartis: Consultancy; Gilead and Novartis: Other: Grant reviews; Ajax, Abbvie, Constellation, Zenalis, Celgene, Roche, and Prelude: Other: research support; Qiagen: Other: supervisory board member. Kucine:Protagonist Therapeutics: Other: Member, Safety monitoring committee.