Clinical and Genetic Characterization Of Patients With C-CBL Mutated Juvenile Myelomonocytic Leukemia By Whole-Exome/Deep Sequencing

Juvenile myelomonocytic leukemia (JMML) is a rare pediatric myeloid neoplasm clinically characterized by excessive proliferation of myelomonocytic cells and hypersensitivity to granulocyte–macrophage colony-stimulating factor (GM-CSF). A cardinal genetic feature of JMML is frequent somatic and/or germline mutations of RAS pathway genes involved in GM-CSF signal transduction, such as NRAS, KRAS, PTPN11, NF1, and c-CBL, which are found in a mutually exclusive manner in >80% affected children.

We examined 108 children (71 boys and 37 girls) diagnosed with JMML in institutions throughout Japan. Written informed consent for sample collection was obtained from the parents of the patients. Molecular analysis, including whole-exome sequencing, was approved by the Ethics Committees of Nagoya University Graduate School of Medicine and Graduate School of Medicine, the University of Tokyo, in accordance with the Declaration of Helsinki. JMML diagnoses were based on internationally accepted criteria.

Using exome sequencing and target deep sequencing, we identified 17 (7 boys and 10 girls) patients with c-CBLmutations. The median age at diagnosis was 11 months (range, 1-67 months). Seven of these 17 (41%) patients underwent allogeneic hematopoietic stem cell transplantation (HSCT).

Comparison of the clinical characteristics between patients with c-CBL mutations (n = 17) and those without c-CBL mutations (n = 91) revealed a statistically significant male predominance restricted to the patients without c-CBL mutations [7/17 (41%) vs. 64/91 (70%); p = 0.02]. The genetic alterations of c-CBLobserved in the 17 patients were as follows: 12 point mutations, 1 splice site mutation, and 4 deletions [c.1105del66: p.Glu369_Asp390del (n = 1); c.1217del22: p.Thr406fs (n = 1); c.1096-110del643: p.Glu366_Phe468del (n = 1); and c.1177_1227+86del: p.Ile393_Gln409del (n = 1)]. We could confirm heterozygous germline mutations in all the 5 patients (100%) whose germline sample was available [buccal smear and nail (n = 2), buccal smear (n = 1), and CD3+T cell (n = 2)].

Deep sequencing using a next generation sequencing platform enabled precise estimation of the mutated allele frequencies of each mutation, and we categorized the patients with c-CBL mutations into 3 distinctive groups: (A) homozygous (allele frequencies >86%; n = 10), (B) heterozygous (35%–42%; n = 5), and (C)small clone mutations (19% and 7%; n = 2).

While the majority of point mutations and splice site mutations are homozygous [11/13 (85%)], which is consistent with previous reports, all 4 deletion mutations showed heterozygosity (p = 0.0037). Surprisingly, both patients with small clone mutations harbored other RAS pathway mutations simultaneously with higher mutated allele frequencies (%) [PTPN11 (c.181G>T, p.Asp61Tyr; 46%; n = 1) and KRAS (c.38G>A, p.Gly13Asp; 42%; n = 1)], suggesting that c-CBL mutations occurred as secondary genetic events in these patients. Any patient with JMML with a c-CBL mutation does not have SETBP1 and JAK3 mutations, which we recently identified as secondary mutations in JMML (Sakaguchi et.al., Nature Genetics2013).

Unexpectedly, central nervous system complications were observed in 3 of the 17 (18%) patients (Moyamoya disease, n = 2, and acute disseminated encephalomyelitis, n = 1). Although 4 patients survived without post HSCT event, late graft rejection (n = 1), relapse as acute myeloid leukemia (n = 1), and death due to unknown reasons (n = 1) was observed among the 7 patients who underwent HSCT. Nine of 10 patients with JMML survived without HSCT. Consequently, the probability of 5-year transplantation-free survival [95% confidence interval (CI)] of the 17 patients with c-CBL mutations was significantly superior to that of the other 91 patients without c-CBL mutations [38% (9%–69%) vs. 14% (5%–26%), p < 0.001].

JMML patients with c-CBL mutations have genetic and clinical heterogeneity. However, this subgroup of JMML showed better survival outcome compared with c-CBL wild-type JMML, although with several characteristic clinical events precluding patients’ quality of life. Further clinical research is warranted to elucidate determining factors for clinical heterogeneity of c-CBL mutated JMML patients.

Makishima:AA & MDS international foundation: Research Funding; Scott Hamilton CARES grant: Research Funding. Maciejewski:NIH: Research Funding; Aplastic anemia&MDS International Foundation: Research Funding.