Early clonality studies suggested that JMML arose at the level of at least an immature myeloid precursor cell. More recent data suggest that JMML may arise in a pluripotent stem cell with involvement of the myeloid, erythroid, and megakaryocyte lineages, as well as B lymphocytes and T lymphocytes. This is supported by finding pathogenic mutations in immature hematopoietic cells identified by flow cytometry.
Somatic mutations that augment signaling through the Ras path way occur in approximately 90% to 95% of JMML patients. These include biallelic inactivation of NF1 (15%), activating mutations in NRAS or KRAS (25%), mutations in the PTPN11 gene (35%), and mutations in CBL (10% to 15%). Less frequent but more recently reported mutations occur in the genes RRAS, RRAS2, RAC2 and SH2B3. This synopsis reflects decades of research, beginning with the seminal observation that JMML is associated with NF1. A rough comparison of incidence rates suggests that NF1 carries a 500-fold increased risk of acquiring JMML. At a molecular level, these children inherit a defective allele of the NF1 gene, which encodes a negative regulator of Ras named neurofibromin. In JMML, the normal allele is lost as a somatic event in the initiating cell, most often by loss of heterozygosity in which the overall copy number remains unchanged (uniparental isodisomy). This completely ablates neurofibromin and thereby increases Ras activity.
Such interplay of inherited and somatic mutations is also relevant to the other JMML genes. Germline mutations in PTPN11 cause Noonan syndrome (NS), a common genetic condition that overlaps phenotypically with NF1 and sometimes includes a mild JMML-like myeloproliferative syndrome. This finding led directly to the discovery of somatic PTPN11 mutations in sporadic JMML, a lesion now recognized as the most common pathogenic mutation in this dis ease. Furthermore, rare germline mutations in KRAS have also been associated with related genetic syndromes including NS and car diofaciocutaneous syndrome. The PTPN11 and KRAS mutations present in the germline generally have less severe biochemical consequences than those in sporadic cases. This suggests that strong signal activation is not tolerated during development. Accordingly, the most common NS-associated PTPN11 alleles have relatively weak effects on signal transduction, and the myeloproliferative syn drome in these patients is usually transient. However, a more recent and comprehensive analysis has suggested that a cohort of NS patients may inherit more deleterious mutations and develop fatal neonatal JMML. Thus, cytoreductive therapy or HCT may be considered for select NS patients with severe disease.
Similar to NF1, mutations in CBL are most often heterozygous in the germline, with subsequent reduction to homozygosity in hematopoietic cells. However, recent reports demonstrate that JMML patients can harbor somatic CBL mutations, similar to adults with MPNs. The more typical heterozygous germline mutations appear to cause a genetic syndrome with features that can overlap with NS, termed CBL syndrome or NS-like disorder with or without JMML. The phenotypic manifestations of NSLL are variable and incompletely penetrant, and they include cryptorchidism, hearing loss, and skeletal abnormalities. Importantly, major vascular abnormalities have been described in patients with CBL-mutant JMML, and this could also reflect an NSLL phenotype. As expected for a heterozygous mutation, transmission is autosomal dominant, although 50% of cases arise spontaneously. In contrast to the loss of function mutations in NF1, the mutations in CBL selectively inactivate the ubiquitin ligase activity of the Cbl protein while leaving the molecule otherwise intact. When homozygous, this alteration eliminates the negative regulatory activity of Cbl while sparing positive signaling functions.
These so-called Ras pathway or driver mutations are largely mutually exclusive, supporting the idea that each provides a similar function in JMML. However, patients with two such lesions (approximately 10% of patients have co-occurring Ras pathway mutations) or duplication of the mutant allele have been reported, typically co-occurring in the same cells. This likely reflects clonal evolution and positive selection for increased Ras signaling even beyond that provided by the initiating mutation. Growing evidence demonstrates that negative feedback prevents the Ras pathway from being fully activated by a single lesion, leading to a dosage effect of mutant alleles on signaling outputs. Consistent with predictions from model systems, cases with multiple Ras pathway mutations have a more aggressive clinical course.
Genomic studies have also begun to address the question of whether mutations outside the Ras pathway contribute to the pathogenesis of JMML. A wide variety of so-called secondary mutations have now been recognized. They are primarily associated with relapse of JMML after HCT, or with transformation to AML. However, they often can be detected at a low level in the blood or BM when JMML is first diagnosed. This suggests that clonal evolution depends on chemotherapy resistance and eventually leukemic transformation. This is supported by the striking observation that secondary mutations of any kind have a major prognostic impact, even when they are present in a very small proportion of hematopoietic cells at diagnosis.
In the few JMML cases in which clonal history has be inferred, mutations appear to be acquired sequentially, beginning with a Ras pathway lesion. This linear evolutionary path contrasts with the highly branched patterns typical of high-grade neoplasms. Thus, despite the discovery of a variety of secondary mutations in JMML, it remains a disease with much less genetic complexity than most cancers. This may have important implications for therapy, as any individual patient is likely to harbor a relatively small number of distinct subclones that need to be eradicated to achieve cure.
The cellular function of cooperating mutations in JMML is under active investigation. Most can be categorized into three groups. Some amplify signaling beyond the level provided by the initial Ras pathway mutation. As described earlier, some second mutations directly alter core Ras pathway genes. Janus-activated kinase (JAK)/signal transducer and activator of transcription (STAT) signaling is enhanced by activating mutations in JAK3 or loss-of-function mutations in SH2B3, which encodes the negative regulatory adaptor protein LNK. Mutations in RRAS, RRAS2, and RAC2 represent a novel signaling complex in JMML that is implicated in regulating the PI3K/Akt pathway. Dominant point mutations in SETBP1 remain some what unexplored, but may augment signal transduction by inhibiting the phosphatase PP2A, or regulate gene transcription and strengthen self-renewal programs. Other secondary lesions have broad effects on gene expression, through inactivation of the PRC2 polycomb repressor complex or alteration of spliceosome components. Many of these genes and cellular functions are also implicated in the pathogenesis of other myeloid neoplasms such as CMML or AML.
Several genome-wide studies have now been published documenting the role of altered DNA methylation as a predictor of the outcome in JMML.
Patients with a hypermethylated profile are typically present at an older age, have higher levels of fetal hemoglobin, and more often have more than one somatic alteration in their JMML cells. In multivariable analyses, DNA methylation has been the most sig nificant predictor of outcome with hypermethylated patients experi encing the highest rates of relapse post-HSCT. In contrast, patients with a hypomethylated profile tend to be younger at presentation, have normal levels of fetal hemoglobin and invariably have only one somatic mutation. These patients are more likely to be cured with HSCT but of interest may also be the most likely to experience spontaneous resolution in the absence of transplant. International efforts are now underway to establish consensus definitions of altered DNA methylation and develop techniques to bring DNA methylation testing into the clinic in order to prospectively test the predictive nature of DNA methylation. The exact mechanism that altered DNA methylation plays in this disease has yet to be elucidated and is the focus of ongoing investigations.
