Molecular Lesions Associated with Loss of Heterozygosity Identified in CMML.

Abstract 416

Chronic myelomonocytic leukemia (CMML) is characterized by monocyte/monoblast proliferation, dysplastic features, progression to leukemia and poor prognosis. With the exception of rare cases of CMML with balanced PDGFRa or PDGFRb translocations, the molecular pathogenesis of the majority of CMML cases remains elusive. To date, somatic mutations pathogenic for CMML have not been identified, and large tyrosine kinase sequencing projects have been negative. The recent application of SNP-A as a karyotyping tool has allowed for more precise analyses of chromosomal defects and detection of copy-neutral loss of heterozygosity (CN-LOH). Previously, various recurrent areas of CN-LOH have been found in myeloid malignancies, but these lesions were particularly frequent in CMML. We have examined 68 patients with CMML and AML with antecedent CMML for recurrent areas of LOH, mapped mutations in genes contained within corresponding minimally affected regions, and examined their impact on clinical outcomes. We focused on Cbl, TET2 and RAS mutations and the resultant clinical and pathomorphologic phenotypes and outcomes associated with individual mutations. SNP-A-based karyotyping showed the presence of chromosomal aberrations in a larger proportion of cases than did metaphase cytogenetics: recurrent areas of somatic UPD were found in 49% of patients, with the most common shared lesions including UPD4q (n=6), UPD7q (n=6) and UPD11q (n=4). Initial identification of recurrent UPD11q and UPD4q and micro-deletions defining the smallest commonly affected areas led to the discovery of new mutations in CMML, including c-Cbl and TET2. In sum, we identified c-Cbl in 13%, TET2 in 49%, and RAS mutations in 10% of patients with CMML and sAML derived from CMML. LOH involving chromosome 7 was present in 18% of patients. All c-Cbl mutations were somatic and in the RING finger domain or linker sequence; 3/6 mutations were homozygous and one case with del11q harbored a hemizygous mutation. TET2 alterations were identified in 23 patients; in 26% of patients, both alleles were affected. However, no clinical differences were found between heterozygous and homozygous cases of TET2 mutations, suggesting a dominant negative effect. We also identified patients who harbored both TET2 and c-Cbl or RAS mutations demonstrating a multi-step pathogenesis of CMML. In addition, aberrant methylation at the CpG islands of promoters of TET2 (2 sites), c-Cbl (2 sites), b-Cbl (2 sites) and RAS (7 sites) was found to be relatively infrequent among patients. Intricate analysis of clinical and phenotypic features did not reveal pathognomonic phenotypes; however TET2 and c-Cbl mutations were associated with higher WBC (p=.027) or thrombocytopenia (p=.026), respectively and RAS mutations were associated with advanced disease (p=.027). Patients with LOH7q were included in our analysis as a separate group because they likely carried mutations in a putative candidate gene on 7q. In general, megakaryocyte nuclei displayed aberrant STAT5 staining in 31% of CMML cases (n=32) and correlated with morphologic dysplasia. Aberrant pSTAT5 staining suggestive of downstream pathways involving proliferative signals was present in 43% of TET2 mutated patients and 67% of c-Cbl mutants. None of the RAS mutants displayed aberrant STAT5 staining. Thus, aberrant STAT5 activation is not an obligatory feature of TET2 and RAS mutant cases, as opposed to the majority of c-Cbl mutant cases. There was no significant difference in survival between patients with TET2, c-Cbl and RAS mutations or those with LOH7q as compared to those without. Nevertheless, TET2 and c-Cbl mutant cases trended towards less favorable survival within lower-risk (<5% blasts) CMML, while no differences were seen in advanced cases, likely due to the uniformly poor survival in this group. Regardless of the presence of putative mutations, patients with LOH7q, including UPD7, shared a poor prognosis (7 vs. 11mo).

In sum, our results characterize CMML as a disorder frequently associated with acquired chromosomal lesions, activated STAT5 signaling, genetic mutations in RAS, ubiquitin modification and TET2 pathways. Mutations present in these patients may help to clarify pathogenic pathways, and thereby develop directed therapies.