Frequent Detection of C-Cbl Homozygous Mutation in Secondary Myelogeneous Leukemia Transformed from MDS/MPD with Chromosome 11q Uniparental Disomy

Abstract

Two types of acquired loss of heterozygosity are possible in cancer: deletions and copy-neutral uniparental disomy (UPD). Conventionally, copy number losses are identified using metaphase cytogenetics while detection of UPD is accomplished by microsatellite and copy number analysis and as such, is not often used clinically. Recently, introduction of single nucleotide polymorphism microarrays (SNP-A) have allowed for the systematic and sensitive detection of UPD in hematological malignancies and other cancers. In this study, we have applied Affymetrics 250K and 6.0 SNP-A technology to detect previously cryptic chromosomal changes, particularly UPD, in a cohort of 301 patients with myelodysplastic syndromes (MDS), overlap MDS/myeloproliferative disorders (MPD), MPD, and primary and secondary acute myeloid leukemia (AML). When appropriate, germ line DNA was analyzed to confirm somatic nature of the suspected lesions. We show that UPD is a common chromosomal defect in myeloid malignancies, particularly in chronic myelomonocytic leukemia (CMML; 52%) and MDS/MPD-unclassifiable (49%). Furthermore, we demonstrate that mapping minimally overlapping segmental UPD regions can help target the search for both known and unknown pathogenic mutations. Chromosomes frequently affected by UPD include 1p (N=12), 4q (N=11), 6p (N=9), 7q (N=9), 9p (N=11), 13 (N=11), 17 (N=11), and 21 (N=7). The chromosome arm most often affected was 11q, occurring in 15/301 patients, 8 of which had MDS/MPDu, CMML or AML evolving from these conditions. These patients with UPD11q appear to display several common clinical phenotypic trends, including history of MDS/MPD, the presence of monocytic blasts or increased numbers of differentiated monocytes, propensity to transformation, and poor prognosis. Given the prevalence of UPD on chromosome 11q, we screened for candidate genes located in this region. Among our UPD11q cohort, the lesions of 12/15 patients were located in the region of the c-Cbl gene encoding the E3 ubiquitin ligase involved in the degradation of active protein tyrosine kinase receptors. Direct genomic sequencing of c-Cbl in these patients revealed the presence of 3 unique missense mutations, all occurring within or directly adjacent to the RING finger domain responsible for ubiquitination activity. In total, 7/12 patients with UPD11q showed c-Cbl mutation. One mutation, occurring in 2/7 patients, resulted in the substitution of an arginine with either glutamine or proline at position 420 (R420Q/P) located just outside the RING domain. However, we also found 2 additional, newly-identified missense mutations, both affecting the cysteines of the RING finger in the remaining 5 patients. In 2/5 patients, residue 384 was altered by substitution of a tyrosine. In the other 3 patients, residue 404 was altered by substitution of either a tyrosine (in 1 patient) or serine (in 2 patients). When additional 71 patients with similar phenotypic features but negative for UPD11q were screened, 2 novel c-Cbl mutations in RING finger domain (heterozygous) and Linker sequence (monoallelic in deletion 11q) were identified to a total of 9 cases affected by c-Cbl mutations. Analysis of clinical/immunological/pathological phenotype of these patients revealed the history of blast transformation in 77%, presence of monocytosis (over 1000/ul) or monocytic blasts in 88%, poor prognosis in 100% (5 years over all survival; 0%), some degree of marrow fibrosis in 100% and c-kit positivity in 77% of cases. We conclude that invariant mutations in c-Cbl E3 ubiquitin ligase may explain the pathogenesis of a clonal process or subsequent AML transformation in a unique subset of MDS/MPD, including CMML.