SNP-A technology, with its improved resolution and ability to detect uniparental disomy, increases the overall detection rate of chromosomal abnormalities and complements metaphase cytogenetics (MC) in the delineation of chromosomal lesions associated with hematological malignancies. Recently, we and others have shown that in MDS, MDS/MPD and MDS-derived AML, previously unrecognized chromosomal defects detected by SNP-A have similar impact on prognostic parameters including overall survival as those lesions detected by MC.(7
) In addition to its potential clinical implications, SNP-A karyotyping is also an excellent investigative tool, facilitating delineation of invariant chromosomal defects and, in turn, corresponding genes which may be implicated in the pathogenesis of hematological malignancies.
Using 250K SNP-A and CNAG v3.0 software, we have identified in a large cohort of patients with MDS, MDS/MPD, MPD, and AML, a significant proportion of UPD, particularly in MDS/MPD. Often times, overlapping regions of UPD correlated with the presence of homozygous gene mutations, including FLT3
-ITD in 7/7 cases with UPD13q, accounting for 30% of all cases positive for a FLT3
-ITD mutation (N=23), and JAK2 V617F mutations in all MDS/MPD and MPD cases characterized by UPD9p. In addition, we identified c-MPL
point mutations in 4 patients with UPD1p. c-MPL
mutations were associated with MDS/MPD, and we believe that this is the first report of biallelic c-MPL or NRAS mutations occurring as a result of acquired UPD. Although not formally tested here, one could also hypothesize that, in accordance with previous studies,(11
) there are homozygous gene mutations affecting either RUNX1
in those patients with acquired UPD on chromosomes 21 (N=6) or 19 (N=2) respectively.
Systematic mapping of copy-neutral LOH has allowed for the identification of other frequently shared areas of UPD that are currently not attributable to any known gene mutations in myeloid malignancies. Identification of novel mutations in these regions may explain the pathophysiology of those individual cases affected. Examples of these commonly affected areas include segments on chromosomes 4q, 6p, 7q, 14, 17 and 11q. Various genes potentially involved in the pathogenesis of leukemia are located in these regions including EIF4E, CCNA2 and FGF2 on 4q, DUSP22 and PAC4 on 6p, BRAF, CUL1, and EZH2 on 7q, TNFAIP2 and AKT1 on 14, and p53 and GRB2 on 17.
When we examined patients with UPD11q, we noted several common clinical phenotypic trends, including history of MDS/MPD, the presence of monocytic blasts or increased numbers of differentiated monocytes, reticulin fibrosis and propensity to AML transformation. Monoallelic mutations in the alpha-helix linker domain of c-Cbl
disrupt binding of c-Cbl to E2 ubiquitin-conjugating enzymes and thus, deregulate proper ubiquitylation and degradation of phosphorylated protein tyrosine kinase receptors in animal models(32
) and in a small number of patients with AML.(27
) We hypothesized that mutated, dysfunctional c-Cbl could lead to deregulated activity along proliferative transduction pathways consistent with the phenotype of MDS/MPD. For example, other TK receptors including CSF1R, PDGFRβ and EGFR have all been described to be inactivated by members of the Cbl ubiquitin ligase family either directly or via binding to Grb2.(33
In addition to 2 patients harboring biallelic R420Q/P mutations previously reported, we identified new, biallelic mutations of c-Cbl in 5 additional patients which cause single amino acid substitutions affecting the highly-conserved cysteine residues at positions 384 and 404 of the RING domain. Analysis of non-clonal T-cells from these cases confirmed the acquired nature of these mutations. However, unlike previous reports, sequencing targeted to identify RING and linker domain monoallelic mutations in heterozygous configuration did not identify patients with monoallelic c-Cbl. Based on our observation of only biallelic mutations in our MDS/MPD patients, we hypothesize that the pathogenic role of c-Cbl mutations may differ in AML versus MDS/MPD. Alternatively or additionally, the discrepancy between our findings and those previously reported in AML may be complicated by technical problems which arise when distinguishing between true monoallelic mutations in one clone and a mixture of abnormal clonal cells harboring biallelic lesions (e.g., due to UPD) and nonclonal, wild-type cell contributions. We have shown that patients with c-Cbl mutations all have corresponding UPD11q; perhaps SNP-A applied to the previously described AML patients would also reveal UPD11q.
Patients with c-Cbl
mutations showed a great deal of diversity with regard to clinical phenotype (). While c-Cbl mutations have been previously linked to primary AML only,(29
) all patients positive for c-Cbl
mutations in this study had developed from a previous hematologic disease. In addition, some patients with c-Cbl
mutations have additional chromosomal aberrations and/or genetic mutations that could modify the resultant clinical phenotype. For example, one patient (Patient 2, ) was JAK2 V617F+. Another two patients (Patients 6 and 12, ) were NPM1+. In addition, related genes Cbl-b
(3q13.11) and Cbl-c
(19q13.31) may harbor mutations with pathogenetic significance which could explain other cases of myeloid malignancies at the molecular level. These results serve to illustrate that advanced myeloid malignancies likely have many cooperating genetic and epigenetic lesions, and that high resolution SNP arrays provide insight into sorting out affected molecular pathways.
Systematic analysis of UPD also provides many important clues as to the pathogenesis that may operate in MDS, MDS/MPD, or AML. We have noted that certain chromosomes are as frequently affected by UPD as deletion (e.g. 7 and 21). However, UPD may suggest a different mechanism and type of gene affected (e.g., duplication of an activating mutation) compared to deletion, which can lead to haploinsufficiency or “unmasking” of a pre-existing inactivating mutation in a tumor suppressor gene. This may explain why some chromosomes are more frequently affected by deletion than UPD. For example, while we have identified 35 cases with deletions involving the minimally affected region on chromosome 5q, only 1 example of UPD was found in the corresponding area, suggesting that affected genes in this region do not provide an additional selection advantage if duplicated.
It should also be noted, however, that not all UPD is likely to be pathogenic but may simply serve as a marker of clonality and/or underlying previous DNA damage.(1
) To enable analysis of as many samples as possible and determine clinical applicability of SNP-A, most patient samples were run using whole bone marrow and/or blood. Most often the admixture of non-clonal with clonal cells is not an issue in samples with advanced MDS, AML and CMML as the percentages of non-clonal cells are low. However, in low grade MDS, it is possible that the admixture of clonal cells with non-clonal cells may be much lower and thus, some UPD may have been undetected by our analysis.
In sum, our investigations provide a proof of principle that delineation of shared areas of UPD identified by SNP-A may facilitate the identification of genes affected by pathogenic mutations and potentially explain clinical phenotype. Segmental UPD appears to be a common clonal lesion in myeloid malignancies. As more sensitive techniques and study of additional myeloid malignancy cases accrue, comparison of individual UPD lesions with current clinical, molecular, genetic, and cytogenetic prognostic schemes in larger cohorts of patients will likely provide additional important diagnostic, prognostic, and translational biomarkers.