Kawamata, Norihiko

Abstract Myeloproliferative disorders (MPD) are a group of heterogeneous diseases that include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). They are characterized by increased hematopoiesis leading to elevated numbers of non-lymphoid cells and/or platelets in the peripheral blood. Beside thrombotic and hemorrhagic complications, MPD may evolve into secondary acute myeloid leukemia (sAML). Recently characterized markers suggest an opportunity to diagnose and identify subpopulations of MPD patients. In particular, altered expression and point mutations of PRV-1, MPL and JAK2 were commonly found in MPD, as well as deletions on chromosome 20q (del20q). Acquired uniparental disomy (UPD) on chromosome 1p (1pUPD) and 9p (9pUPD) leading to copy-neutral loss of heterozygosity (LOH) is a further mechanism found in MPD which often leads to homozygous activated mutations of MPL and JAK2, respectively. However, the molecular mechanisms involved in the transformation process to sAML remains unclear. Using standard metaphase cytogenetics (MC), chromosomal abnormalities are found in only a proportion of patients with MPD. We hypothesized that with new precise methods more genomic lesions can be uncovered that may be associated with leukemic transformation. To address this issue, we used 250K single nucleotide polymorphisms (SNP) Chip arrays to study chromosomal lesions in 40 sAML samples from patients who evolved from MPD; 7 had preexisting PV, 25 PMF, and 8 ET. Moreover, 43 additional samples of MPD (10 PV, 17 ET, and 16 PMF) were included in this study. SNP-chip analysis showed major chromosomal changes in almost all the sAML samples including monosomy 16, deletions of 1q-, 3p-, 6p-, 5q-, 7q-, 9q-, 12p-, 6q-, 3q-, 17p-, 19q-, and 20q- as well as trisomy 2, 3, 8, 9, 12, 15, 19, 21, and 22. We validated these data by MC. However, numerous new genomic alterations which contained potentially interesting genes that might contribute to leukemic transformation were detected by SNP Chip Array in patient samples with normal karyotype. Moreover, UPD was very frequent: 44% (19/43) of MPD and 53 % (21/40) sAML samples. 1pUPD occurred in 5 patients with MPD (1PV, 4 PMF; 12 %) compared to 5 patients with sAML (1 PV, 4 PMF; 13 %). 9pUPD was found in 16 MPD patients (8 PV, 7 PMF, 1 ET; 37%) and 6 sAML patients (3 PV, 2 PMF, 1ET; 15 %). All patients with 9pUPD proved to be positive for the JAK2 V617F mutation seen by allele specific PCR. Interestingly, the MPD samples only had UPD on 1p, 9p, and 12q. In contrast, sAML samples showed additional UPD regions on 7q, 11q, 12q, 16p, 17p, 19q, and 21q. Beside the evaluation of the non-matching groups of patients with MPD and sAML, we also evaluated 4 patients during their PMF and sAML stages by SNP Chip. The sAML samples acquired additional genomic changes including trisomy 8, 10, 14, 19, duplication on 3q and 6p, and heterozygote deletion on 18q. In contrast, 1pUPD, 9pUPD, and 12qUPD were detected in both MPD and sAML matching samples, suggesting that these changes do not play an immediate role in causing transformation. In conclusion, we detected chromosomal regions possessing genes which may be involved in the leukemic transformation of MPD patients.

Abstract Philadelphia chromosome–negative myeloproliferative neoplasms (MPNs) including polycythemia vera, essential thrombocythemia, and primary myelofibrosis show an inherent tendency for transformation into leukemia (MPN-blast phase), which is hypothesized to be accompanied by acquisition of additional genomic lesions. We, therefore, examined chromosomal abnormalities by high-resolution single nucleotide polymorphism (SNP) array in 88 MPN patients, as well as 71 cases with MPN-blast phase, and correlated these findings with their clinical parameters. Frequent genomic alterations were found in MPN after leukemic transformation with up to 3-fold more genomic changes per sample compared with samples in chronic phase (P < .001). We identified commonly altered regions involved in disease progression including not only established targets (ETV6, TP53, and RUNX1) but also new candidate genes on 7q, 16q, 19p, and 21q. Moreover, trisomy 8 or amplification of 8q24 (MYC) was almost exclusively detected in JAK2V617F− cases with MPN-blast phase. Remarkably, copy number–neutral loss of heterozygosity (CNN-LOH) on either 7q or 9p including homozygous JAK2V617F was related to decreased survival after leukemic transformation (P = .01 and P = .016, respectively). Our high-density SNP-array analysis of MPN genomes in the chronic compared with leukemic stage identified novel target genes and provided prognostic insights associated with the evolution to leukemia.