Weiss, Tamara

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 Abstract 3895 Poster Board III-831 A genome-wide single nucleotide polymorphism (SNP) screen led to the identification of 11q aUPD in patients diagnosed with various subtypes of myeloproliferative neoplasms (MPN), e.g. chronic myelomonocytic leukemia (CMML), atypical chronic myeloid leukemia (aCML) and myelofibrosis (MF) (Grand et al., Blood 2009;113:6182). Further molecular analyses revealed acquired activating point and length mutations in CBL exons 8 and 9 in 10% of CMML, 8% of aCML and 6% of MF cases. Most variants were missense substitutions in the RING or linker domains that abrogated CBL ubiquitin ligase activity and conferred a proliferative advantage to 32D cells overexpressing FLT3. In this study, 160 patients with BCR-ABL and JAK2 V617F negative MPNs were screened for CBL mutations by PCR and direct sequencing. Eighteen known (Y371H, L380P [2x], C381R, C381Y [2x], C384Y, C396Y, H398P, H398Q, W408C, P417H, F418L, R420Q [5x]) and four new (F378L, G397V, I423N, V430M) missense mutations affecting fourteen residues were identified in 20 patients. Two patients harbored two different mutations. The clinical phenotype could be characterized more precisely in 17 patients. Median age was 68 years (range 59–85) with a slight female predominance (f, n=10; m, n=7). Striking hematological features were leukocytosis (14/17; 82%; median 29,000/μl, range 4,500-141,000) with continuously left-shifted granulopoiesis (blasts, promyelocytes, myelocytes, metamyelocytes) in 85% and elevated monocytes (median 2,500/μl, range 630-10,656) >1,000/μL in 88% (15/17) of patients. Eosinophilia (>1,500/μL) was rare (3/17, 18%). Anemia (normal values: f, Hb <12g/dL; m, Hb <14g/dL) was present in all 17 patients (f, median 10g/dL, range 8.7-11.8; m, median 11.2g/dL, range 8.6-12.9). Platelets did not exceed 300,000/μL in any patient while 11/17 (65%) patients presented with thrombocytopenia (median 125,000/μL, range 18,000-271,000). Splenomegaly was present in 11/17 patients (65%) and LDH was elevated (median 304U/L, range 189-729) in 9/17 patients (52%). Bone marrow histology and immunohistochemistry were available from 12 patients. Relevant features were hypercellularity, marked granulopoiesis and microlobulated megakaryocytes without clusters in 11/12 patients (92%), respectively. Increased fibres were seen in 8/12 (67%) patients of whom one showed severe fibrosis. Clinical follow-up was available from 17 patients. Thirteen patients (76%) have died because of progression to secondary acute myeloid leukemia/blast phase (n=7), cytopenia-related complications (n=2) or for unknown reasons (n=4) after a median of 23 months (range 3-60) following diagnosis. In conclusion, point mutations of CBL exons 8 and 9 are present in approximately 6-12% of BCR-ABL and JAK2 V617F negative MPNs. They are associated with a distinct clinical and hematological phenotype presenting with myeloproliferative features allowing diagnosis of a proliferative subtype of CMML rather than aCML or MF in the majority of cases. Patients with left-shifted leukocytosis, monocytosis, anemia and lack of thrombocytosis who are negative for BCR-ABL and point or length mutations of JAK2 should be routinely screened for CBL mutations. Disclosures: No relevant conflicts of interest to declare.

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.