W515L Mutation Research Articles

Prognostic Irrelevance of Ring Sideroblast Percentage in World Health Organization Defined Myelodysplastic Syndromes without Excess Blasts,

Abstract 3803 Background:Ring sideroblasts (RS) represent abnormal mitochondrial iron deposits that are commonly present in myelodysplastic syndromes (MDS). The presence of ≥15% RS, by definition (World Health Organization-WHO) is necessary for a diagnosis of a MDS-RS; with refractory anemia with ring sideroblasts (RARS) being a specific morphologic category. RS can also be seen with other morphologic categories such as; refractory cytopenias with multilineage dysplasia (RCMD), MDS-unclassifiable (MDS-U) and refractory anemia with excess blasts (RAEB-1/2). Amongst these categories, RARS is generally believed to have the best survival rate with the lowest risk of leukemic transformation and this has been attributed to the absence of associated dysplasia in more than one lineage. However, it has not been systematically shown whether or not the exact percentage of RS provides additional prognostic information in the context of an accurate morphologic categorization and risk stratification by karyotype or the revised IPSS (IPSS-R). Methods:200 patients with primary MDS without excess blasts and ≥1% RS were seen at the Mayo Clinic from 1997 through 2007. All patients underwent bone marrow (BM) examination and cytogenetic evaluation at diagnosis and the pathology slides, including iron stains, were centrally re-reviewed to accurately quantify bone marrow (BM) RS percentage and to confirm WHO morphologic categories. Molecular profiling included analysis for IDH1, IDH2, JAK2 and MPL mutations. For the purposes of this study patients were divided into 4 categories based on the RS percentage; <5%, 5–14%, 15–50% and >50%. In addition, each patient was assigned IPSS and IPSS-R prognostic scores at diagnosis and risk stratified by karyotype according to the IPSS-R cytogenetic risk categories. Cox proportional regression method was used for multivariable analysis. Results:Of the overall 200 study patients, 140 (70%) were male and median age was 71 years (range, 17–90 years). At presentation, 34 (17%) patients were red cell transfusion-dependent. There were 56 (28%) patients with <5% RS (RCMD-52, MDS-U-14), 32 (16%) with 5–14% RS (RCMD-29, MDS-U-3), 79 (39%) with 15–50% RS (RARS-43, RCMD-29, MDS-U-2) and 33 (16%) with >50% RS (RARS-13, RCMD-17, MDS-U-3). Three patients (2%) were IDH1 mutant (RARS-1, RCMD-2), 17 (8%) IDH2 mutant (RARS-1, RCMD-14, MDS-U-2), 5 JAK2 V617F (3%) mutant (RARS-2, RCMD-3) and one patient with RCMD had the MPL W515L mutation. At a median follow-up of 33 months, 156 (73%) deaths and 24 (12%) leukemic transformations were documented. Median survivals were 63 months for MDS with >50% RS, 43 months for MDS with 15–50% RS, 35 months for MDS with 5–14% RS, and 14 months for MDS with <5% RS (p=0.005). In univariate analysis, additional risk factors included decreased hemoglobin, decreased platelet count, increased circulating blasts, WHO morphologic categories, red cell transfusion need, IPSS, IPSS-R and cytogenetic risk categories per IPSS-R. On a multivariable analysis only the IPSS-R prognostic score (p<0.0001) and the WHO morphologic categories (p=0.02 for RARS) retained significance. Univariate analysis disclosed significantly inferior leukemia-free survival (LFS) in patients with RCMD, thrombocytopenia, increased circulating blasts, and poor IPSS and IPSS-R prognostic scores. On multivariable analysis once again only the IPSS-R prognostic scores (p=0.02) and WHO morphologic categories (p=0.02) retained their significance. In other words, RS percentage, independent of WHO classification, as a continuous or categorical variable (i.e.: <5%, 5–14%, 15–50% & >50%) did not affect either overall survival or LFS. Conclusions:In MDS without excess blasts, once an accurate WHO morphologic categorization is made based on the presence or absence of multi-lineage dysplasia, there is no additional prognostic value for quantifying bone marrow ring sideroblasts. WHO classification and IPSS-R prognostic scores remain the most important factors in assessing patients with MDS. Disclosures:No relevant conflicts of interest to declare.

Cytogenetics, JAK2 and MPL mutations in polycythemia vera, primary myelofibrosis and essential thrombocythemia

BackgroundThe detection of molecular and cytogenetic alterations is important for the diagnosis, prognosis and classification of myeloproliferative neoplasms.ObjectivesThe aim of this study was to detect the following mutations: JAK2 V617F, JAK2 exon 12 and MPL W515K/L, besides chromosomal abnormalities. Furthermore, molecular and cytogenetic alterations were correlated with the leukocyte and platelet counts, hemoglobin levels and age in all patients and with the degree of fibrosis in primary myelofibrosis cases.MethodsTwenty cases of polycythemia vera, 17 of essential thrombocythemia and 21 of primary myelofibrosis were selected in the Hematology Department of the Universidade Federal de São Paulo (UNIFESP) between February 2008 and December 2009. The JAK2 V617F, JAK2 exon 12 mutations, MPL W515K and MPL W515L mutations were investigated by real-time PCR and direct sequencing. G-band karyotyping and fluorescence in situ hybridization were used to detect chromosomal abnormalities.ResultsChromosomal abnormalities were observed only in polycythemia vera (11.8%) and primary myelofibrosis cases (17.6%), without correlation to clinical data. Chromosomal abnormalities were not detected by fluorescence in situ hybridization. The JAK2 V617F mutation was observed in polycythemia vera (90%), primary myelofibrosis (42.8%) and essential thrombocythemia (47%). Patients with JAK2 V617F-negative polycythemia vera had lower platelet and leukocyte counts compared to V617F-positive polycythemia vera (p-value = 0.0001 and p-value = 0.023, respectively). JAK2 V617F-positive and MPL W515L-positive primary myelofibrosis cases had a higher degree of fibrosis than V617F-negative cases (p-value = 0.022). JAK2 exon 12 mutations were not detected in polycythemia vera patients. The MPL W515L mutation was observed in one case of primary myelofibrosis and in one of essential thrombocythemia. The MPL W515K mutation was not found in patients with essential thrombocythemia or primary myelofibrosis. The MPL W515L-positive patient with primary myelofibrosis had more severe anemia than other patients with primary myelofibrosis.ConclusionsThis study demonstrates that karyotyping for JAK2 and MPL mutations is useful in the diagnosis of myeloproliferative neoplasms. The precise pathogenetic contribution of these alterations is still unclear. However, this study adds more information about the pathophysiology of polycythemia vera, essential thrombocythemia and primary myelofibrosis.

The analysis of JAK2 and MPL mutations and JAK2 single nucleotide polymorphisms in MPN patients by MassARRAY assay

Recent studies have shown that JAK2 V617F, MPL W515L/K and JAK2 exon 12 mutations underlie the major molecular pathogenesis of myeloproliferative disorders (MPN). Allele-Specific Polymerase Chain Reaction (AS-PCR), direct sequencing and MassARRAY assay were used to ascertain the real prevalence of these mutations and the influence of genetic susceptibility in Chinese MPN patients. The positive rate of JAK2 V617F in polycythaemia vera (PV), essential thrombocythaemia (ET) and primary myelofibrosis (PMF) was 82.0%, 36.6% and 51.1% respectively. One ET patient and two PMF patients harboured the MPL W515L mutation and three PV patients harboured JAK2 exon 12 mutations. All of these patients were confirmed as JAK2 V617F negative. Clinical data demonstrated that PV patients with JAK2 exon 12 mutations were younger, had higher haemoglobin levels and white blood cell counts than PV patients with JAK2 V617F. In addition, through analysis of 4 polymorphic loci of JAK2 gene, no significant difference of distribution frequency was found among PV, ET and PMF patients. Distribution frequency of haplotype also was not significantly different among PV, ET and PMF patients. We conclude that JAK2 V617F is a major molecular pathogenesis in Chinese MPN patients. MPL W515L mutation and JAK2 exon 12 mutations can also be found in JAK2 V617F negative MPN patients.

Clinical utility of routine MPL exon 10 analysis in the diagnosis of essential thrombocythaemia and primary myelofibrosis

Approximately 50% of essential thrombocythaemia and primary myelo-fibrosis patients do not have a JAK2 V617F mutation. Up to 5% of these are reported to have a MPL exon 10 mutation but testing for MPL is not routine as there are multiple mutation types. The ability to routinely assess both JAK2 and MPL mutations would be beneficial in the differential diagnosis of unexplained thrombocytosis or myelofibrosis. We developed and applied a high resolution melt (HRM) assay, capable of detecting all known MPL mutations in a single analysis, for the detection of MPL exon 10 mutations. We assessed 175 ET and PMF patients, including 67 that were JAK2 V617F-negative by real time polymerase chain reaction (PCR). Overall, 19/175 (11%) patients had a MPL exon 10 mutation, of whom 16 were JAK2 V617F-negative (16/67; 24%). MPL mutation types were W515L (11), W515K (4), W515R (2) and W515A (1). One patient had both W515L and S505N MPL mutations and these were present in the same haemopoietic colonies. Real time PCR for JAK2 V617F analysis and HRM for MPL exon 10 status identified one or more clonal marker in 71% of patients. This combined genetic approach increases the sensitivity of meeting the World Health Organization diagnostic criteria for these myeloproliferative neoplasms.

Rapid real‐time PCR assay for detection of MPL W515L mutation in patients with chronic myeloproliferative disorders

Clinical diagnosis of the myeloproliferative disorders (MPD) has previously been based on clinical data and bone marrow morphology due to lack of specific molecular markers. The discovery of JAK2 V617F mutation has shed light on understanding of the molecular pathways involved in the pathogenesis of the myeloproliferative disorders. The thrombopoietin receptor gene (MPL) is expressed in megakaryocytes and exhibits the gain of function point mutation in approximately 5% of MPDs. Several research groups have used real-time PCR to detect and quantify the presence of JAK2 V617F mutation. We report here a highly specific real-time assay based on the TaqMan((R)) technology to detect the MPL W515L mutation with high sensitivity from the patient's blood. This assay can be easily performed together with the JAK2 V617F mutation assay on the same real-time PCR reaction plate.

Screening forMPLmutations in essential thrombocythemia and primary myelofibrosis: normal Mpl expression and absence of constitutive STAT3 and STAT5 activation inMPLW515L-positive platelets

To evaluate the frequency of MPL W515L, W515K and S505N mutations in essential thrombocythemia (ET) and primary myelofibrosis (PMF) and to determine whether MPLW515L leads to impaired Mpl expression, constitutive STAT3 and STAT5 activation and enhanced response to thrombopoietin (TPO). Mutation detection was performed by allele-specific PCR and sequencing. Platelet Mpl expression was evaluated by flow cytometry, immunoblotting and real-time RT-PCR. Activation of STAT3 and STAT5 before and after stimulation with increasing concentrations of TPO was studied by immunoblotting. Plasma TPO was measured by ELISA. MPLW515L was detected in 1 of 100 patients with ET and 1 of 11 with PMF. Platelets from the PMF patient showed 100% mutant allele, which was <50% in platelets from the ET patient, who also showed the mutation in granulocytes, monocytes and B cells. Mpl surface and total protein expression were normal, and TPO levels were mildly increased in the MPLW515L-positive ET patient, while MPL transcripts did not differ from controls in both MPLW515L-positive patients. Constitutive STAT3 and STAT5 phosphorylation was absent and dose response to TPO-induced phosphorylation was not enhanced. The low frequency of MPL mutations in this cohort is in agreement with previous studies. The finding of normal Mpl levels in MPLW515L-positive platelets indicates this mutation does not lead to dysregulated Mpl expression, as frequently shown for myeloproliferative neoplasms. The lack of spontaneous STAT3 and STAT5 activation and the normal response to TPO is unexpected as MPLW515L leads to constitutive receptor activation and hypersensitivity to TPO in experimental models.

Screening and monitoring of MPL W515L mutation with real-time PCR in patients with myelofibrosis undergoing allogeneic-SCT

Monitoring of minimal residual disease (MRD) after allogeneic (allo)-SCT for myelofibrosis (MF) allows recognizing the depth of remission and thus guides application of appropriate therapeutic interventions. MPL W515L/K mutations, which are detected in 5-10% of JAK2V617F-negative patients, may be useful for this purpose. Using a highly sensitive quantitative PCR method, we tested 90 patients with MF who underwent allo-SCT for the presence of MPL W515L/K mutations. Two patients with primary MF were found to harbor MPLW515L while no patient was positive for MPLW515K mutation. Both patients were JAK2V617F negative and cleared the mutation rapidly after allo-SCT and remained negative for a median follow-up of 19 months. The results of molecular monitoring correlated well with other remission parameters such as normalization of peripheral blood counts and morphology and complete donor chimerism. We conclude that MPLW515L can be cleared after allo-SCT and hence may be used as an MRD marker in a proportion of JAK2V617F-negative MF patients.

TET2 Mutations Are Frequent in RARS-T.

Abstract 3794Poster Board III-730Refractory anemia with ring sideroblasts and thrombocytosis (RARS-T) has been considered a provisional subtype within the diagnostic entity of myelodysplastic/myeloproliferative neoplasms (MDS/MPN). Since JAK2 V617F and MPL W515L mutations are present in a significant proportion of RARS-T patients, many investigators consider this entity to be more closely related to classical MPN. However, a significant minority of patients with RARS-T do not display either JAK2 V617F or MPL W515L mutations. We have studied a cohort of patients with RARS-T (N=20) characterized by the presence of ring sideroblasts, reticulin fibrosis and thrombocytosis (>450×109/L), that lack obvious causes of secondary thrombocytosis. While 8/20 patients harbored the JAK2 V617F, and 3/20 the MPL W515L mutations, the molecular pathogenesis for the remaining 9 patients was unexplained. Activation of JAK2 and MPL is associated with aberrant phospho-STAT5. Cases positive only for phospho-STAT5 may harbor other related, so far unidentified mutations. Many groups have recently observed a frequent area of somatic uniparental disomy (UPD) at 4q24, most commonly encountered in patients with chronic myelomonocytic leukemia (CMML), MDS/MPN, some typical MDS, and secondary acute myeloid leukemia (sAML). Overlapping microdeletions and UPD on 4q24 pointed towards possible mutations in the TET2 gene; such mutations were subsequently found in myeloid malignancies, most significantly MPN and MDS/MPN. Based on these findings, and the established correlation of RARS-T with JAK2 V617F and MPL W515L mutations, we evaluated the mutational status of TET2 in RARS-T patients. SNP-A allowed detection of copy neutral loss of heterozygosity (CN-LOH), such as UPD9p, which is associated with the JAK2 V617F mutation, and UPD1p, associated with MPL W515L. SNP-A facilitated detection of previously cryptic lesions; 11/20 patients showed an abnormal SNP-A-based karyotype (only 3 of these defects were also detected by MC). The new lesions seen by SNP-A included various UPD, such as, 1p, 2p, 3q, 6p, 8p, 9p and 10p. The presence of UPD9p/1p was consistent with homozygous JAK2 V617F/MPL W515L mutations. Likely, duplication of mutated alleles constituted a further permissive event during clinical evolution. However, none of the patients showed a somatic LOH at 4q24, suggesting that biallelic TET2 mutations were not involved in the pathogenesis of RARS-T. Simultaneously, lack of UPD11q suggested that CBL mutations were absent. Indeed, Cbl ring finger domain mutational screening revealed no mutations. An aberrant phospho-STAT5 staining pattern was present in all cases that were positive for either JAK2 V617F or MPL W515L mutations (N=10). However, 4 patients demonstrated abnormal megakaryocytic STAT5 phosphorylation, despite the absence of both JAK2 V617F and MPL W515L mutations. Within this group, a monoallelic TET2 mutation, delC 1480Sfs, was identified. In addition, we found a group of 5 patients without either JAK2 V617F or MPL W515L mutations, and also without association of the aberrant phospho-STAT5 staining. One of these patients had a monoallelic TET2 V1718L mutation; interestingly, another patient's specimen showed two novel non-synonymous SNPs: Y867H and P1723S. In total, 2/19 (11%) patients harbored TET2 mutations. These findings indicate involvement of TET2 mutations in RARS-T pathogenesis. RARS-T cases with MPN-associated mutations may not show obligatory phospho-STAT5 staining. The majority of patients were characterized by lack of splenomegaly, decreased white blood cell counts, increased thrombocytosis, and a normal karyotype. In summary, the majority of RARS-T patients harbor JAK2 V617F and MPL W515L mutations that strongly activate STAT5 phosphorylation. We described herein the third most common mutation in RARS-T, which can occur with or without abnormal STAT5 activation. Disclosures:No relevant conflicts of interest to declare.

Acute Panmyelosis with Myelofibrosis: Clinicopathological and Molecular Features of a Rare Malignant Bone Marrow Disease.

Abstract 3101Poster Board III-38 BackgroundAcute panmyelosis with myelofibrosis (APMF) is listed among the category of “AML, not otherwise specified” in the actual WHO classification of hematopoietic tumors. Since specific genetic alterations have not yet been defined, this myeloid neoplasm (MN) remains clinicopathologically assigned. The WHO classification addresses the difficulties concerning the differential diagnosis between APMF, myelodysplastic syndromes (MDS) with excess of blasts and concomitant fibrosis (RAEB-F) and acute megakaryoblastic leukaemia (AMGL). MethodsOur files were searched for patients with rapid-onset MN without a prior history of a myeloproliferative neoplasm or a MDS fulfilling the 2008 WHO criteria of APMF. An age- and gender-matched control group of RAEB-F cases with multilineage dysplasia was retrieved for comparison. Our approach included:i) the careful review of morphological, clinical and laboratory data with special regard to the proportion of immature CD34 + precursors/blasts in bone marrow (BM) biopsies; ii) the grading of BM fibrosis according to the European consensus guidelines; iii) the analysis of the JAK2-V617F, the MPL W515L and the GATA1 mutational status, the cytoplasmic localization of nucleophosphamin1 (NPM1) in BM blasts, and the expression the inhibitor of differentiation protein-2 (Id2) in hematopoietic and stroma cells; iv) the evaluation of the prognostic relevance of these parameters. ResultsFifty-four patients (32 males, 22 females) with a median age of 66, 9 years from our institutions were diagnosed APMF. Sufficient DNA for allele specific PCR for JAK2 genotyping was obtained from paraffin-embedded EDTA-decalcified BMB specimens from 22 APMF and 14 RAEB-F patients. In addition, an analysis of MPL W515L and GATA1 mutations was performed on 15 APMF cases. The relevant clinicopathological and molecular data are summarized in the table.APMFRAEB-FP valueMedianSurvival, median (months)0,0006,5 (*CI: 5,6-7,49)36,3 (CI: 0,9-71,7)MCV, mean (fl)<0,00190,29 (CI: 88,3-92,3)98,3 (CI: 95,2-101,3)LDH, mean ( U/l)<0,001401,8 (CI: 341,6-461,9258,3 (CI: 226,2-290,4)BM fibrosis, (grade)<0,0012,5 (CI: 2,4- 2,7)1,8 (CI: 1,6-2)BM Blasts (%)<0,00131,4 (CI: 27,2-35,5)13,3 (CI: 11,3-14,9)Aberrant NPM1 (%)n.s.12,97%JAK2V617 F + (%)n.s.36%21%MPL W515L +(%)0n.d.GATA1 + (%)0n.d.*CI: 95% confidence intervalSemiquantitative evaluation revealed a decreased expression level of Id2 in erythropoiesis of the APMF samples, while Id2 was increased in the stromal compartment. Within the total APMF cohort, patients aged ≤60 years had a longer OS (p=0,013). A comparison between the Kaplan-Meier curves of the APMF patients who underwent hematopietic cell transplantation (HCT) with those treated by conventional chemotherapy clearly showed that HCT significantly improved OS (median survival 6,1 vs. 11,1 months, p=0,005). Within the non-transplanted group, a bone marrow blast count ≤25% was associated with a longer OS (p=0,006). ConclusionsOur results point to significant differences between APMF and RAEB-F with regard to OS, MCV, LDH, BM fibrosis and BM blast cells. We detected a higher frequency of mutated JAK2 V616F in the MB samples than generally reported in the literature for other categories of AML or MDS. However, the JAK2 V617F mutation status or an aberrant cytoplasmic NPM1 localization were both not predictive of outcome. In contrast to data published for AMGL, no association between a MPL W515L mutation and APMF was observed. Survival was significantly worse in APMF than in RAEB-F. A rapid diagnosis based on a multiparameter approach and a proper management including the option of HCT even in elderly patients seems mandatory for APMF patients. Targeting the fibrotic BM microenvironment which is characterized by an increased Id2 expression may be considered for future therapeutic strategies. DisclosuresSkoda:Genentech Inc.: Consultancy.

MPL Mutation Profile in JAK2 Mutation-Negative Patients with Myeloproliferative Neoplasms.

Abstract 1891Poster Board I-914Myeloproliferative neoplasms (MPNs) are multipotent hematopoietic stem cell neoplasms characterized by excess production of various blood cells. Mutations in the thrombopoietin receptor gene (MPL) have been reported in JAK2 V617F-negative patients with MPNs. We evaluated the prevalence of MPL mutations relative to those of JAK2 mutations at V617 or in exons 12 through 15 in a large number of patients with suspected MPNs. A total of 2790 patients with suspected MPNs referred to our institution were first screened for JAK2 V617F and exon 12–15 mutations. Patients with no JAK2 mutations detected were then tested for MPL mutations in exons 10 and 11 by means of a sensitive MPL reverse-transcription-PCR-based assay with direct bidirectional sequencing. All JAK2 and MPL mutation assays were performed on plasma RNA, rather than DNA isolated from blood or bone marrow cells. Of the 2790 patients, 529 (18.96%) had a V617F mutation; 12 (0.43%) had small insertion/deletions in exon 12; and 7 (0.25%) had other JAK2 mutations, including point mutations in exons 13–15 and an exon 14 splice mutation. MPL mutations were identified in 66 of the 2242 (2.94%) JAK2 mutation-negative patients (2.37% of all tested patients). W515L was the dominant MPL mutant detected in 46 patients (70%) including two patients with homozygous W515L mutation. The other W515 variants (W515K, W515R, W515S, W515G, W515A, W515*) comprised 16% (N=11) of the MPL mutations. The remaining MPL mutations (n =9, 14%) were detected at other locations in exon 10 and 11. Two of these were novel exon 10 deletion/insertion mutations and two were unreported exon 11 point mutations. The exon 10 T496-A497ALVI (4AA) homozygous insertion was detected in one patient with a confirmed diagnosis of idiopathic myelofibrosis and the W515-P518 del/ins (KT) mutation was detected in another patient with unspecified MPN. The two novel MPL exon 11 point mutations were D545G and D545N. The S505N mutation (n=2) and V507I (n=1), R514K (n=1), and A519V (n=1) were also detected. Furthermore, three unreported silent polymorphisms/mutations (T496, L543, and D534) were detected. In conclusion, our results demonstrate that for every 100 V617F mutations detected in patients with MPNs, there are 2.3 JAK2 exon 12 mutations, 1.3 JAK2 exon 13–15 mutations, and 12.5 MPL mutations. Thus, MPL mutation detection should be performed on all JAK2 V617F-negative patients with suspected MPNs. In addition, our findings indicate that >20% of MPL mutations would have been missed if only W515L and W515K were analyzed; thus, sequencing of both exon 10 and 11 may be beneficial. Disclosures:No relevant conflicts of interest to declare.

SNP-Array Profiling Identifies Complex Aberrations and Candidate Genes in Myeloproliferative Neoplasms with Leukemic Transformation.

Abstract 2608▪▪This icon denotes an abstract that is clinically relevant.Poster Board II-584 Background:Myeloproliferative neoplasms (MPN) represent a heterogeneous group of acquired hematopoietic stem cell disorders. Clonality leads to exceeding production of myeloid cells resulting in an inherent tendency for thrombotic and hemorrhagic complications as well as transformation into acute myeloid leukemia (sAML). While vascular complications predominantly account for the morbidity in essential thrombocythemia (ET) and polycythemia vera (PV), the mortality of MPN is significantly related to leukemic transformation. Secondary AML occurs more frequently in primary and secondary myelofibrosis (PMF and SMF) than in ET and PV, and the risk for leukemic transformation increases with the duration of the disease. The molecular basis underlying the progression of MPN is poorly understood. Clonal evolution due to genomic instability is considered to play an important role. Aim:To identify genomic lesions associated with leukemic transformation, we applied 250K single-nucleotide polymorphism (SNP) arrays that allow for genome-wide screening of both copy-number alterations (CNAs) and copy-neutral runs of homozygosity (ROH) at high resolution. Method:An unpaired SNP-array analysis of 23 sAML samples was performed [former diagnosis: ET, n=5; PV, n=7; PMF, n=9; SMF, n=2;]. An own set of 30 reference samples was used for normalization. CNAs and ROH were analyzed by CNAG 3.0 software. Aberrations were compared with the 250K SNP-array dataset of 151 MPN patients [ET, n=45; PV, n=45; PMF, n=47, SMF, n=14]. In one sAML patient corresponding SNP-array data from the time of ET diagnosis were available. Results:CNAs were present in 15 of 23 (65%) sAML patients. Thirty-five percent of cases (n=8) exhibited complex genomic aberrations with up to 20 CNAs in one patient (range 5–20). The most frequent larger (>5 Mb) CNAs were trisomy 8 (n=7), gain of 1q, loss of 5q, and deletion in 6p25-pter (16.2–26.7 Mb) and 20q11-q13 (13.6–16.9 Mb) in three cases each, followed by gain in 3q24-qter (51.7 and 54.1 Mb) in two patients. Of note, one case with deletion in 17p12-pter (64.4 Mb) encompassing TP53 and a second with gain in 21q22.12-qter (11.8 Mb) were identified; in the latter one the proximal breakpoint of the gain was located at RUNX1. Smaller CNAs (<5 Mb) were restricted to single cases with four cases exhibiting micro-deletions ranging from 0.7 to 2.7 Mb in size. Interestingly, three chromosomal regions harbour single genes: 11p11.2 (FOLH1), 18q21.2 (TCF4), and 21q22.12 (RUNX1). ROH comprising the terminal end of the chromosome were detectable in 13 of 23 (57%) sAML cases. The most frequent ROH included the JAK2 locus in 9p24 (n=6; 15.6–38.7 Mb), followed by ROH in 17p13-pter (16.3 and 17.7 Mb) covering TP53 and an overlapping segment in 1p32-pter (53 Mb) affecting MPL in two cases each. All cases with 9p ROH were JAK2 V617F mutated, whereas the MPL W515L mutation was found in one of the two 1p ROH cases. Moreover, sequencing analyses in both patients with ROH in 17p revealed TP53 missense mutations in exon 7 and exon 8, respectively. In addition, non-recurrent ROH covering the long arm of chromosome 7, 11, and 21 as well as ROH in the chromosomal segments 14q32-qter (12.6 Mb) and 17q31-qter (31 Mb) were identified. In one sAML patient SNP-array data performed at the time of ET were available for comparative analysis. While 20q deletion was present as sole aberration in ET, complex genomic aberrations (7 CNAs) were identified after development of sAML. Merging the results from our recent 250K SNP-array analysis (Stegelmann et al., Blood 2008; 112: Abstract #2794) on 61 PMF and SMF cases with data from this study, we were able to identify a second case with micro-deletion in 12q24. The commonly deleted region of both cases is 1.3 Mb in size and encompasses TCF1 as a novel recurrent aberration in MPN. Conclusion:In summary, our data on a large series of well-defined sAML cases that evolved from MPN demonstrate that 250K SNP-array profiling is an excellent tool to identify genomic aberrations. In contrast to MPN, genomic alterations in sAML are characterized by a marked complexity reflecting both genomic instability and genetic heterogeneity of sAML. However, in our study several regions of interest including recurrently affected candidate genes such as TCF1, RUNX1, and TP53 were identified that need to be further investigated on a single gene level. Disclosures:No relevant conflicts of interest to declare.

The Analysis of JAK2 and MPL Mutation as Well as JAK2 SNPs in MPN Patients by MassARRAY Assay.

Abstract 4989 IntroductionRecent studies have shown that JAK2 V617F, MPL W515L/K and JAK2 exon 12 mutations underlie the major molecular pathogenesis of myeloproliferative disorders (MPN). MethodsTo ascertain the real prevalence of these mutations and the influence of genetic susceptibility in Chinese MPN patients, we applied Allele-Specific Polymerase Chain Reaction (AS-PCR), directly sequencing and MassARRAY assay into our study. ResultsThe positive rate of JAK2 V617F in polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) was 82.0%, 36.6% and 51.1% individually. We also found one ET patient, two PMF patients harboring MPL W515L mutation, and three PV patients harboring JAK2 exon 12 mutations. All of these patients were confirmed as JAK2 V617F negative. Moreover, clinical data demonstrated that PV patients with JAK2 exon 12 mutations had higher hemoglobin and lower age as well as WBC than PV patients with JAK2 V617F. In addition, through analysis of 4 polymorphic loci of JAK2 gene, no significant difference of distribution frequency was found among PV, ET and PMF patients. Distribution frequency of haplotype was not found to have significant difference among PV, ET and PMF patients either. ConclusionWe conclude that JAK2 V617F is major molecular pathogenesis in Chinese MPN patients. MPL W515L mutation and JAK2 exon 12 mutations can also be found in JAK2 V617F negative MPN patients. DisclosuresNo relevant conflicts of interest to declare.

Molecular characterization of chronic myeloproliferative neoplasias in México

By using novel molecular markers, it is possible to gain information on both the classification and the pathophysiology of the chronic myeloproliferative neoplasia (MPN). In a group of 36 Mexican mestizo patients with MPN, we studied five molecular markers: The BCR/ABL1 fusion gene, the JAK2 V617F mutation, the JAK2 exon 12 mutations, the MPL W515L mutation and the MPL W515K mutation; 17 patients with essential thrombocythemia (ET), eight with polycythemia vera (PV), four with primary mielofibrosis (MF), five with undifferentiated MPN, one with primary erythrocytosis and one with familial thrombocytosis. Patients with the BCR/ABL1 fusion gene were excluded. Twelve individuals with the JAK2 V617F mutation were found; 11 of them had been clinically classified as PV and one had been classified as MF. One patient with the MPL W515L was identified with a clinical picture of ET. No individuals with either the MPL W515K mutation or the JAK2 exon 12 mutations were identified. Of the 17 individuals with ET, six (35%) had the JAK2 V617F mutation and one (6%) was found to have the MPL W515L mutation. Of the eight individuals with PV, five displayed the JAK2 V617F mutation, whereas of the four patients with MF, one had the JAK2 V617F mutation. The most consistent relationship was that between PV and the JAK2 V617F mutation (p=0·08). In the diagnosis and classification of the MPN, in addition to the newly identified molecular markers, clinical and laboratory data are still very important.

Ligand-independent Thrombopoietin Mutant Receptor Requires Cell Surface Localization for Endogenous Activity

The activating W515L mutation in the thrombopoietin receptor (MPL) has been identified in primary myelofibrosis and essential thrombocythemia. MPL belongs to a subset of the cytokine receptor superfamily that requires the JAK2 kinase for signaling. We examined whether the ligand-independent MPL(W515L) mutant could signal intracellularly. Addition of the endoplasmic reticulum (ER) retention KDEL sequence to the receptor C terminus efficiently locked MPL(W515L) within its natural ER/Golgi maturation pathway. In contrast to cells expressing the parental MPL(W515L), MPL(W515L)-KDEL-expressing FDC-P1 cells were unable to grow autonomously and to produce tumors in nude mice. When observed, tumor nodules resulted from in vivo selection of cells leaking the receptor at their surface. JAK2 co-immunoprecipitated with MPL(W515L)-KDEL but was not phosphorylated. We generated disulfide-bonded MPL(W515L) homodimers by the S402C substitution, both in the normal and KDEL context. Unlike MPL(W515L)-KDEL, MPL(W515L-S402C)-KDEL signaled constitutively and exhibited cell surface localization. These data establish that MPL(W515L) with appended JAK2 matures through the ER/Golgi system in an inactive conformation and suggest that the MPL(W515L)/JAK2 complex requires membrane localization for JAK2 phosphorylation, resulting in autonomous receptor signaling.

Acquired 1p Uniparental Disomy Is Associated with Biallelic MPL W515L Mutation in RARS-T.

Refractory anemia with ringed sideroblasts and thrombocytosis (RARS-T) has been considered a provisional subtype within the diagnostic entity of myelodysplastic/myeloproliferative diseases (MDS/MPD). Since JAK2 V617F mutation is present in a significant proportion of RARS-T patients (Szpurka et al. Blood, 2006), many investigators consider this entity to be more closely related to classical MPD. However, a significant minority of patients with RARS-T do not display a JAK2 V617F mutation. We have studied a cohort of patients with RARS-T (N=18) characterized by the presence of ringed sideroblasts, reticulin fibrosis and thrombocytosis (>600×109/L), that lack obvious causes of secondary thrombocytosis. While 8/18 patients harbored a JAK2 V617F mutation, a molecular pathogenesis for the remaining patients was unexplained. The successful application of SNP-A to characterize the genomic lesions in MDS prompted us to use this technology to study RARS-T. SNP-A allows detection of copy neutral loss of heterozygosity such as UPD9p which is associated with JAK2 V617F mutation. SNP-A facilitated detection of previously cryptic lesions; 9/18 patients showed an abnormal SNP-A-based karyotype often involving multiple lesions (only 5 of these defects were detected by metaphase cytogenetics). The new lesions seen by SNP-A included gains of chromosome 11p, 20q and 21q; deletion of 2p and various areas of UPD including 1p, 9p, 6p, 2p and 8p. SNP-A allowed identification of seemingly invariant UPD1p in 4/18 patients. As this region includes the Mpl gene, we analyzed patients for the presence of MPL W515L/K mutations which have been described in MPD. We did not find any patients with MPL W515K, however MPL W515L mutation was present in 2/4 RARS-T patients with UPD1p; another patient showed monoallelic MPL W515L variant. In addition, 1 patient with UPD1p harbored both JAK2 V617F and MPL W515L mutations. To further delineate the molecular lesion we analyzed all patients for the presence of abnormal STAT5 activation. An aberrant phospho-STAT5 staining pattern was present in all cases that were positive for either JAK2 V617F or MPL W515L mutations (N=10); unexplained STAT5 activation was found in only 4 cases, pointing towards a molecular defect involving this pathway. In these 4 patients, and in 1 additional with UPD1p who did not harbor MPL W515L mutation, we searched for other genes which might explain the pathogenesis of this disease by potentially causing aberrant activation of STAT5. We sequenced Jak1T478S, Jak1V623A and Ntrk1S677N as well as the transmembrane, juxtamembrane and kinase domain of Tie1, Epha2 and Ephb2 genes, but no mutation was found. In addition, we found a group of phospho-STAT5-negative patients (N=4) that showed typical genetic features of myelodysplasia e.g. del(5), +8 and partial loss of chromosome X; these cases are probably best considered to be of MDS origin rather than MPD. To our knowledge, our work is the first description of biallelic MPL W515L mutation and UPD1p found in RARS-T patients. This data is important for understanding the clonal selection process and pathophysiology of activating mutations in MDS/MPD. Overall, our studies demonstrate that somatic UPD1p is associated with homozygous MPL W515L mutation in MDS/MPD cases. Localizing areas of somatic UPD by SNP-A may help identify candidate genes within the shared regions that are likely targets for mutations.

The Overall Analysis of JAK2 and MPL Mutation Status in Chinese MPD Patients by MassARRAY Assay

In recent three years the new notable progress associated with myeloproliferative disorder (MPD) was the identification of JAK2 and MPL mutations, which have been confirmed to be the major molecular mechanism and marker of BCR/ABL negative MPD. Multiple techniques including allele-specific PCR, conventional DNA sequencing, pyrosequencing as well as BsaXI restriction analysis have been used to detect these mutations. The previous data of Shanghai Group had demonstrated the sensitivity and preliminary result of JAK2 V617F in 162 Chinese patients with MPD using MassARRAY assay. To generally identify the frequency of these mutations in Chinese MPD patients, we continued to employ this technique-MassARRAY assay to detect JAK2 V617F, JAK2 K539L as well as MPL W515L mutation in a larger scale of Chinese MPD patients. A total of 204 Chinese MPD patients were enrolled in our study. These patients were referred to polycythemia vera (PV) (n=55), essential thrombocythemia (ET) (n=110), primary myelofibrosis (PMF) (n=29) and hypereosinophilic syndrome (HES) (n=10). 2 ml peripheral blood was obtained with informed consent and genomic DNA was isolated. The diagnosis of MPD was established according to the 2001 WHO diagnostic criteria. Finally, 187 patient samples with good signal can be analyzed for JAK2 V617F mutation. Among the available 101 signals of ET patients, 30 were heterozygous mutation and 9 were homozygous mutation. Among 52 PV patients, 25 were heterozygous mutation and 14 were homozygous mutation. Among 25 PMF patients, 9 were heterozygous mutation and 5 were homozygous mutation. None of HES patients was found harboring JAK2 V617F. In addition, we identified a PV patient harboring JAK2 K539L but not V617F mutation (1/52, 1.9%), and an ET patient harboring MPL W515L mutation (1/101, 1%). Both results were further confirmed by sequence analysis. Hence, we concluded that JAK2 V617F underlie the major molecular pathogenesis of Chinese MPD patients especially PV, however, JAK2 K539L and MPL W515L are rare events in these patients.

Frequency and clinical features of the JAK2 V617F mutation in pediatric patients with sporadic essential thrombocythemia

Pediatric essential thrombocythemia (ET) is a rare and heterogenous disease entity. While several recent studies have focused on the role of the JAK2 V617F mutation in pediatric ET, the frequency of pediatric ET cases with this mutation and the associated clinical features remain unclear. We examined six childhood cases who had been diagnosed with ET according to WHO criteria (onset age: 0.2-14 years) for the presence of the JAK2 V617F mutation, MPLW515L mutation and JAK2 exon 12 mutations. Two sensitive PCR-based methods were used for the JAK2 V617F genotyping. We also examined the expression of polycythemia rubra vera-1 (PRV-1), which is a diagnostic marker for clonal ET. We found that three of the six cases had the JAK2 V617F mutation and that all six cases expressed PRV-1 in their peripheral granulocytes. Neither MPL W515L mutation nor JAK2 exon 12 mutations was detected in the patients without JAK2 V617F mutation. The two patients who developed thrombocythemia during infancy were JAK2 V617F-negative. These findings suggest that the JAK2 V617F mutation is not rare in childhood sporadic ET cases, and that these cases might be older and myeloproliferative features.

UPD1p indicates the presence of MPL W515L mutation in RARS-T, a mechanism analogous to UPD9p and JAK2 V617F mutation

UPD1p indicates the presence of MPL W515L mutation in RARS-T, a mechanism analogous to UPD9p and JAK2 V617F mutation

MPL W515L mutation in patients with myeloproliferative diseases

18019 Background: The BCR-ABL negative chronic myeloproliferative disorders (MPD) include polycythemia vera (PV), essential thrombocytosis (ET), and myelofibrosis with idopathic myelofibrosis (IMF). Although clonal hematopoiesis was observed in these disorders more than three decades ago, the molecular etiology of these disorders was not known until recently when several groups reported a somatic mutation in the JAK2 tyrosine kinase (JAK2 V617F) in most patients with PV and in a subset of patients with ET and IMF. As JAK2 V617F, MPL W515L is a novel acquired mutation that induces constitutive cytokine-independent activation of the JAK-STAT pathway in MPD. The discovery of this mutation provides a novel mechanism for activation of signal transduction in hematopoietic malignancies. Methods: To investigate its prevalence in Chinese patients with MPD, we introduced allele-specific PCR (AS- PCR) combined with sequence analysis to simultaneously screen MPL W515L and JAK2 V617F mutations in 190 MPD patients. Results: The results indicated that MPL W515L mutation was found to be harbored in only one of 102 ET patients (1.0%) and was not detected in PV, IMF, chronic myelogenous leukemia (CML) patients. Sixty eight BCR/ABL-negative MPD patients (46.3%) were found harbouring JAK2 V617F mutation (PV, 62.5% ET, 42.1% IMF 38.1%). Furthermore, MPL W515L and JAK2 V617F mutations were not detected in patients of acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), myelodysplastic syndromes (MDS) and CML. Conclusions: MPL W515L mutations may contribute to the primary molecular pathogenesis mechanism of Chinese patients with ET. No significant financial relationships to disclose.

Phospho-STAT5 expression pattern with the MPL W515L mutation is similar to that seen in chronic myeloproliferative disorders with JAK2 V617F

Abnormal nuclear megakaryocytic staining for phospho-STAT5 (pSTAT5) correlates with JAK2 V617F mutational status in non-chronic myelogenous leukemia chronic myeloproliferative disorders. However, a proportion of wild-type JAK2 non-chronic myelogenous leukemia chronic myeloproliferative disorders cases also demonstrate this abnormal pSTAT5 expression pattern. We report a patient with a JAK2 V617F-negative myeloproliferative/myelodysplastic syndrome who had abnormal megakaryocytic pSTAT5 expression and a MPL W515L mutation. The patient was a 71-year-old man with anemia and thrombocythemia on laboratory examination. His peripheral blood smear demonstrated occasional dysplastic neutrophils. Bone marrow biopsy revealed hypercellular marrow with features consistent with myeloproliferative/myelodysplastic syndrome. Immunohistochemistry for pSTAT5 showed abnormal nuclear megakaryocyte positivity. Cytogenetic analysis revealed a normal karyotype, fluorescence in situ hybridization for BCR-ABL was negative, and JAK2 genotyping demonstrated wild-type JAK2. However, MPL genotyping showed a MPL W515L mutation. Abnormal nuclear megakaryocytic staining for pSTAT5 expression, previously associated with the JAK2 V617F mutation, is also associated with MPL W515L, likely reflecting activation of the JAK-STAT signaling pathway.