Characterization of 35 new cases with four different MPLW515 mutations and essential thrombocytosis or primary myelofibrosis

Abstract

F e r r a t a S t o r t i F o u n d a t i o nMPLW515 mutated patients is given in Table 1. In the total cohort with JAK2V617wt ET, any MPLW515 mutation was detected in 19 out of 324 patients (5.9%).In 104 JAK2V617wt PMF, a total of 10 MPLW515 mutations was detected (9.6%). In contrast, in the JAK2V617F mutated cases (32 ET; 89 PMF), no MPLW515 mutation was detected. Sequencing of the 35 MPLW515 mutations revealed four different MPLW515 subtypes: 20 patients had a W515L mutation, 13 a W515K, one case showed a so far not described W515A mutation leading to a tryptophane to alanine exchange, and another case a novel W515R associated to a tryptophane to arginine exchange ( Figure 1). Although the functional relevance of these two new mutations still has to be evaluated, it has to be hypothesized that the replacement of a large amino acid by a smaller one probably alters the protein structure in both novel mutation subtypes. Mutation/wild type ratios of greater than 1.0 indicated that at least some cells in the respective patient showed loss of the wildtype allele (LOH). Such high mutation ratios were detected in 13/35 (37%) mutated cases. Eight of these cases with high ratios were at advanced stage with a disease history of 2-9 years. Mutation ratios greater than 1% for the W515 were more frequent in PMF/s-AML following PMF (6/8 cases; 75%) when compared to ET (7/26; 27%) (Online Supplementary Table S1a). The mutation/wild type ratios in the remaining 22 patients were between 0.1 and 0.9% (median: 0.3%) (Online Supplementary Table S1b). Based on the applied method, cells with LOH could not be excluded in these low level cases because unapparent homozygousity based on dilution of homozygous cells with wild type cells may be present. However, the low level cases at least had less cells with LOH and thus the ratios may be important. This allows the hypothesis that higher proportions of W515 mutated alleles in total could indicate progression of disease. High mutation ratios were more frequent in the W515K (9/13; 69%) than in W515L (3/20; 15%) (p=0.034) corresponding to the results of Vannucchi et al. 7 and Beer et al. 8 Thus, the W515K mutation seems more often associated with loss of the wildtype allele. Karyotypes were available in 12 MPLW515 mutated cases. Eleven had a normal karyotype; one case had a del(5)(q14q34) and a del(13)(q12q22). It is remarkable that at least at the microscopic level, no LOH of chromosome 1p, where MPL is located, was detectable. This issue has to be investigated with more sophisticated techniques like SNP-array analyses.The frequency of the MPLW515 mutation in our cohort corresponded to previous studies with 5.3% in the JAK2V617wt ET and 9.6% in JAK2V617wt PMF.1,2 In contrast to previous findings, in our small cohort of 121 JAK2V617F mutated patients with ET and PMF there was no case with an MPLW515, whereas others found such a coexistence in up to 22% of MPL mutated MF cases. 1,3,9 Finally, the W515 mutations have so far been identified in ET and PMF only.1 Based on the new potential of...

Impact of isolated germline JAK2V617I mutation on human hematopoiesis

“…Similarly, an inherited mutation of MPL causes hereditary thrombocytosis, but when arising as a somatic mutation, it is associated with both ET and myelofibrosis phenotypes. 46,47 Thus, the findings presented here are consistent with a model of MPN pathogenesis in which JAK2 mutation may cause an MPN phenotype as a "single hit." However, an obvious difference between germline and somatic JAK2 mutations is that the latter occurs in a single cell, which must then undergo clonal expansion because it has been estimated that JAK2V617F needs to be present in at least 1% to 2% of HSCs before a clinical MPN phenotype emerges.…”

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

“…6 Both MPLW515L-positive patients were JAK2V617F negative consistent with earlier findings. 15,16 Importantly, despite the relevant mutant allele burden in both patients before allo-SCT, MPLW515L positive cells were not detectable very early after allo-SCT. In both patients, the mutation remained undetectable during the whole follow-up in well agreement with the absence of clinical or molecular relapse.…”

“…10 Those mutations are reported in B10% of MF and 4% of ET patients, most being negative for the JAK2V617F. 6,[13][14][15][16] However, coexistence of the two mutations is possible as cases of monoclonal or biclonal expression of both of them have been already published. 17,18 To detect MPLW515L/K mutations, genotyping techniques using allele-specific PCR were first used with qualitative or semiquantitative results and a 3-5% sensitivity level.…”

“…20,21 Melting curve analysis using LightCycler assay was also reported to be sensitive for mutant allele burden of at least 5%. 16 Several Taqman PCR techniques were recently reported and confer maximally 0.1% sensitivity; some of them use DNA from granulocytes rather than whole peripheral blood. 7,8 In our study, we optimized previously reported Q-PCR assays to be used for MRD assessment after allo-SCT.…”

JAK2 V617F and MPL W515L/K Mutations in Korean Patients with Essential Thrombocythemia

“…Missense mutations in the MPL gene, which encodes the thrombopoietin receptor MIM 159530 have also been reported in a small proportion (19%) of patients with ET and primary myelofibrosis [3][4][5][6]. MPL mutations usually affect the W515 residue in exon 10 [3,7].…”