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We have previously identified NME2 (Nm23-H2) as a tumor antigen in a patient with chronic myeloid leukemia (CML). Here we investigated the association between NME2 and Bcr-Abl. NME2 protein was highly overexpressed in the cytoplasm of peripheral blood mononuclear cells from 29/30 patients with CML at diagnosis and 10/10 patients resistant to imatinib. Protein was overexpressed in the absence of increased levels of mRNA and was limited to Bcr-Abl + populations, being absent from Bcr-Abl - patient cells, normal donors and 14/15 acute myeloid leukemia (AML) samples. Furthermore, the Bcr-Abl dependent overexpression of NME2 protein was reversed specifically by tyrosine kinase inhibitor (TKI) treatment of Ba/F3 expressing wild-type and TKI-sensitive, but not TKI-resistant, mutants of Bcr-Abl. The post-transcriptional up-regulation of the tumor antigen NME2 is therefore a common and specific property of CML closely associated with Bcr-Abl activity.

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Sequential genotyping for phenotype-driver mutations in JAK2 (exon 14), CALR (exon 9), and MPL (exon 10) is recommended in patients with myeloproliferative neoplasms. Yet, atypical JAK2-and MPL-mutations were described in some triple-negative patients. Whether noncanonical and/or concomitant JAK2-and MPL-mutations exist in myelofibrosis (MF) regardless of phenotype-driver mutations is not yet elucidated. For this, next-generation sequencing (NGS) was performed using blood genomic DNA from 128 MF patients (primary MF, n = 93; post-ET-MF, n = 18; post-PV-MF, n = 17). While no atypical JAK2-or MPL-mutations were seen in 24 CALR-positive samples, two JAK2-mutations [c.3323A > G, p.N1108S; c.3188G > A, p.R1063H] were detected in two of the 21 (9.5%) triple-negative patients. Twelve of the 82 (14.6%) JAK2V617Fpositive cases had coexisting germline JAK2-mutations [JAK2R1063H, n = 6; JAK2R893T, n = 1; JAK2T525A, n = 1] or at least one somatic MPL-mutation [MPLY591D, n = 3; MPLW515 L, n = 2; MPLE335K, n = 1]. Overall, MPL-mutations always coexisted with JAK2V617F and/or other MPL-mutations. None of the JAK2V617F plus a second JAK2-mutation carried a TET2-mutation but all patients with JAK2V617F plus an MPL-mutation harbored a somatic TET2-mutation. Four genomic clusters could be identified in the JAK2V617F-positive cohort. Cluster-I (10%) (noncanonical JAK2 mutated (mut) + TET2 wildtype (wt) ) were younger and had less proliferative disease compared with cluster-IV (5%) (TET2 mut + MPL mut ). In conclusion, recurrent concomitant classical and/or noncanonical JAK2-and MPL-mutations could be detected by NGS in 15.7% of JAK2V617F-and MPLW515-positive MF patients with genotype-phenotype associations. Many of the germline and/or somatic mutations might act as "Significantly Mutated Genes" contributing to the pathogenesis and phenotypic heterogeneity. A cost-effective NGS-based approach might be an important step towards patient-tailored medicine. K E Y W O R D S germline mutations, JAK2 mutation, MPL mutation, myelofibrosis, noncanonical mutations, somatic mutations