ALBERT

Description: An acquired gain-of-function mutation in the Janus kinase 2 (JAK2-V617F) is frequently found in patients with myeloproliferative disorders (MPDs). To test the hypothesis that JAK2-V617F is the disease-initiating mutation, we examined whether all cells of clonal origin carry the JAK2-V617F mutation. Using allele-specific polymerase chain reaction (PCR) assays for the JAK2 mutation and for the X-chromosomal clonality markers IDS and MPP1, we found that the percentage of granulocytes and platelets with JAK2-V617F was often markedly lower than the percentage of clonal granulocytes determined by IDS or MPP1 clonality assays in female patients. Using deletions of chromosome 20q (del20q) as an autosomal, X-chromosome–independent clonality marker, we found a similar discrepancy between the percentage of cells carrying JAK2-V617F and del20q. Our results suggest that in a proportion of patients with MPDs, JAK2-V617F occurs on the background of clonal hematopoiesis caused by a somatic mutation in an as-yet-unknown gene.

Description: An acquired somatic mutation in the JAK2 gene (JAK2-V617F) is frequently found in patients with myeloproliferative disorders (MPD). In most studies the JAK2-V617F mutation has been analyzed at single time point. Here we performed a retrospective single center study on 73 MPD patients (36 polycythemia vera (PV), 29 essential thrombocythemia (ET), and 8 primary myelofibrosis (PMF)) from whom at least two blood samples (mean=5, range 2–17) were available with an interval of at least 8 months. The mean follow-up period was 35±17 months (range 8–78 months). The allelic ratio of JAK2-V617F (%T) was determined in DNA from purified peripheral blood granulocytes by allele-specific PCR. In 73 MPD patients studied, 53 (73%) carried the JAK2-V617F mutation (32 PV, 18 ET, 3 PMF). None of the 20 patients negative for JAK2-V617F acquired the JAK2-V617F mutation during the observation period (n=20, mean number of samples=4). In the majority of the JAK2-V617F positive patients (35/53; 66%) the JAK2-V617F allelic ratios remained remarkably stable during the follow-up period (variation ±5%T) (Figure 1 A and B). In 10/53 patients (19%) we observed an increase and in 8/53 (15%) a decrease in JAK2-V617F allelic ratio greater than 5%T. Interestingly, 3/10 patients (1 ET and 2 PV) who showed increase of JAK2-V617F developed secondary myelofibrosis. Twenty six patients (49%) received cytoreductive treatment (hydroxyurea: 24, interferon alpha: 1, anagrelide: 1). Cytoreduction with hydroxyurea did not significantly reduce the JAK2-V617F allelic ratios (Figure 1A) compared to untreated patients (Figure 1B). The only molecular remission was seen in one patient treated with interferon alpha (Figure 1 A). In one patient without cytoreduction JAK2-V617F became undetectable because of transformation to acute myeloid leukemia with blast cells negative for JAK2-V617F. A second patient treated with hydroxyurea showed a pronounced decrease of JAK2-V617F (–47%T in 6 months), but no clinical or laboratory signs of leukemic transformation were present. We conclude that the amount of JAK2-V617F remains very stable in a majority of JAK2-V617F positive patients. Prospective studies will help to elucidate whether increasing JAK2-V617F allelic ratios can predict secondary myelofibrosis or decreasing allelic ratios in absence of cytoreductive therapy (e.g. interferon alpha) are early signs of leukemic transformation. Figure 1A. Figure 1A. Figure 1B. Figure 1B.

Description: We studied the lineage distribution of JAK2 mutations in peripheral blood of 8 polycythemia vera (PV) patients with exon 12 mutations and in 21 PV patients with JAK2-V617F. Using a quantitative allele discrimination assay, we detected exon 12 mutations in purified granulocytes, monocytes, and platelets of 8 patients studied, but lymphoid cells showed variable involvement and the mutation was absent in T cells. Endogenous erythroid colonies grew in all patients analyzed. One patient displayed erythroid colonies homozygous for the exon 12 mutation with evidence for mitotic recombination on chromosome 9p. In some patients with exon 12 mutations or JAK2-V617F, a proportion of endogenous erythroid colonies were negative for both JAK2 mutations. One patient carried 2 independent clones: one with an exon 12 mutation and a second with JAK2-V617F. The finding of clonal heterogeneity is compatible with the hypothesis that additional clonal events are involved in the pathogenesis of PV.

Description: About 95% of patients with polycythemia vera (PV) carry the unique V617F mutation in JAK2 exon 14, which encodes a portion of the JH2 auto-inhibitory domain of the Jak2 kinase. Mutations in exon 12 have been recently reported in JAK2 (V617F)-negative patients with PV or idiopathic erythrocytosis. We searched for exon 12 mutations in 168 patients with JAK2 (V617F)-negative myeloproliferative disorders. The 2001 WHO criteria were employed for diagnosis. Of the 168 patients studied, 47 had sporadic PV, 11 had familial PV, 75 had essential thrombocythemia (ET), and 35 had primary myelofibrosis (PM). Seventeen patients with PV, including 15/47 sporadic cases and 2/11 familial cases, were found to carry deletions (n=15) or duplications (n=2) of exon 12 in circulating granulocytes but not in T-lymphocytes. None of the 110 patients with ET or PM was found to be positive. Mutations were detected by sequencing, and were then confirmed by sub-cloning in bacteria in 7/17 cases. Four of the 8 mutations detected were novel, while the most frequent ones were N542–E543del and E543–D544del. Mutations spanned from base 1606 to 1640, and the two duplications modified the rest of the sequence by adding 33 bp. In terms of protein, deletions predicted aminoacid changes spanning from phenylalanine 537 to aspartic acid 544, while duplications predicted changes from phenylalanine 547 onwards within the JH2 pseudokinase domain. Three categories of molecular lesions were identified: those involving a K539L substitution; those involving the E543del; and aminoacid duplications involving a substitution of phenylalanine 547. At clinical onset, 16/17 (94%) patients carrying a JAK2 exon 12 mutation had low serum erythropoietin (Epo) levels, indicating a combination of absolute erythrocytosis and suppressed endogenous Epo production. Moreover, 12/17 patients had erythrocytosis associated with normal white blood cell and platelet counts, i.e., isolated erythrocytosis. This frequency (71%) was significantly higher than that observed in 92 patients diagnosed with JAK2 (V617F)-positive PV at the Department of Hematology, IRCCS Policlinico San Matteo, Pavia, Italy (P12 x 109/L) and/or thrombocytosis (PLT〉400 x 109/L), and only 22% of them had isolated erythrocytosis. Both patients with familial PV carrying an exon 12 mutation had an affected sibling with JAK2 (V617F)-positive PV. While the former showed isolated erythrocytosis, their JAK2 (V617F)-positive siblings had also thrombocytosis. In conclusion: several somatic mutations of JAK2 exon 12 - mostly 6 bp deletions - can be found in patients with a myeloproliferative disorder that is mainly characterized by erythrocytosis associated with low serum Epo levels; a genetic predisposition to acquisition of different JAK2 mutations is inherited in families with myeloproliferative disorders, and the mutation type (exon 12 vs exon 14) contributes to determining their variable clinical phenotype.

Description: Acute myeloid leukemia (AML) is a common complication of myeloproliferative disorders (MPDs). The role of the JAK2-V617F mutation in this process is unknown. We performed a retrospective analysis of DNA samples from MPD patients with secondary AML. We analysed DNA samples taken at the time of transformation to AML from 54 MPD patients (24 PV, 21 ET, 9 IMF). In addition, DNA samples taken at diagnosis of MPD were obtained in 21 of these patients. DNA was extracted from bone marrow or peripheral blood films, purified granulocytes or frozen cells. FACS sorting of blast cells, T cells and neutrophils was performed in some of the samples. The allelic ratio of JAK2-V617F was determined by allele-specific quantitative PCR (AS-PCR). We obtained AS-PCR data on 52/54 samples taken at the time of transformation (96%), whereas 2 samples did not yield PCR products: 24/52 samples were negative for JAK2-V617F (46%) and 28/52 were positive (54%). For 14/24 negative patients (58%) we had additional DNA samples taken at the time of MPD diagnosis and interestingly, 5 of these 14 patients (36%) were positive for JAK2-V617F at this earlier time point before AML transformation. This suggests that in these patients the JAK2-V617F positive clone was lost during the evolution to AML. Furthermore, comparison of the JAK2-V617F allelic ratios with the percentage of blast cells in patient samples positive at transformation revealed 8/28 cases where the JAK2-V617F allelic ratio was markedly lower than the percentage of blasts, e.g. 8%T-allele and 52% myeloid blast cells. In these patients a JAK2-V617F negative AML clone most likely co-exists with a JAK2-V617F positive MPD clone. To address the question whether the AML clone arose independently from the JAK2-V617F clone, we analyzed loss of heterozygosity on chromosome 9p (9pLOH) in one informative patient who displayed a high allelic ratio of mutant JAK2 at diagnosis (94%T). The CD15+ cells from this patient showed 9pLOH at diagnosis, as demonstrated with two independent microsatellite markers. In contrast, the FACS sorted blast cells at the time of transformation contained both parental alleles in the 9p region and were JAK2-V617F negative by AS-PCR. This excludes the possibility that the AML clone lost the JAK2V617F in the process of undergoing mitotic recombination at a stage heterozygous for JAK2-V617F. Analysis of additional patients is under way. In summary, we found in a cohort of 54 MPD patients, 13 patients initially positive for JAK2-V617F that transformed into JAK2-V617F negative AML. Although not confirmed in the one patient analyzed, we cannot exclude that other patients the JAK2-V617F positive MPD clone lost the JAK2 mutation during the process of transformation. Alternatively, the AML clone could have developed de novo from a JAK2-V617F negative progenitor or stem cell. The latter model has difficulties explaining the high incidence of de novo AML (8/54 patients), unless the JAK2-V617F negative progenitor already carried an as yet unknown mutation and was part of the MPD clone.

Description: Introduction While the key transforming genetic events occur in the developing cancerous cell, this cell is dependent on its environmental context and interaction for competitive outgrowth and subsequent tumor-development. Myelofibrosis (MF) represents a model cancer disease with stepwise development from a chronic state that depends on microenvironmental interactions to a more aggressive disease. Engraftment of primary MF patient cells in murine xenograft models is poor (Wang et al., JCI 2012) and is possibly explained by the lack of supportive microenvironmental factors. Thrombopoietin (TPO) has been implicated in the pathogenesis of MF (Schepers et al., Cell Stem Cell 2013, Dadfarnia et al., Blood 2014, Abdel-Wahab et al., Annu Rev Med 2009). Also, the interaction between human hematopoietic cells and SIRPα expressed on mouse macrophages is critical for human engraftment in xenografts (Takenaka et al., Nature Immunology 2007). We hypothesized that the constitutive expression of human TPO and human SIRPα may promote the development of the human MF clone in mouse xenografts. Methods Purified peripheral blood CD34+ cells were collected from six patients with primary MF or post-PV/ET MF and low to intermediate 2 risk disease according to the dynamic international prognostic scoring system (DIPSS). Four patients carried a JAK2-V617F mutation and two patients carried a calreticulin (CALR) mutation. CD34+ cells were intrahepatically transplanted into sublethally irradiated newborn humanSIRPα-transgenic/humanTPO-knockin Rag2-/- gamma-/- (TPO-SIRPα) mice (Rongvaux et al., Ann Rev. Immunol 2013). NSG mice were used as controls and injected with the same number of CD34+ cells. Two to three mice were injected with ≥1 million CD34+ cells from the same patient sample each. Mice were sacrificed 12-16 weeks after transplantation and human engraftment and hematopoietic cell lineage distribution was assessed by flow cytometry using human specific antibodies. Tissues were collected for immunohistochemistry, assessment of fibrosis and spleen weight. DNA was extracted from whole bone marrow and a qualitative PCR was performed to determine the presence of the JAK2-V617F or CALR-mutations. Results Three out of six samples generated a human graft of ≥20% human CD45+ cells, while the three other samples generated engraftment of 0.1-3%. The human graft was mainly composed of myeloid cells and monocytic differentiation was observed. In 2/2 experiments analysed, a JAK2-V617F and a CALR type 2 mutation were detected in the bone marrow of engrafted mice transplanted with the respective patient sample. Development of fibrosis was not observed three months post-transplantation, presumably due to the short observation time. Spleen weight was significantly increased in mice engrafted with human MF and was the consequence of increased murine extramedullary hematopoiesis. We then aimed to identify factors that could predict human MF engraftment in TPO-SIRPα mice. While neither the DIPSS, nor the presence of myeloid precursors in the peripheral blood (blasts excluded) were predictive of human MF engraftment, the presence of blasts in the peripheral blood significantly correlated with engraftment potential. Importantly, none of the patients developed acute leukemia during follow-up. Finally, preliminary evidence suggests that TPO-SIRPα mice are more supportive of human MF engraftment than NSG mice. Conclusions This is the first xenograft model that supports robust engraftment of human peripheral blood MF cells and further supports a role for TPO in the pathogenesis of MF. In contrast to previous models TPO-SIRPα mice strongly promote myeloid rather than lymphoid engraftment. The tight correlation between the presence of peripheral blood blasts and the human MF engraftment potential suggests that human MF stem cells reside in the blast population. In summary, the xenograft model presented here constitutes a powerful tool to assess heterogeneity regarding MF biology, microenvironmental dependence of the MF clone and likely also therapeutic response of MF in vivo. Disclosures No relevant conflicts of interest to declare.

Description: We searched for JAK2 exon 12 mutations in patients with JAK2 (V617F)-negative myeloproliferative disorders. Seventeen patients with polycythemia vera (PV), including 15 sporadic cases and 2 familial cases, carried deletions or duplications of exon 12 in circulating granulocytes but not in T lymphocytes. Two of the 8 mutations detected were novel, and the most frequent ones were N542-E543del and E543-D544del. Most patients with PV carrying an exon 12 mutation had isolated erythrocytosis at clinical onset, unlike patients with JAK2 (V617F)-positive PV, most of whom had also elevations in white blood cell and/or platelet counts. Both patients with familial PV carrying an exon 12 mutation had an affected sibling with JAK2 (V617F)-positive PV. Thus, several somatic mutations of JAK2 exon 12 can be found in a myeloproliferative disorder that is mainly characterized by erythrocytosis. Moreover, a genetic predisposition to acquisition of different JAK2 mutations is inherited in families with myeloproliferative disorders.