Aurélie Chauveau

Clinical and molecular response to interferon-α therapy in essential thrombocythemia patients with CALR mutations

Myeloproliferative neoplasms are clonal disorders characterized by the presence of several gene mutations associated with particular hematologic parameters, clinical evolution, and prognosis. Few therapeutic options are available, among which interferon α (IFNα) presents interesting properties like the ability to induce hematologic responses (HRs) and molecular responses (MRs) in patients with JAK2 mutation. We report on the response to IFNα therapy in a cohort of 31 essential thrombocythemia (ET) patients with CALR mutations (mean follow-up of 11.8 years). HR was achieved in all patients. Median CALR mutant allelic burden (%CALR) significantly decreased from 41% at baseline to 26% after treatment, and 2 patients even achieved complete MR. In contrast, %CALR was not significantly modified in ET patients treated with hydroxyurea or aspirin only. Next-generation sequencing identified additional mutations in 6 patients (affecting TET2, ASXL1, IDH2, and TP53 genes). The presence of additional mutations was associated with poorer MR on CALR mutant clones, with only minor or no MRs in this subset of patients. Analysis of the evolution of the different variant allele frequencies showed that the mutated clones had a differential sensitivity to IFNα in a given patient, but no new mutation emerged during treatment. In all, this study shows that IFNα induces high rates of HRs and MRs in CALR-mutated ET, and that the presence of additional nondriver mutations may influence the MR to therapy.

Genetic basis of Congenital Erythrocytosis mutation update and online databases

Congenital erythrocytosis (CE), or congenital polycythemia, represents a rare and heterogeneous clinical entity. It is caused by deregulated red blood cell production where erythrocyte overproduction results in elevated hemoglobin and hematocrit levels. Primary congenital familial erythrocytosis is associated with low erythropoietin (Epo) levels and results from mutations in the Epo receptor gene (EPOR). Secondary CE arises from conditions causing tissue hypoxia and results in increased Epo production. These include hemoglobin variants with increased affinity for oxygen (HBB, HBA mutations), decreased production of 2,3‐bisphosphoglycerate due to BPGM mutations, or mutations in the genes involved in the hypoxia sensing pathway (VHL, EPAS1, and EGLN1). Depending on the affected gene, CE can be inherited either in an autosomal dominant or recessive mode, with sporadic cases arising de novo. Despite recent important discoveries in the molecular pathogenesis of CE, the molecular causes remain to be identified in about 70% of the patients. With the objective of collecting all the published and unpublished cases of CE the COST action MPN&MPNr‐Euronet developed a comprehensive Internet‐based database focusing on the registration of clinical history, hematological, biochemical, and molecular data (http://www.erythrocytosis.org/). In addition, unreported mutations are also curated in the corresponding Leiden Open Variation Database.

Molecular characterization and follow-up of five CML patients with new BCR–ABL1 fusion transcripts

We report five chronic myeloid leukaemia (CML) patients in whom we identified and characterized undescribed BCR–ABL1 fusion transcripts. We investigated the precise features of the molecular rearrangements and the minimal residual disease follow‐up for these five patients. Three resulted from new rearrangements between the BCR and ABL1 sequences (the breakpoints being located within BCR exon 13 in two cases and within BCR exon 18 in one case). The other two cases revealed a complex e8‐[ins]‐a2 fusion transcript involving a third partner gene, PRDM12 and SPECC1L, respectively. Moreover, single nucleotide polymorphism‐array analysis performed in the latter two cases showed copy number alterations shared by the two patients, thus identifying genes that were deleted during rearrangement and suggesting their potential role in CML pathogenesis. Interestingly, we highlight that the prognosis of alterations, such as the presence of an e8a2 transcript or the deletion of various genes, which have been controversial, may be definitively erased by the introduction of tyrosine kinase inhibitors (TKIs).

Clinical and biological characterization of MPN patients harboring two driver mutations, a French intergroup of myeloproliferative neoplasms (FIM) study

To the Editor: Mutations in “driver” genes (JAK2, MPL, or CALR) are found in the vast majority of Philadelphia-negative myeloproliferative neoplasms (MPN): JAK2V617F or exon 12 mutations in polycythemia vera (PV), JAK2V617F, CALR exon 9, or MPL exon 10 mutations in essential thrombocythemia (ET) or primary myelofibrosis (PMF). In the latter, phenotypic presentation and evolutive course vary according to the mutation, eg, higher platelet counts in CALR mutated ET patients and higher hemoglobin, white blood cell counts and higher risk of thrombosis in JAK2-mutated ET. Driver mutations were initially described as mutually exclusive, but can co-exist in rare “doubly mutated” (DM) patients. We have previously shown that double mutations were more frequently encountered in patients with low (<5%) JAK2V617F allelic burdens. However, due to the limited reports of DM patients, it is unclear how co-occurrence of driver mutations affects the presentation or evolution of MPN. In order to confirm DM frequency in a multicentric fashion, two additional cohorts of JAK2-mutated MPN were screened for CALR mutations: 134 JAK2V617F-mutated ET from the University Hospital of Cr eteil (France) and a cohort of 31 MPN with a low JAK2V617F allele burden (<2%) from the University Hospital of Nantes (France). Five of 49 patients with a JAK2V617F allele burden <5% also had a CALR mutation (10.2%), similar to our previous findings (14.3%) versus none of the 119 patients with JAK2V617F>5%. This confirms the relatively high frequency of DM in patients with low JAK2V617F burden. In addition, clinical and biological data were gathered on 21 DM patients discovered fortuitously in 4 other French centers. Overall, 47 cases of DM patients (Supporting Information Figure 1) were analyzed, of which 28 harbored a JAK2V617F allele burden equal to or below 1% (60%) while only 2 (5%) had a JAK2V617F allele burden above 5%. Among the 47 DM patients, 40 were diagnosed with ET, 5 with PMF, 1 with PV, and 1 with MDS/MPN-RS-T. Thirty-two patients (68%) presented with JAK2V617F and CALR mutations, 11 (23%) with JAK2V617F and MPL mutations, 2 with CALR and MPL mutations, 1 PV patient had JAK2V617F and JAK2 exon 12 mutations, and 1 patient had an association of two different CALR mutations. In order to know whether a double mutation impacts the clinical presentation or the evolutive course, the 40 DM patients with ET were compared to a control cohort comprising 577 “classical” ET patients from OBENE cohort, NCT02897297. Patients’ characteristics were compared using Fisher exact test for categorical variables as well as Mann and Whitney and Kruskal–Wallis (followed by Dunn tests for multiples comparisons) tests for continuous variables. For multiple tests, P-values were adjusted with Hochberg’s method. Survival estimates were obtained with the Kaplan–Meier method, and statistical analyses were performed with Log-rank test and Cox model. DM-ET patients were more often males (M/F sex ratio 1.4 [23/17] vs. 0.68 [233/344], P 5 .045) and significantly older than control ET patients (median: 72 vs. 61 years old, P< .001), especially compared to JAK2-singly mutated (SM) (63 years old, P 5 .006), CALR-SM (59 years old, P< .001) or triple-negative (56 years old, P< .001) ET patients. For clinical comparisons, the 37 DM-ET patients with a JAK2V617F mutation (29 with JAK2-CALR and 8 with JAK2-MPL) were compared to ageand sex-matched ET patients (Supporting Information Table 1): each CALR-JAK2V617F DM patient was matched with 3 JAK2V617F and 2 CALR; MPL-JAK2V617F DM with 3 JAK2V617F and 1 MPL-SM ET patients. At diagnosis, DM-ET and CALR or MPL-SM matched control patients had similar hemoglobin and platelet counts, but lower hemoglobin and higher platelet counts than JAK2-SM controls (Figure 1A,B). Leukocyte counts of JAK2-CALR DM-ET patients were intermediate between those of CALR and JAK2-mutated controls, without statistically significant difference (Figure 1C). Splenomegaly at diagnosis was observed in 17% (4/24) of JAK2-CALR DM-ET patients and none of the 6 JAK2-MPL DM-ET for whom this data was available. These frequencies were not significantly different from those observed in respective JAK2, CALR, or MPL SM control groups (Supporting Information Table 1). A history of thrombosis before or at diagnosis was found in 3/16 (19%) of JAK2-CALR DM-ET, which is equivalent to the proportion observed in CALR mutated controls (21%), but lower than that in JAK2 mutated controls (41%), without reaching statistical significance (P5 .27). The JAK2V617F allele burden was much lower in DM-ET patients than in matched controls (median 1% vs. 26%, P< .001, Supporting Information Figure 2A). The relatively frequent discovery of low JAK2V617F burdens in DM patients might be explained by a preexisting clonal hematopoiesis of indeterminate potential (CHIP), followed by the acquisition of a second oncogenic event (CALR or MPL mutation) leading to the MPN phenotype. This would be supported by

Absence of CALR mutations in JAK2-negative polycythemia

Polycythemia vera (PV) is a myeloproliferative neoplasm (MPN) responsible for increased hematopoiesis, mainly affecting the erythroid lineage. The diagnosis of PV has been simplified enormously by the description of JAK2 mutations, affecting the canonical V617 in exon 14 in more than 95% of cases,

Sequential analysis of 18 genes in polycythemia vera and essential thrombocythemia reveals an association between mutational status and clinical outcome

Philadelphia‐negative classical myeloproliferative neoplasms (MPN) are clonal diseases characterized by driver mutations of JAK2, MPL, or CALR. Additional mutations may occur in epigenetic regulators, signaling, or splicing genes that may be useful in the prognostic assessment of MPN patients. In primary myelofibrosis, molecular‐based prognostic scoring systems have been recently proposed, but few data are available to date for polycythemia vera (PV) and essential thrombocythemia (ET). In this study, we used a next generation sequencing‐based 18‐gene panel in 50 JAK2V617F positive PV and JAK2V617F positive ET patients from an institutional cohort investigated at diagnosis and at 3‐year follow‐up (3y). Disease progression at 3y was defined by a composite criterion. Patients (28 PV and 22 ET) were included according to their clinical status, with or without disease progression. At diagnosis, we found 28 additional mutations in 21 of the 50 patients. Patients with disease progression were more likely to have at least one additional mutation. There was no difference between PV and ET. All patients with two or more additional mutations exhibited disease progression at 3y. No novel mutations appeared at 3y. The allele burden increase by at least one mutation at 3y was more frequent in patients with disease progression. Our data suggest that screening for additional mutations in PV and ET could identify patients at a higher risk of disease progression.

Absence of CALR mutation among a cohort of 394 unselected patients with a first episode of unprovoked venous thromboembolism

Absence of CALR mutation among a cohort of 394 unselected patients with a first episode of unprovoked venous thromboembolism -

A new point mutation in EPOR inducing a short deletion in congenital erythrocytosis

JIS, AF, JQ and DS performed the research and analysed the data. JIS wrote the paper. SB hosted the research and provided experimental expertise. KLY designed the research study. All authors reviewed and approved the final manuscript. Jonathan I. Sive Andrew Feber Dean Smith John Quinn Stephan Beck Kwee Yong Department of Haematology, University College London, and Department of Medical Genomics, UCL Cancer Institute, University College London, London, UK E-mail: jonathan.sive@uclh.nhs.uk

Outcome of Ph negative myeloproliferative neoplasms transforming to accelerated or leukemic phase

Abstract Myeloproliferative neoplasms (MPN) are chronic disorders that can sometimes evolve into accelerated or leukemic phases. We retrospectively identified 122 patients with such blastic phases. The overall median survival was four months: 10.2 months for patients treated with intensive treatments compared to three months for best supportive care (p = .005). Azacytidine, intensive chemotherapies, or allogeneic stem cell transplantation gave the highest median survivals with 9, 10.2, and 19.4 months, respectively. Accelerated phases (AP) had a longer median survival compared to acute leukemia (4.8 months vs. 3.1 months; p = .02). In this retrospective and observational study, we observe that the longest survivals are seen in patients eligible for intensive treatments. Azacytidine shows interesting results in patients non-fit for intensive chemotherapy. Supportive care should probably be restricted to elderly patients and those with unfavorable karyotype. An early diagnosis of AP could also result in a better survival rate.

Acute myeloid leukaemia (FAB AML‐M4Eo) with cryptic insertion of cbfb resulting in cbfb‐Myh11 fusion

Inv(16)(p13q22) and t(16;16)(p13;q22) are cytogenetic hallmarks of acute myelomonoblastic leukaemia, most of them associated with abnormal bone marrow eosinophils [acute myeloid leukaemia French–American–British classification M4 with eosinophilia (FAB AML‐M4Eo)] and a relatively favourable clinical course. They generate a 5′CBFB‐3′MYH11 fusion gene. However, in a few cases, although RT‐PCR identified a CBFB‐MYH11 transcript, normal karyotype and/or fluorescent in situ hybridization (FISH) analyses using commercially available probes are found. We identified a 32‐year‐old woman with AML‐M4Eo and normal karyotype and FISH results. Using two libraries of Bacterial Artificial Chromosome clones on 16p13 and 16q22, FISH analyses identified an insertion of 16q22 material in band 16p13, generating a CBFB‐MYH11 type A transcript. Although very rare, insertions should be searched for in patients with discordant cytological and cytogenetic features because of the therapeutic consequences. Copyright