Insertion and/or deletion mutations of the CALR gene have recently been demonstrated to be the second most common driver mutations in the myeloproliferative neoplasms (MPNs) of essential thrombocythemia (ET) and primary myelofibrosis (PMF). Given the diagnostic and emerging prognostic significance of these mutations, in addition to the geographical heterogeneity reported, the incidence of CALR mutations was determined in an Irish cohort of patients with MPNs with a view to incorporate this analysis into a prospective screening program. A series of 202 patients with known or suspected ET and PMF were screened for the presence of CALR mutations. CALR mutations were detected in 58 patients. Type 1 and Type 1-like deletion mutations were the most common (n=40) followed by Type 2 and Type 2-like insertion mutations (n=17). The CALR mutation profile in Irish ET and PMF patients appears similar to that in other European populations. Establishment of this mutational profile allows the introduction of a rational, molecular diagnostic algorithm in cases of suspected ET and PMF that will improve clinical management.

BACKGROUND

The discovery of calreticulin (CALR) has shown it to be the second most frequent mutation after the Janus Kinase 2 (JAK2) mutation in myeloproliferative neoplasms (MPNs). Its structure indicates various functions, of which two are to ensure calcium homeostasis and proper folding of other target proteins. Over 36 types of CALR mutations have been identified, all causing a recurrent frameshift in the C-terminal domain affecting CALR's localisation and calcium-binding function.

OBJECTIVE

To screen a cohort of 89 patients suspected of having an MPN for the CALR mutations.

METHODS

Capillary and gel electrophoresis were used in conjunction as confirmatory tests to screen the cohort of patients.

RESULTS

Of three samples containing a type 1 CALR mutation, two were heterozygous and one homozygous for a 52-base pair deletion in CALR.

CONCLUSIONS

Most studies report CALR mutations to be present only in patients with primary myelofibrosis or essential thrombocythaemia, with mutual exclusivity to JAK2 mutations. The findings of this study indicate that JAK2 and CALR mutations are no longer considered mutually exclusive. Similarly, patients with a polycythaemia vera phenotype could also carry a CALR mutation.

We investigated the variation of CALR-mutant burden during follow-up in 105 CALR-mutant MPN and compared it to the variation of JAK2-mutant burden in 226 JAK2-mutant MPN.The median allele burden at last evaluation was significantly higher than at first evaluation in essential thrombocythemia (ET) (49.5% vs 45%, P < .001) but not in primary myelofibrosis (PMF) (52% vs 51%, P 0.398). Median values of slope were positive both in ET (0.071) and in PMF (0.032). In CALR-mutant ET there was a difference between natural and therapy-related slope (P 0.006).In the JAK2-mutated cohort, the median allele burden at last evaluation was not different respect to that at first evaluation, neither in ET (22.9% vs 23.2%, P = 0.216) nor in PMF (50.5% vs 45.0%, P = 0.809), despite a positive slope. Multivariate analysis to evaluate the effect of mutation (CALR vs JAK2) on the slope of mutant burden in not treated pts with a positive slope adjusting for diagnosis (ET vs PMF) showed a trend toward a higher increase of mutant burden in CALR vs JAK2 (β = 0.19, P = 0.061) with no difference between diagnosis (P = 0.419). The findings of this study suggest that clonal expansion in CALR-mutant MPN is faster than that observed in JAK2-mutant MPN.

BACKGROUND

Classical BCR-ABL1-negative myeloproliferative neoplasms (MPNs) including polycythemia vera, essential thrombocythemia (ET), and primary myelofibrosis frequently harbor JAK2, MPL, and CALR somatic mutations.

METHODS

AS-PCR for JAK2 V617F, pyrosequencing for MPL W515L/K, and PCR-fragment analysis for CALR exon 9 mutations were established to analyze genomic DNA isolated from peripheral blood samples of 58 newly diagnosed ET patients in Thailand.

RESULTS

JAK2 V617F was detected in 41 patients (71%) and CALR exon 9 mutation was positive in eight patients (14%), whereas no mutation of MPL W515L/K was observed in this study. Patients with CALR mutation were older (p = 0.023) and exhibited lower number of platelet count (p = 0.041) than patients without CALR mutation. Two previously known CALR mutation types were identified in this study (six patients with CALR-type 1 and two patients with CALR-type 2). Additionally, no co-existence of JAK2 V617F and CALR mutations was identified in this work.

CONCLUSIONS

We reported the frequency of JAK2 V617F, MPL W515L/K, and CALR mutations in Thai patients with ET. Clinical and hematological phenotypes of patients were associated with JAK2 and CALR mutation statuses. The combination of laboratory testing for the detection of JAK2, CALR, and MPL mutations is necessary to improve the diagnosis and classification of BCR-ABL1-negative MPN.

We investigated the changes in chromosomal abnormalities in myeloproliferative neoplasm (MPN) patients during long-term follow-up. In total, 28 MPN patients (22 with primary myelofibrosis and 6 with polycythemia vera) were included. Among them, 25 patients underwent serial bone marrow (BM) biopsies during disease progression, and 3 patients had cytogenetic abnormalities at initial diagnosis but lacked follow-up BM biopsies. JAK2, CALR, and MPL mutation analyses were performed. Targeted sequencing analysis was conducted in 11 patients. Among the 28 patients, 21 (75.0%) had cytogenetic abnormalities either at diagnosis (8/26) or during follow-up. The median time from the initial analysis to the appearance of additional cytogenetic abnormalities was 8.4 years. Among the chromosomal abnormalities at initial diagnosis, trisomy 8 (3/26, 11.5%) was the most frequent, followed by gain of 1q, del(20q), and del(9q) (each in 2/26). Among all chromosomal abnormalities, including those that occurred during follow-up, the most frequent was del(20q) and +1q (8/28, 28.6%), followed by del(6p) (14.3%) and trisomy 8 (10.7%). Del(20q) was more frequent in CALR-mutated patients (4/6, 66.7%) than in JAK2-mutated patients (3/19, 15.8%, P = 0.016). The presence of cytogenetic abnormalities at initial diagnosis was associated with poor prognosis. Cytogenetic evolution may provide interesting insights into the disease course.

Myeloproliferative neoplasms (MPNs) driver mutations are usually found in JAK2, MPL, and CALR genes; however, 10%-15% of cases are triple negative (TN). A previous study showed lower rate of JAK2 V617F in primary myelofibrosis patients exposed to low doses of ionizing radiation (IR) from Chernobyl accident. To examine distinct driver mutations, we enrolled 281 Ukrainian IR-exposed and unexposed MPN patients. Genomic DNA was obtained from peripheral blood leukocytes. JAK2 V617F, MPL W515, types 1- and 2-like CALR mutations were identified by Sanger Sequencing and real time polymerase chain reaction. Chromosomal alterations were assessed by oligo-SNP microarray platform. Additional genetic variants were identified by whole exome and targeted sequencing. Statistical significance was evaluated by Fisher's exact test and Wilcoxon's rank sum test (R, version 3.4.2). IR-exposed MPN patients exhibited a different genetic profile vs unexposed: lower rate of JAK2 V617F (58.4% vs 75.4%, P = .0077), higher rate of type 1-like CALR mutation (12.2% vs 3.1%, P = .0056), higher rate of TN cases (27.8% vs 16.2%, P = .0366), higher rate of potentially pathogenic sequence variants (mean numbers: 4.8 vs 3.1, P = .0242). Furthermore, we identified several potential drivers specific to IR-exposed TN MPN patients: ATM p.S1691R with copy-neutral loss of heterozygosity at 11q; EZH2 p.D659G at 7q and SUZ12 p.V71 M at 17q with copy number loss. Thus, IR-exposed MPN patients represent a group with distinct genomic characteristics worthy of further study.

Essential thrombocythemia (ET) is a BCL-ABL1-negative myeloproliferative neoplasm. We have reported that increased activated B cells can facilitate platelet production mediated by cytokines regardless JAK2 mutational status in ET. Recently, calreticulin (CALR) mutations were discovered in ~30% JAK2/MPL-unmutated ET and primary myelofibrosis. Here we sought to screen for CALR mutations and to evaluate B cell immune profiles in a cohort of adult Taiwanese ET patients. B cell populations, granulocytes/monocytes membrane-bound B cell-activating factor (mBAFF) levels, B cells toll-like receptor 4 (TLR4) expression and intracellular levels of interleukin (IL)-1β/IL-6 and the expression of CD69, CD80, and CD86 were quantified by flow cytometry. Serum BAFF concentration was measured by ELISA. 48 healthy adults were used for comparison. 19 (35.2%) of 54 ET patients harbored 8 types of CALR exon 9 mutations including 4 (7.4%) patients with concomitant JAK2V617F mutations. Compared to JAK2V617F mutation, CALR mutations correlated with younger age at diagnosis (p=0.04), higher platelet count (p=0.004), lower hemoglobin level (p=0.013) and lower leukocyte count (p=0.013). Multivariate analysis adjusted for age, sex, follow-up period and hematological parameters confirmed that increased activated B cells were universally present in JAK2-mutated, CALR-mutated and triple-negative ET patients when compared to healthy adults. JAK2- and CALR-mutated ET have significantly higher fraction of B cells with TLR4 expression when compared to triple-negative ET (p=0.019 and 0.02, respectively). CALR-mutated ET had significantly higher number of CD69-positive activated B cells when compared to triple-negative ET (p=0.035). In conclusion, increased B cell activation is present in ET patients across different mutational subgroups.

Mutations of the multifunctional protein calreticulin (CALR) are recognised as one of the main driver alterations involved in the pathogenesis of Philadelphia negative myeloproliferative neoplasms (Ph^- MPN) and also represent a major diagnostic criterion in the most recent World Health Organization classification of myeloid neoplasms. Nowadays, quantitative assessment of the driver mutations is gaining importance, as recent studies demonstrated the clinical relevance of the mutation load reflecting the size of the mutant clone. Here, we performed for the first time a manual and automated quantitative assessment of the CALR mutation load at protein level using CAL2, a recently developed CALR mutation specific monoclonal antibody, on a cohort of 117 patients with essential thrombocythemia (ET) or primary myelofibrosis (PMF) and compared the CALR protein mutation loads with the CALR mutation load values established by a molecular assay. Eighteen different CALR mutations were detected in the cohort of the 91 CALR mutant cases. Mutation loads of the CALR mutations were between 13% and 94% with mean value in PMF cases significantly higher than ET cases (49.94 vs 41.09; t-test, p=0.004). Cases without CALR mutation (n=26) showed no or only minimal labelling with the CAL2 antibody, while all 18 different types of CALR mutations were associated with CAL2 labelling. The CALR mutation load showed a significant correlation (p=0.03) with the occurrence of major thrombotic events, with higher mutation load in patients presenting with these complications. We report a 100% concordance between the mutation status determined by immunohistochemistry and the CALR molecular assay, and we extend the applicability of this approach to 16 rare CALR mutations previously not analysed at protein level.

OBJECTIVE

To explore the coexistence of ASXL1 and CALR gene mutations in patients with essential thrombocytheima (ET) and with primary myelofibrosis(PMF), and to compare the differences of clinical characteristics between ET and PMF patients carrying ASXL1 and CALR mutations, and ET and PMF patients carrying solitary gene mutation, and ET and PMF patients without any mutations.

METHODS

The mutations of ASXL1 gene at exon 12, CALR gene at exon 9 and MPL gene at exon 10 in 263 essential ET patients and 29 PMF patients were detected by PCR amplification followed by direct sequencing of genomic DNA. The JAK2V617F mutations were used by allele specific PCR detection.

RESULTS

72.6%(212/292)of patients harbored at least one mutation. The incidences of ASXL1 and CALR mutations were 5.8% and 30.5%, respectively. The frequencies of JAK2V617F and MPL mutations were 39.0% and 2.4%, respectively. 5.1%(15/292) of patients had double mutations, including ASXL1 and CALR(n=11), ASXL1 and JAK2V617F(n=2), MPL and CALR(n=1) and ASXL1 and MPL(n=1). The frequency of concurrent ASXL1 and CALR mutations was found to be high. Significant difference was found on hemoglobin levels and platelet counts between CALR and ASXL1 mutations and single mutation (P<0.05),however, the difference on leukocyte counts and median age was not found. Compared with negative patients, the presence of ASXL1 and CALR mutations was found to be significantly correlative with lower hemoglobin level (P=0.045), lower leukocyte count (P=0.002) and with higher platelet counts(P=0.001), but the difference of median age was not found.

CONCLUSIONS

The frequency of concurrent ASXL1 and CALR mutations is higher in ET patients. The coexistence of ASXL1 and CALR gene mutations significantly associated with lower hemoglobin level and higher platelet count.

OBJECTIVE

To detect the JAK2, CALR and MPL gene mutations in patients with BCR/ABL1 negative chronic myeloproliferative diseases（BCR/ABL1-CMPD）and to evaluate their diagnostic value.

METHODS

Two hundred and eight cases of BCR/ABL1-CMPD comprising of 146 cases of essential thrombocythemia（ET）, 37 cases of polycythemia vera（PV）and 25 cases of primary myelofibrosis（PMF）from March 2012 to December 2015 were enrolled in the BCR/ABL1-CMPD, while 124 cases of secondary thrombocythemia and 73 cases of secondary polycythemia were enrolled in the control group. The genomic DNA and total RNA Were isolated from bone marrow or peripheral blood, then the exons 12 to 20 of JAK2 gene, exon 10 of MPL gene and exons 3 to 9 of CALR gene were analyzed by using DNA sequencing.

RESULTS

among 146 ET patients, the JAK2, CALR or MPL mutations were found in: 138 cases（94.5%）including 86 cases with JAK2V617F mutation（58.9%）and 2 cases（1.4%）with exon 12 of JAK2 mutations. CALR mutations were detected in 41 cases（28.1%）, among them type 1（c.1092_1143del）in 22 cases, type 2（c.1154_1155insTTGTC）in 11 cases, and type 5（c. 1091_1142del）, type 8（c.1104_1137del）, type 41(c.1107_1137del）, type 42(c.1125_1125del）in one case respectively. In addition, 4 cases were detected withother mutations of the CALR gene（c.1107_1115del, c.1111_1144 del, c.1101 A>C, c.1112_1117del）. Moreover, 9 cases harbored MPL mutations（6.2%）. Secondly, 31 patients were detected with JAK2V617F mutation（83.8%）in 37 cases of PV, and JAK2 exon 12 mutations were found in 2 cases（5.4%）. Besides, CALR mutations were detected in 2 cases（5.4%）, including 1 case of type I, the other of novel mutation of CALR. Thirdly, 19 in 25 cases of PMF were detected with JAK2V617F mutation（76%）, 2 cases with CALR mutations（8%）. 4 patients（16%）, JAK2, CALR or MPL mutations were not detected, but among them 3 cases were found harboring other genetic abnormalities. Fourthly, no mutations of JAK2, MPL and CALR genes were detected in 124 patients with secondary thrombocytosis and 73 cases with secondary polycythemia.

CONCLUSIONS

Combined detection of JAK2, CALR and MPL gene mutations can cover the vast majority of patients with BCR/ABL1-negative myeloproliferative neoplasms. For higher frequencies of the mutations of CALR in ET patients, CALR mutation can be used as a new diagnostic marker in ET patients with JAK2 and MPL wild type.

The identification of acquired CALR mutations in patients with essential thrombocythaemia (ET) or myelofibrosis (MF) has meant that disease-initiating mutations can now be detected in about 90% of all patients with a myeloproliferative neoplasm (MPN). Here, we show that only those CALR mutations that cause a +1 frameshift, thereby altering the carboxy-terminus of calreticulin, promote cytokine independence in vitro; in-frame deletions were not functional, and are unlikely to be the pathogenetic mutation underlying some MPN cases. Expression of the thrombopoietin receptor, MPL, was also necessary for factor-independence. Although the CALR mutations are considered to occur only in JAK2 V617F-negative cases and in a heterozygous state, progenitor genotyping revealed that this is not always true. Notably, CALR mutation-positive MPNs can be polyclonal: in one case, two distinct CALR mutation-positive subpopulations could be identified; in another, separate populations of JAK2 V617F-positive and CALR-mutated cells were present. Mitotic recombination involving chromosome 19 in a third instance resulted in the emergence of a CALR mutation-homozygous subclone. Collectively, our studies demonstrate that occasional patients with CALR mutation-positive ET or MF carry other MPN-initiating genetic mutations (including JAK2 V617F), acquire "secondary mutations" before or after the CALR mutation, or evolve over time to being CALR mutation-homozygous.

BACKGROUND

Despite recent advances in the investigation of myeloproliferative neoplasms (MPN), the impact of genetic heterogeneity on its molecular pathogenesis has not been fully elucidated. Thus, in this study, we aim to characterize the genetic complexity in Korean patients with polycythemia vera (PV) and essential thrombocythemia (ET).

METHODS

We conducted association studies using 84 single-nucleotide polymorphisms (SNPs) in 229 patients (96 with PV and 133 with ET) and 170 controls. Further, whole-genome sequencing was performed in six patients (two with JAK2 V617F and four with wild-type JAK2), and putative somatic mutations were validated in a further 69 ET patients. Clinical and laboratory characteristics were also analyzed.

RESULTS

Several germline SNPs and the 46 haplotype were significantly associated with PV and ET. Three somatic mutations in MPDZ, IQCH, and CALR genes were selected and validated. The frequency of the CALR mutation was 58.0% (40/69) in ET patients, who did not carry JAK2/MPL mutations. Moreover, compared with JAK2 V617F-positive patients, those with CALR mutations showed lower hemoglobin and hematocrit levels (P = 0.004 and P = 0.002, respectively), higher platelet counts (P =0.008), and a lower frequency of cytoreductive therapy (P = 0.014).

CONCLUSIONS

This study was the first comprehensive investigation of the genetic characteristics of Korean patients with PV and ET. We found that somatic mutations and the 46 haplotype contribute to PV and ET pathogenesis in Korean patients.

The true frequency of the JAK2 46/1 haplotype in patients of myeloproliferative neoplasms (MPN) with CALR mutations was unknown. Totally 187 MPN cases with diagnosis of polycythemia vera (PV) and essential thrombocythemia (ET) were recruited. The frequency of 46/1 haplotype was significantly higher in JAK2V617F-positive PV (51%, p < 0.001) and ET (41%, p = 0.005) compared to normal controls. The exact location of JAK2V617F mutation was located at the cis-46/1 haplotype in 86.4% (32/37) PV patients and 87.5% (28/32) ET patients, respectively. Among the 51 patients of ET without JAK2V617F mutation, 38 (75%) patients harbored CALR mutations and 3 patients had MPL mutation. The frequency of 46/1 haplotype in the 38 ET patients with CALR mutations was 27%, which is not significantly different from that of normal control (p value = 0.879). Compared to non-46/1 haplotype, the presence of 46/1 haplotype had a trend to have higher white blood cell count in JAK2V617F-mutated PV and ET patients but not in CALR-mutated ET. We conclude that the 46/1 haplotype could have functioning effect but only in the context of JAK2V617F mutation.

The detection of somatic mutations in the 9 exon of the calreticulin gene (CALR) is regulated by the clinical recommendations as a diagnostic criterion for chronic Ph-negative myeloproliferative neoplasms (MPN). Some methods of nucleic acids testing are used to identify CALR gene mutations with different requirements for special skills of personnel and expensive equipment. The purpose of this work is to compare the results of the detection of CALR gene mutations in venous blood samples by allele-specific RT-PCR with subsequent electrophoresis, fragment analysis and Sanger- or pyro- sequencing. We used 1284 blood samples of patients with suspected MPN and 20 blood donor samples. Mutations in the CALR gene of the I and II type were identified using PCR-RT with the original primers and TaqMan probes. Also, all samples were tested for mutations in the CALR gene by electrophoretic detection of PCR results in an agarose gel. The use of allele-specific RT-PCR followed by electrophoretic detection made it possible to determine clinically significant mutations in the CALR gene in 81 venous blood samples of JAK2- and MPL-negative patients, including 42 cases of type I mutation, 33 cases of type II mutation and 8 rare CALR mutations. Mutations in the 9 exon of the CALR gene were not detected in any of the 20 blood donor samples or in 121 blood samples of patients with polycythemia vera. In randomly selected 20 negative samples, CALR gene mutations were also not detected using Sanger sequencing. All positive samples were confirmed by fragment analysis, as well as with Sanger- sequencing and pyro- sequencing. The described combined approach to detect mutations of the CALR gene in peripheral blood samples can be used in clinical diagnostic laboratories that have a standard set of equipment for electrophoresis of nucleic acids and a PCR-RT. We also propose a confirmatory test based on the pyrosequencing of DNA using the system of genetic analysis "PyroMark Q24".

Chromosome region maintenance 1 (CRM-1) and calreticulin (CALR) are two proteins that act as exportins for some nuclear receptors, in addition to other critical functions for cellular homeostasis. In several cancer types, CRM-1 and CALR are upregulated suggesting an imbalance in their functions. However, the regulation of CRM-1 and CALR, and their biological implications, are not completely known. Here, we evaluated the interplay between the levels of CRM-1 and CALR, and estrogen receptor alpha (ERα) status, in breast cancer cells. CRM-1 and CALR were upregulated in mammary tumors relative to normal mammary tissue. Furthermore, the mRNA and protein levels of CRM-1 and CALR were higher in breast cancer cells lacking ERα, in comparison with those that express ERα. Additionally, both proteins were distributed in the nucleus and cytoplasm in the two cell types. Importantly, we identified novel interactions for these exportins. First, we showed an interaction between CRM-1 and CALR, and then we identified that SUN1 and SUN2, two proteins localized in the nuclear envelop, were able to interact specifically with CRM-1, but not CALR. Interestingly, SUN1 and SUN2 expression seemed to be decreased in breast cancer, thereby affecting the interactions of these proteins with CRM-1, and possibly its actions as an exportin. Thus, our data suggest that expression levels for CRM-1 and CALR, the interaction between these exportins, and specific interactions of SUN1 and SUN2 with CRM-1 but not CALR, may be central elements in nucleo-cytoplasmic transport. Furthermore, deregulation of these elements may have serious implications in the progression of breast and other types of cancer.

Congenital erythrocytosis (CE) is a rare and heterogeneous disease. The high oxygen affinity hemoglobin (Hb) variants are the most common cause of CE. Herein, we report a Korean patient with isolated erythrocytosis. A 25-year-old man was referred to our hospital for evaluation of high Hb level (Hb 20.4 g/dL, hematocrit 58%, reticulocyte count 2.90%, white blood cell count 6.83×10⁹/L, and platelet count 195×10⁹/L). Bone marrow biopsy revealed normocellular marrow without myeloproliferative features. JAK2 (V617F, exon 12), CALR (exon 9), and MPL W515K/L mutations were not detected. P₅₀ (partial pressure at which Hb is half saturated with oxygen), which is an indicator of left-shift of oxygen dissociation curve (high oxygen affinity state), was 14.3 mm Hg (reference value 22.6-29.4 mm Hg). He was suspected to have CE. Mutation analysis of the HBB gene revealed the known Hb variant, Hb Heathrow [β103(G5)Phe→Leu]. This is the first report of Hb Heathrow in Asian.

Of BCR-ABL negative myeloproliferative neoplasm (MPN) patients, 3-14 % display a concomitant monoclonal gammopathy (MGUS). Nonetheless, literature on co-occurring MPN and MGUS is scarce, the molecular underpinnings are unknown and it is unclear whether patients require a specific management. Here, we compared the clinical and genetic features of MPN patients with and without concomitant MGUS. Of 114 MPN patients prospectively studied by serum immunofixation (median age, 67 years; 36.0 % essential thrombocythemia [ET], 24.6 % polycythemia vera [PV], 11.4 % secondary myelofibrosis [sMF], 28.1 % primary myelofibrois [PMF]; 73.7 % JAK2 V617F positive), 10 (9 %) harbored an M-protein. No relevant clinical differences existed between MPN patients with or without M-protein. Seven additional MPN/MGUS patients were retrospectively identified in our MPN registry, yielding a total of 17 patients (7 ET, 3 PV, 3 sMF, 4 PMF). One patient developed multiple myeloma (MM) and one smoldering MM. Seven of 12 patients analyzed carried mutations (e.g. in ASXL1 or TET2) in addition to those in JAK2 or CALR, and 4 of 10 patients showed aberrant cytogenetics. M-protein was mainly IgG (12/17), followed by IgM (4/17). In the two patients that underwent allogeneic stem cell transplantation mutant JAK2 and M-protein were no longer detectable post-transplant. In conclusion, MGUS prevalence in our cohort was in the range of previous reports and at most slightly higher than expected in the general population. MGUS presence did not correlate with a specific MPN entity, clinical features or genetic alterations. Our observations suggest that there is no strong clinical or biological relationship between the occurrence of MGUS and MPN.

Keywords: Essential thrombocythemia; MGUS; Monoclonal gammopathy; Myeloproliferative neoplasm; Polycythemia vera; Primary myelofibrosis.

Some myeloproliferative neoplasm (MPN) patients harbor JAK2(V617F) mutation, and CALR mutations were recently discovered in wild type (WT) JAK2(V617F). We evaluated the frequency and type of CALR mutations, and clinical and hematological characteristics in WT JAK2(V617F) and MPL(W515K/L) MPN patients. Sixty-five patients were included: 21 with primary myelofibrosis (PMF), 21 with myelofibrosis post-essential thrombocythemia (MPET) and 23 with essential thrombocythemia (ET). Screening for JAK2(V617F) and MPL(W515K/L) were performed using real-time PCR, while CALR mutations were analyzed by fragment analysis and Sanger sequencing. JAK2(V617F) was the most frequent mutation (54.5%) and one patient (1.5%) harbored MPL(W515L). CALR mutations were present in 38.1% of PMF, 12.5% of ET and 33.3% of MPET patients. Five types of CALR mutations were detected, among which type 1 (32.1%) and type 2 (21.4%) were found to be the most common. A novel CALR mutation in a PMF patient was found. Patients carrying CALR mutations had higher platelet count and less presence of splenomegaly than JAK2(V617F), while triple negatives had higher C-reactive protein levels than CALR mutant carriers. Screening for CALR mutations and its correlation with clinical features could be useful for the characterization of MPN patients and result in its incorporation into a new prognostic score.

Frameshift mutations in the calreticulin (CALR) gene are present in 30% of essential thrombocythemia and myelofibrosis patients. The two most frequent mutations are CALR del52 (type 1, approximately 60%) and CALR ins5 (type 2, around 30%), but many other rarer mutations exist accounting each for less than 2% of all CALR mutations. Most of them are structurally classified as type 1-like and type 2-like CALR mutations according to the absence or presence of a residual wild-type calcium-binding motif and the modification of the alpha-helix structure. Yet, several key questions remain unanswered, especially the reason of such low frequencies of these other mutations. In an attempt to investigate specific pathogenic differences between type 1-like and type 2-like CALR mutations and del52 and ins5, we modeled two type 1-like (del34 and del46) and one type 2-like (del19) mutations in cell lines and in mice. All CALR mutants constitutively activate JAK2 and STAT5/3/1 in a similar way in the presence of the thrombopoietin receptor (MPL) and induced cytokine-independent cell growth but to a lesser extent with rare mutants over time. This correlates with reduced expression levels of rare CALR mutants compared to del52 and ins5. Lethally irradiated mice that were engrafted with bone marrow transduced with the different CALR mutations developed thrombocytosis, but to a much lesser extent with ins5 and the type 2-like CALR mutation. In contrast to type 2-like mice, type 1-like mice developed marked myelofibrosis and splenomegaly 10 months after engraftment. Similar to del52, type 1-like CALR mutations induced an expansion at an early stage of hematopoiesis compared to ins5 and type 2-like mutation. Thus, type 1-like and type 2-like CALR mutants structurally and functionally resemble del52 and ins5 mutants, respectively.

Objective: To explore the relationship between driver gene mutation (JAK2, MPL and CALR) and disease type in BCR-ABL negative myeloproliferative neoplasms (MPNs) including primary myeloid fibrosis (PMF), essential thrombocytosis (ET) and polycythemia vera (PV). Methods: A total of 32 MPN related genes were detected by high-throughput sequencing in 156 MPN patients. The relationships between disease type and patients' general performance, the characteristics of driver gene mutations, concomitant gene mutations were analyzed. Results: In the population with JAK2 V617F positive mutation, the proportion of patients over 60 years old in PMF was higher than that with ET or PV. By high-throughput sequencing, 22 concomitant gene mutations were detected in 46 patients with JAK2, MPL or CALR mutations, including 4 (8.3%) in PV, 20 (29.4%) in ET, and 22 (55.0%) in PMF. DNMT3A mutation was detected only in patients with PV, while splicing factor related genes including SF3B1, SRSF2 and U2AF1 were only accompanied by PMF. According to the variation allele frequency (VAF) value of JAK2 V617F mutation, the VAF value associated with PV was the highest (68.15%), followed by PMF (37.7%) and ET (23%). However, there were significant differences in the incidence of JAK2 V617F homozygous among 3 different diseases. In patients with JAK2 mutation, the proportion of other gene mutations in PV and ET was significantly lower than that in PMF. Conclusions: Under the condition of common driver gene mutations (JAK2, MPL and CALR), patients' age, VAF value and homozygous state, concomitant gene mutations are closely related to different disease type. These correlations help to improve clinical understanding of disease characteristics and risk assessment.

The discovery of the activating Janus kinase (JAK)2V617F mutation in 2005 in most patients with the classic Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) spurred intense interest in research into these disorders, culminating in the identification of activating mutations in MPL in 2006 and indels in the gene encoding calreticulin (CALR) in 2013, thus providing additional mechanistic explanations for the universal activation of JAK-signal transducer and activator of transcription (JAK-STAT) observed in these conditions, and the success of the JAK1/2 inhibitor ruxolitinib, which first received regulatory approval in 2011. The field has continued to advance rapidly since then, and the past 2 years have witnessed important changes to the classification of MPN and diagnostic criteria for polycythemia vera (PV), novel insights into the mechanisms of bone marrow fibrosis in primary myelofibrosis (PMF), increasing appreciation of the biologic differences between essential thrombocythemia (ET), prefibrotic and overt PMF, and between primary and post-PV/ET myelofibrosis (MF). Additionally, the mechanisms through which mutant CALR drives JAK-STAT pathway activation and oncogenic transformation are now better understood. Although mastocytosis is no longer included under the broad heading of MPN in the 2016 revision to the World Health Organization classification, an important milestone in mastocytosis research was reached in 2017 with the regulatory approval of midostaurin for patients with advanced systemic mastocytosis (AdvSM). In this article, we review the major recent developments in the areas of PV, ET, and MF, and also briefly summarize the literature on midostaurin and other KIT inhibitors for patients with AdvSM.

The 2016 revised WHO classification of tumors of hematopoietic and lymphoid tissues has incorporated novel molecular markers, such as calreticulin (CALR) mutations, for the diagnosis of myeloproliferative neoplasms (MPNs). Typically, CALR mutations are detected in 25%-30% of patients with essential thrombocythemia (ET) or primary myelofibrosis (PMF) and can lead to frameshifts that produce proteins with a novel C-terminal. In addition, the CALR mutation plays a crucial role in the MPN pathogenesis. The second major revision comprises the change in RBC parameters for polycythemia diagnosis; additionally, it emphasizes BM biopsy for the diagnosis of PV. Previously, PV was often underdiagnosed when considering the Hb levels of >18.5 g/dl for males and >16.5 g/dl for females; thus, the 2016 revision lowered these levels to >16.5 g/dl for males and >16.0 g/l for females. The third major revision is the introduction of a novel entity "prefibrotic/early" PMF (prePMF) to PMF. Although megakaryocytic proliferation and atypia were observed in in BM biopsy specimens of prePMF, these were not accompanied by reticulin fibrosis>> grade 1. Thus, the inferior prognosis of prePMF was reported in comparison with "true" ET.

Background & aims: Myeloproliferative neoplasms (MPN) are the most frequent cause of non-tumoral non-cirrhotic splanchnic vein thrombosis (NC-SVT). Diagnosis of MPN is based on blood cell count alterations, bone marrow histology and detection of specific gene mutations. Next generation sequencing (NGS) allows the simultaneous evaluation of multiple genes implicated in myeloid clonal pathology. The aim of this study was to evaluate the potential role of NGS in the etiology of NC-SVT.

Methods: DNA samples from 80 patients, (75 with idiopathic or exclusively local factor (Idiop/loc-NC-SVT) and 5 with MPN and NC-SVT (SVT-MPN) negative for JAK2 (V617F and exon 12), CALR and MPL mutations by classic techniques) were analyzed by NGS. Mutations involved in myeloid disorders different from JAK2, CALR and MPL genes were categorized as High Molecular Risk (HMR)-variants or Variants of Unknown Significance (VUS).

Results: In 2/5 triple-negative SVT-MPN cases (40%) a mutation in exon 12 of JAK2 was identified. JAK2-exon 12 mutation was also identified in 1/75 patients with idiop/loc-NC-SVT. Moreover, 28/74 (37.8%) of the remaining idiop/loc-NC-SVT had at least one HMR variant. Sixty-two patients with idiop/loc-NC-SVT were not receiving long-term anticoagulation and five of them (8.1%) had recurrent NC-SVT. This cumulative incidence was significantly higher in patients with HMR-variants than in those without.

Conclusions: NGS identified JAK2-exon12 mutations not previously detected by conventional techniques. In addition, NGS detected HMR-variants in approximately one third of patients with idiop/loc-NC-SVT. These patients seem to have a higher risk of splanchnic rethrombosis. NGS might be a useful diagnostic tool in NC-SVT.

Keywords: Budd-chiari syndrome; NGS (next generation sequencing); myeloproliferative neoplasms; non-cirrhotic splanchnic vein thrombosis; portal vein thrombosis.