JAK2-negative Myeloproliferative Neoplasms Research Articles

Different CALR mutation subtypes in essential thrombocythemia and primary myelofibrosis patients without JAK2 mutation

Discovery of calreticulin (CALR) mutations are considered essential in understanding the molecular basis of myeloproliferative neoplasms (MPNs). These mutations have been demonstrated to be the second driver mutation in MPNs especially essential thrombocythemia (ET) and primary myelofibrosis (PMF). The diagnostic and prognostic significance of these mutations, in addition to the geographical and ethnic heterogeneity, were previously reported. Molecular profiling also guides the selection of therapy for patients with MPNs. Therefore, we investigated the frequency and clinical and demographic characteristics of CALR mutations in Egyptian patients with MPNs. We investigated CALR mutations in 84 Egyptian patients with MPNs using florescent polymerase chain reaction followed by fragment analysis. Then, we analyzed the relationship between the mutations and patient clinical data. The frequency of CALR mutations was 13% (type 2 mutations, 55%; mix or complex mutations, 27%; type 1 mutations, 18%). CALR mutations were most frequent in ET (21.4%), followed by PMF (15.4%) and were not found in polycythemia vera (PV). Clinically, patients with mutated CALR had significantly lower levels of leukocytes (P = 0.042) and higher platelet count (P = 0.04) compared to those with nonmutated CALR and lower risk of thrombosis although a statistical difference was not detected. CALR mutations were found frequently in Egyptian MPNs patients with ET and PMF especially in those without JAK2 mutation. Patients with type 2 and complex mutations were more frequent in our cohort than the previously known published data. Thus, CALR is a potentially valuable marker for stratification of JAK2-negative MPN patients.

Bone Marrow Morphology in Myeloproliferative Neoplasms – Relevance in the Molecular Era

Background: Myeloproliferative neoplasms (MPNs) are clonal hematopoietic disorders. The mutation BCR-ABL is sine qua non for diagnosis of chronic myeloid leukemia and JAK2 V617F is present in majority of cases of polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (MF). Recent 2016 classification consigns importance to BM morphology as mutations are not universal. Identification of specific features would aid diagnosis of JAK2V617F negative MPN. This study aims to assess utility of peripheral blood and bone marrow morphology in myeloproliferative neoplasms in correlation with BCR-ABL and JAK2V617F. Methods: A retrospective 7 years study of peripheral blood, bone marrow aspirate (BMA) and bone marrow biopsy (BMB) of 44 cases of myeloproliferative neoplasms with BCR-ABL and JAK2 V617F mutation analysis. Results: Forty four cases of MPNs including 24 (54.5%) cases of CML, 7 (15.9% ) cases of PV, 5 (11.3%) cases of MF, 3 (6.8%) cases of ET, and 5 (11.3%) cases of MPN-U. MPV and LDH were significantly higher in MF in comparison to PV and ET. Higher haemoglobin (Hb) and platelets were seen in PV and ET. Dwarf MKs with hypolobated, cloud like nuclei in CML (fig 1), large MKs with staghorn nuclei were seen in ET. normal sized MKs with cloud like hypolobated forms in PV(fig2) and cloud like atypical dysmorphic nuclei with dense clustering were seen in MF. Conclusion: Hb, mean platelet volume (MPV), and LDH in conjunction with MK morphology aids in the diagnosis of MPNs. These findings will enable accurate diagnosis in JAK2 negative MPNs and benefit low resource center.

Clinicopathological differences exist between CALR- and JAK2-mutated myeloproliferative neoplasms despite a similar molecular landscape: data from targeted next-generation sequencing in the diagnostic laboratory.

Mutations in CALR have recently been detected in JAK2-negative myeloproliferative neoplasms (MPNs) and are key pathological drivers in these diseases. CALR-mutated MPNs are shown to have numerous clinicopathological differences to JAK2-mutated MPNs. The basis of these differences is poorly understood. It is unknown whether these differences result directly from any differences in intracellular signalling abnormalities induced by JAK2/CALR mutations or whether they relate to other phenomena such as a differing spectrum of genetic lesions between the two groups. We aimed to review the clinicopathological and molecular features of CALR- and JAK2-mutated MPNs from samples referred for diagnostic testing using a custom-designed targeted next-generation sequencing (NGS) panel. Eighty-nine CALR-mutated cases were compared with 70 JAK2-mutated cases. CALR-mutated MPNs showed higher platelet counts and a female predominance as compared to JAK2-mutated MPNs in our cohort. We have also observed differences between CALR mutation subtypes in terms of disease phenotype, mutational frequency and allelic burden. Type 1 CALR mutations were found to be more common in myelofibrosis, associated with a higher frequency and number of additional mutations and a higher mutant allelic burden as compared to type 2 CALR mutations. Despite these biological differences, our molecular characterisation suggests that CALR- and JAK2-mutated MPNs are broadly similar in terms of the quantity, frequency and spectrum of co-occurring mutations and therefore observed biological differences are likely to not be heavily influenced by the nature and quantity of co-mutated genes.

Cosmic Mutational Analysis in Suspected Myeloproliferative Neoplasms Using Next Generation Sequencing with a Fifty Gene Panel

Abstract Introduction: As per the 2008 World Health Organization (WHO) classification, Myeloproliferative neoplasms (MPN) are subclassified into eight clinicopathological groups.1 The discovery of activating JAK2 mutations revolutionized the approach to diagnosis of MPN. Recently there have been studies suggesting an increasing number of mutations distinct from JAK2 associated with MPN. The new mutations being studied are MPL with a mutation frequency of 1-5% commonly seen in Essential Thrombocytosis (ET) and Primary Myelofibrosis (PMF).2 IDH1 has a mutational frequency of 21% for blast phase of MPN and 4% for PMF.3 Other mutations that have been found to be coexistent with MPNs are EZH2, TP53 and TET2.4 Methods: A total of 79 cases with clinical suspicion of MPN were studied over one year. Based on WHO criteria, a total of 20 cases were diagnosed as MPN taking into account complete blood counts, bone marrow and cytogenetic studies with molecular profiling using next generation sequencing (NGS). Out of these, 13 cases were diagnosed as JAK2 positive and 7 as JAK2 negative MPN. All cases were studied for a panel of 50 mutations (Table 1) using NGS (Ion Torrent PGM) and a minimum coverage of 100x was considered to be significant. Table 1. Mutation Panel ABL1 EGFR GNAQ KRAS PTPN11 AKT1 ERBB2 GNAS MET RB11 ALK ERBB4 HNF1A MLH1 RET APC EZH2 HRAS MPL SMAD4 ATM FBXW7 IDH1 NOTCH1 SMARCB1 BRAF FGFR1 IDH2 NPM1 SMO CDH1 FGFR2 JAK2 NRAS SRC CDKN2A FGFR3 JAK3 PDGFRA STK11 CSF1R FLT3 KDR PIK3CA TP53 CTNNB1 GNA11 KIT PTEN VHL Results: Out of 20 MPN cases there were 3 cases of Polycythemia Vera (PV) 8 cases of ET and 9 cases of PMF. From the 14 mutations found in MPN, JAK 2 (65%) was the commonest followed by HRAS (45%), PDGFRA (45%), SMARCB1 (45%), Kit (40%), MET (25%), TP53 (20%), PIK3CA(20%), IDH1 (15%), STK11 (10%), APC (5%), FLT3(5%),PTEN (5%) and PTPN11(5%) (Graph1). Apart from JAK2, statistically significant mutations in the MPN group as compared to the non MPN group were Kit, TP53 and STK11 (Graph 2). Kit showed Single Nucleotide Polymorphism (SNP) with substitution of Adenosine (A) with Cytosine(C) on chr4:55593464 (hg19) and was statistically significant in MPN group as compared to non MPN group (40 % vs16.9 %, p value = 0.016). TP53 mutation showed a C to A SNP on chr17:7577036 (hg19) which was found more often in MPN groups than non MPN (20 % vs. 1.6 %, p value = 0.0018). STK11 mutation showed a C to Guanine (G) SNP on chr19:1223125 and a G insertion at chr19:1221320 and was found to be more associated with MPN than non MPN (10% vs. 1.6%, p value = 0.046) . However all other mutations were statistically insignificant. Graph 1: Total Mutations in MPN versus Non MPN Graph 2: Significant mutations in MPN *p-value: 0.016, **p-value: 0.046, ***p-value: 0.0018 In the MPN group, 13 cases were JAK2 positive and 7 cases were JAK2 negative. There was no statistical significance of presence of mutations between the two groups. In our study there was no significant association of IDH1 in MPN group in comparison to non MPN group ( 15% vs. 11.86%, p value = 0.35), in contrast to earlier studies.3 Conclusion: The mutations in Kit, TP53 and STK11 were found to be significantly more in cases of MPN as compared to non MPN. In future studies, different mutations present in MPN should be identified using NGS which will be crucial to not only diagnose and further characterize MPN cases but also for better understanding of the stepwise pathogenesis leading to cancer development in humans and to develop new targeted therapies. This is the first study of its kind in Indian population, to the best of our knowledge.

Clinical validation of a multipurpose assay for detection and genotyping of CALR mutations in myeloproliferative neoplasms.

To develop a polymerase chain reaction (PCR)-based approach to detect CALR mutations in myeloproliferative neoplasms (MPNs) in a clinical laboratory. DNA was extracted from bone marrow aspirate samples of 67 JAK2 wild-type MPNs (22 with matched peripheral blood), 54 cases of unclassifiable myelodysplastic syndrome/MPN, and 16 cases of atypical chronic myeloid leukemia. We used genomic DNA to detect somatic mutations in exon 9 of CALR and PCR with fluorescently labeled and M13-tagged primers and subjected the products to capillary electrophoresis (CE) followed by Sanger sequencing. Detailed assay performance characteristics were established. We identified CALR mutations in 19 (28.4%) of 67 JAK2-negative MPNs, including 14 type I (52-base pair [bp] deletion), four type II (5-bp insertions), and one type III (18-bp deletion). All mutations were confirmed by Sanger sequencing. Sensitivity studies showed 2.5% and 5% mutation detection levels by CE and Sanger sequencing, respectively, with high reproducibility. This assay allows for rapid, convenient screening for CALR mutations in MPNs, thereby reducing the number of cases that require assessment by Sanger sequencing, reducing labor and improving turnaround time.

CALR, JAK2, and MPL mutation profiles in patients with four different subtypes of myeloproliferative neoplasms: primary myelofibrosis, essential thrombocythemia, polycythemia vera, and myeloproliferative neoplasm, unclassifiable.

We investigated mutation profiles of CALR, JAK2, and MPL in 199 Korean patients with myeloproliferative neoplasms (MPNs). In total, 199 patients with MPN (54 primary myelofibrosis [PMF], 79 essential thrombocythemia [ET], 58 polycythemia vera [PV], and eight MPN-unclassifiable [MPN-U]) and 4 patients with acute panmyelosis with myelofibrosis (APMF) were retrospectively subjected to Sanger sequencing for CALR, JAK2, and MPL. The overall frequency of CALR mutations was 12.6% (type 1 mutation, 16 patients; type 2 mutation, nine patients): most frequent in MPN-U (37.5%), followed by ET (17.7%) and PMF (14.8%). CALR mutations were not found in PV or APMF. CALR and JAK2 or MPL mutations were mutually exclusive. In PMF, the CALR mutations were associated with lower levels of leukocytes, lower bone marrow cellularity, and higher number of megakaryocytes. Patients with CALR-mutated ET more frequently progressed to the accelerated or blast phases compared with patients with JAK2 mutations. CALR mutations were frequently observed in the JAK2-negative MPNs, most frequently in MPN-U. The prognostic significance of CALR mutations likely differs among the MPN subtypes.

Calreticulin Mutations in JAK2-Negative Myeloproliferative Neoplasms. Experience in Our Center

Abstract Introduction: Calreticulin (CALR) is a multifunctional protein regulated by calcium that is located in the endoplasmic reticulum. Recently, mutations in the calreticulin gene have been described in patients with the diagnosis of essential thrombocytemia (ET) and primary myelofibrosis (PMF), mainly in JAK2-negative cases. CALR mutations are localized to exon 9 and generate deletions or insertions that lead to a frameshift change resulting in a mutant protein. The detection of these mutations helps in the actual diagnosis of JAK2 mieloproliferative syndromes (MPN). Our aim is to assess the utility of the determination of these mutations in the management of patients with diagnosis of MPN in our center. Patients and methods: This study includes 94 patients with diagnosis of JAK2-negative MPN retrospectively selected following clinical and analytical criteria between 2008 and 2014 in our center (Table 1, 2). CALR mutations were performed with the use of fluorescent PCR following the methods described by Klampf et al. (NEJM, 2013). Results: 94 patients were analyzed, 77 of them had the diagnosis of TE, 8 of PMF and 9 of others disorders of myelodisplastic/mieloproliferative. 22% of the cases of ET had mutations in CALR (Table 1). In these mutations, a total of 53% were type I mutations (52-bp deletion) and 47% were type II mutations (5-bp insertion). Only one mutation was infrequent, a 46-pb deletion. We have found statistical correlation in the number of platelets depending on the presence of the mutation and in the largest number of platelets in type II mutations. 33% of the cases of PMF had mutations in CALR, all of them type I. Among other diseases not included in MPN, one of them had a type I mutation (data not shown). Conclusions: Our results are close to recently published results regarding the frequency of mutation and as the largest number of platelets in type II mutations with respect to mutation type I. This study confirms the importance of CALR mutations determination in the diagnosis of JAK2-negative ET and PMF with high clinical suspicion. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Calr Gene Mutation in Patients with JAK2-Negative Myeloproliferative Neoplasms

Abstract Methods Genomic DNA from bone marrow and peripheral blood cells were extracted from 40 patients with MPD(male 26,female 14),Aged 13 to 76 years old, JAK2V617F mutation and CALR genetic mutations was identified by PCR- direct sequencing. Results The number of MPD(PV15,ET25) 40 cases in patients with JAK2V617F mutation rate was 60%,among them the polycythemia vera ( PV) positive rate was 66. 7% ( 10 /15) ,and essential thrombocythemia ( ET ) positive rate was 56% ( 14 /25) . The number of JAK2V617F -negative MPD 16 cases in patients with CALR mutation rate was 12. 5% ( 2 /16),Two of the 11 patients with ET were CALR mutation positive(18.2%). Two novel mutation in CALR exon 9,c.1099_1150del ( p. chr19F12915572-12915623del) was detected in patient Tao c.1099-1151delinsT( p.chr19:12915572_12915624delinsT) was detected in patient Xu. These mutation was absent in the controls,Two novel mutation in CALR have not been reported so far. Conclusion CALR gene mutations testing helps to JAK2V617F negative MPD diagnosis, makes the MPD early detection and treatment. Disclosures No relevant conflicts of interest to declare.

Frequency and Molecular Characteristics of Calreticulin Gene (CALR) Mutations in Patients with JAK2-Negative Myeloproliferative Neoplasms

In 2013, Nangalia et al. and Klampfl et al. found a recurrent and abundant mutation in the calreticulin gene (CALR), mutually exclusive with JAK2 and MPL alterations. At present, the data concerning the new mutation, i.e. its prevalence, allele burden and clinical significance, are scarce. We report the incidence and molecular characteristics of CALR mutations in a group of 184 Polish patients with myeloproliferative neoplasms (MPNs). Clinical data analysis revealed significant differences between JAK2 V617F-mutated and CALR-mutated groups. In essential thrombocythemia patients, hemoglobin levels and leukocyte counts were significantly higher in JAK2-positive than in CALR-positive patients (p = 0.023 and p = 0.017, respectively), but the CALR-positive patients had significantly higher platelet counts (p = 0.022). Patients harboring CALR mutations were also younger at the time of diagnosis (p = 0.039). In primary myelofibrosis patients, the degree of anemia was less severe in those who were CALR exon 9 mutation-positive than in those who were JAK2 V617F-positive (p = 0.048). © 2014 S. Karger AG, Basel

Integrated genomic analysis illustrates the central role of JAK-STAT pathway activation in myeloproliferative neoplasm pathogenesis

Genomic studies have identified somatic alterations in the majority of myeloproliferative neoplasms (MPN) patients, including JAK2 mutations in the majority of MPN patients and CALR mutations in JAK2-negative MPN patients. However, the role of JAK-STAT pathway activation in different MPNs, and in patients without JAK2 mutations, has not been definitively delineated. We used expression profiling, single nucleotide polymorphism arrays, and mutational profiling to investigate a well-characterized cohort of MPN patients. MPN patients with homozygous JAK2V617F mutations were characterized by a distinctive transcriptional profile. Notably, a transcriptional signature consistent with activated JAK2 signaling is seen in all MPN patients regardless of clinical phenotype or mutational status. In addition, the activated JAK2 signature was present in patients with somatic CALR mutations. Conversely, we identified a gene expression signature of CALR mutations; this signature was significantly enriched in JAK2-mutant MPN patients consistent with a shared mechanism of transformation by JAK2 and CALR mutations. We also identified a transcriptional signature of TET2 mutations in MPN patent samples. Our data indicate that MPN patients, regardless of diagnosis or JAK2 mutational status, are characterized by a distinct gene expression signature with upregulation of JAK-STAT target genes, demonstrating the central importance of the JAK-STAT pathway in MPN pathogenesis.

FLT3 Mutations in Myeloproliferative Neoplasms

FLT3 is one of the most frequently mutated genes in acute myeloid leukemia. Previous studies have reported FLT3 mutation in as many as 9.2% of myeloproliferative neoplasms (MPNs) and myelodysplastic/myeloproliferative neoplasms (MDS/MPNs), as well as in chronic myelogenous leukemia, that are negative for the JAK2 V617F gene mutation; no FLT3 mutation has been found in JAK2-positive MPNs, suggesting that the mutations are mutually exclusive. The goal of our study is to evaluate the mutational status of the FLT3 gene in patients with an MPN or MDS/MPN, in correlation with the JAK2 mutational status. Patient specimens were retrospectively identified on the basis of MPN or MDS/MPN diagnosis and JAK2 analysis from February 2006 to December 2011. FLT3 mutation analysis was performed on DNA extracted from 152 patients using polymerase chain reaction amplification and analysis of amplicons by gel electrophoresis for internal tandem duplication mutations and by restriction endonuclease digestion fragment analysis for the D835 point mutation. FLT3 mutation analysis was performed on 90 cases of JAK2-negative MPN or MDS/MPN and 62 cases of JAK2-positive MPN. One FLT3 internal tandem duplication mutation was identified in the JAK2-negative group (1.1%), and none were identified in the JAK2-positive group, confirming the absence of FLT3 mutations in JAK2-positive specimens. The FLT3-positive MPN patient was diagnosed with MPN, unclassifiable, and was later found to have myeloid sarcoma; thus, FLT3 mutation was not seen in the usual types of MPN in our series. Our result of 1.1% FLT3 mutations in JAK2-negative MPN and MDS/MPN cases is lower than the 9.2% previously reported.

The spectrum of JAK2-positive myeloproliferative neoplasms.

The discovery of the JAK2V617F mutation triggered an unexpected flowering of basic and clinical studies in the field of myeloproliferative neoplasms (MPNs), resulting after just a few years in an exceptional amount of new information. One important consequence of those new findings was the modification of the World Health Organization classification and diagnostic algorithms for these diseases, which is still based on the original concept developed by William Dameshek in 1951 and keeps distinct entities under the umbrella of classical Philadelphia-negative MPNs. These MPNs are essential thrombocythemia, polycythemia vera, and primary myelofibrosis. Could a new molecular classification be a better tool to manage MPN patients? Several studies have shown that essential thrombocythemia and primary myelofibrosis can be divided into distinct subtypes based on the presence of the JAK2V617F mutation. Can we now define JAK2-positive diseases to depict a distinct entity from JAK2-negative MPNs? This chapter reviews the significance of JAK2 mutation positivity in the diagnosis, prognosis, and therapy of MPNs.

The spectrum of JAK2-positive myeloproliferative neoplasms

Abstract The discovery of the JAK2V617F mutation triggered an unexpected flowering of basic and clinical studies in the field of myeloproliferative neoplasms (MPNs), resulting after just a few years in an exceptional amount of new information. One important consequence of those new findings was the modification of the World Health Organization classification and diagnostic algorithms for these diseases, which is still based on the original concept developed by William Dameshek in 1951 and keeps distinct entities under the umbrella of classical Philadelphia-negative MPNs. These MPNs are essential thrombocythemia, polycythemia vera, and primary myelofibrosis. Could a new molecular classification be a better tool to manage MPN patients? Several studies have shown that essential thrombocythemia and primary myelofibrosis can be divided into distinct subtypes based on the presence of the JAK2V617F mutation. Can we now define JAK2-positive diseases to depict a distinct entity from JAK2-negative MPNs? This chapter reviews the significance of JAK2 mutation positivity in the diagnosis, prognosis, and therapy of MPNs.