CALR Exon Research Articles

High-resolution melting assay for rapid, simultaneous detection of JAK2, MPL and CALR variants

AimsIdentification of recurrent genetic alterations in JAK2, MPL and CALR remains crucial in the diagnosis of Philadelphia-negative myeloproliferative neoplasms (MPNs). Current laboratory testing algorithms may entail batching and/or sequential testing,...

Germline MPL Mutations May be a Rare Cause of “Triple-Negative” Thrombocytosis

Background Rare cases of primary thrombocytosis are related to inherited genetic abnormalities and are called hereditary thrombocytosis (HT). HT has clinical features resembling sporadic essential thrombocythemia (ET) but has usually an autosomal dominant (AD) inheritance with variable penetrance. To date, germline mutation involving the MPL, THPO, JAK2 genes have been associated with HT. Methods The study included 933 patients evaluated at our Division because of persistent isolated thrombocytosis for whom secondary reactive causes were excluded. Patients were screened for the three canonical somatic driver mutations associated with myeloproliferative neoplasms: JAK2 V617F mutation, CALR exon 9 mutations, MPL exon 10 mutations. Triple-negative cases with unexplained isolated thrombocytosis which also resulted negative for non-canonical exon 10 MPL mutations were analyzed using next generation sequencing (NGS) to test a panel of 80 myeloid genes. Results Of 933 patients with isolated thrombocytosis screened, 567 were JAK2-mutated, 255 CALR-mutated, 41 MPL-mutated, 2 double-mutated and 68 triple-negative. Two MPL-mutated patients carried a non-canonical mutation located in exon 10: MPL W515* and MPL V501A. Of the 68 triple-negative patients, 32 were evaluated by NGS and one of them showed another non-canonical mutation of MPL located outside exon 10: MPL R102P. These 3 non-canonical MPL mutations resulted to be germline as they were detected also in DNA extracted from T lymphocytes and from hair roots. Due to the germline nature of the mutations, a study of the family was carried out. In 2 out of 3 pedigrees at least a second relative carried the same germline mutation discovered in the proband; these 2 pedigrees are shown in the figure. Patient MPC17_15 was an 80 years-old patient with a history of thrombocytosis. She resulted JAK2- and CALR- wild-type while HRM screening was positive for exon 10 MPL mutations; Sanger sequencing identified the MPL W515* mutation in DNA from granulocytes, T-lymphocytes and hair roots, confirming its germline nature. Her children showed a normal complete blood count (CBC) and were MPL wild-type, thus confirming a segregation between mutation and clinical phenotype. Patient MPC14_674 (panel A) was a 36 years-old women with a history of mild thrombocytosis never investigated before. She was referred to our clinic by Pediatricians after the delivery because her baby (MPC17_167) showed persistent isolated thrombocytosis (826 x10 9/L) during the first months of life despite the exclusion of the most common reactive causes of thrombocytosis. Sanger sequencing identified the MPL V501A mutation in granulocyte and in T-lymphocyte in both cases. To evaluate the segregation of MPL V501A with the disease phenotype, we evaluated the CBC and MPL mutation in the other relatives. Both proband's father (MPC23_597) and sister (MPC23_602) carried germline heterozygous MPL V501A mutation associated with a PLT count above or at the upper limit of normal range (570 x10 9/L and 355 x10 9/L, respectively). Conversely, the husband (MPC17_495), the second son (MPC18_43) and the mother (MPC23_603) of the proband showed a normal CBC and were MPL wild-type, confirming an AD inheritance. Patient MPC07_130 (panel B) was a 44 years-old women with triple-negative isolated thrombocytosis lasting since 2007. NGS analysis showed the presence of a heterozygous germline MPL R102P mutation. As this mutation is located in exon 3, it was not detected through HRM and Sanger sequencing which target only exon 10. Her son (MPC13_672), 18 years-old, was completely asymptomatic but the CBC showed mild thrombocytosis (472 x 10 9/L). He tested negative for the 3 driver mutations but Sanger sequencing of MPL exon 3 identified the heterozygous germline MPL R102P mutation. Conclusions We identified 3 non-canonical germline MPL mutations in 3 different unrelated subjects with isolated thrombocytosis: MPL W515*, MPL R102P, and MPL V501A. Germline MPL mutations may underly HT and should be evaluated by sequencing of the whole exon 10 of MPL gene or by NGS in cases with isolated thrombocytosis negative for the three canonical somatic driver mutations. Namely, patients with HT might present as sporadic cases, and in the initial workup they might be inappropriately diagnosed with triple-negative ET. A correct diagnosis is pivotal to avoid unnecessary treatments.

Pitfalls of using polymerase chain reaction-based assays for JAK2 and CALR exon 9 variant testing in myeloproliferative neoplasms: Knowing when to go the extra mile!

The BCR::ABL1 negative myeloproliferative neoplasms are sequentially tested for JAK2 p.V617F, followed by CALR exon 9 pathogenic variants. Historically, these variants were thought to be mutually exclusive. However, recent reports indicate coexisting JAK2 p.V617F and CALR exon 9 somatic variants. Analysis of JAK2 p.V617F and CALR exon 9 variant was performed by polymerase chain reaction (PCR)-based assays. Subsequent testing was performed on the Genexus integrated sequencer (ThermoFisher) using the Oncomine myeloid assay GX v2. CALR exon 9 variants were positive in 3 cases, while 2 were positive for JAK2 p.V617F on PCR-based assays. Next-generation sequencing confirmed the JAK2 P.V617F status in all cases. CALR variants resulting in in-frame deletions were identified in 2 cases at a variant allele frequency of 52.16% and 50.91%, while the third case had an intronic CALR variant c.-48G>A at a variant allele frequency of 51.1%. Thus, CALR variants in all 3 cases were interpreted as potentially germline. Of the 228 cases that underwent JAK2 p.V617F and CALR cotesting in the past 2 years, only these 2 cases were positive for both JAK2 p.V617F and CALR exon 9 variants. These cases highlight the importance of understanding the pitfalls of molecular techniques in current practice.

Mutation-Driven S100A8 Overexpression Confers Aberrant Phenotypes in Type 1 CALR-Mutated MPN.

Numerous pathogenic CALR exon 9 mutations have been identified in myeloproliferative neoplasms (MPN), with type 1 (52bp deletion; CALRDEL) and type 2 (5bp insertion; CALRINS) being the most prevalent. Despite the universal pathobiology of MPN driven by various CALR mutants, it is unclear why different CALR mutations result in diverse clinical phenotypes. Through RNA sequencing followed by validation at the protein and mRNA levels, we found that S100A8 was specifically enriched in CALRDEL but not in CALRINS MPN-model cells. The expression of S100a8 could be regulated by STAT3 based on luciferase reporter assay complemented with inhibitor treatment. Pyrosequencing demonstrated relative hypomethylation in two CpG sites within the potential pSTAT3-targeting S100a8 promoter region in CALRDEL cells as compared to CALRINS cells, suggesting that distinct epigenetic alteration could factor into the divergent S100A8 levels in these cells. The functional analysis confirmed that S100A8 non-redundantly contributed to accelerated cellular proliferation and reduced apoptosis in CALRDEL cells. Clinical validation showed significantly enhanced S100A8 expression in CALRDEL-mutated MPN patients compared to CALRINS-mutated cases, and thrombocytosis was less prominent in those with S100A8 upregulation. This study provides indispensable insights into how different CALR mutations discrepantly drive the expression of specific genes that contributes to unique phenotypes in MPN.

Comparison of Clinical and Molecular Features Between Patients With Essential Thrombocythemia and Early/Prefibrotic Primary Myelofibrosis Presenting With Thrombocytosis in Taiwan.

The clinical presentations of essential thrombocythemia (ET) may be quite similar to early/prefibrotic primary myelofibrosis (pre-PMF), especially in pre-PMF presenting with thrombocytosis (pre-PMF-T), but may be associated with a different outcome. It is very important to distinguish these two entities. The aim of this study was to address the clinical and prognostic relevance of distinguishing pre-PMF-T from ET. All patients, including 258 with ET and 105 with pre-PMF-T, received JAK2V617F, MPL (exon 10), and CALR (exon 9) mutation analysis and allele burden measurement for JAK2V617F and CALR mutants. Patients with pre-PMF-T had an older age and higher leukocyte and platelet counts but lower hemoglobin levels than patients with ET. Patients with pre-PMF-T had a shorter overall, leukemia-free, and thrombosis-free survival compared with patients with ET. Patients with ET had a higher rate of cerebral ischemic stroke, whereas patients with pre-PMF-T tended to have splanchnic vein thrombosis. The frequencies of JAK2V617F, CALR, and MPL mutations and CALR allele burden were no different, but JAK2V617F allele burden was significantly higher in pre-PMF-T. Patients with pre-PMF-T with the JAK2V617F mutation had an inferior overall survival and thrombosis-free survival, whereas the status of driver gene mutations did not influence the outcomes of patients with ET. ET and pre-PMF-T were two distinct disease entities and exhibited different clinical phenotype, genotype, and outcomes.

Clinical and molecular characteristics of forty Chinese children with essential thrombocythemia: A single-center, retrospective analysis.

Due to the infrequency of essential thrombocythemia (ET) in children, little is known about its pathophysiological mechanism. To learn about the clinical and molecular features of Chinese children with ET, we retrospectively analysed 40 children with ET in a single center from 2015-2021. More than half of the children (51.3%, 20/39) were asymptomatic at diagnosis. Nearly half of the children (48.7%, 19/39) had microvascular symptoms, including headache, dizziness, stomachache, and paresthesia. Only two cases experienced vascular events. The proportion of children with typical "driver gene mutations" (i.e., JAK2 p.V617F, CALR exon 9, or MPL exon 10 mutation) was low (12.5%, 5/40). The equivalent ratio of children carried atypical driver gene mutations; however, 30 (75%) patients did not harbour driver gene mutations. Children carrying JAK2 p.V617F had lower platelet count (938 × 109 /L vs. 1654 × 109 /L, p=0.031) compared to those without driver gene mutations. Cases harbouring typical driver mutations had higher median WBC counts than those without driver gene mutations (15.14 × 109 /L vs. 8.01 × 109 /L, p=0.015). Compared to those without driver gene mutations, cases carrying typical and atypical driver gene mutations were both younger (median ages were 12, 6, and 7 years old, respectively; p=0.023). The most prevalent non-driver gene mutations and those mutations with prognostic significance in adult counterparts were less common in children with ET compared to adults with ET.

Clinical Features and Outcomes of Pre-Fibrotic Primary Myelofibrosis Patients with Triple-Negative, JAK2V617F and Calr Mutations

The 2016 WHO classification recognized pre-fibrotic primary myelofibrosis (pre-PMF) as a distinct entity. However, little is known about the clinical characteristics and prognosis of pre-PMF patients with JAK2V617F mutations, CALR exon 9 mutations, and triple-negative (TN) mutations. Thrombotic prognostic model specific for pre-PMF is still lacking and the applicability of International Prognostic Score for Thrombosis in Essential Thrombocythemia (IPSET-thrombosis) needs further validation in pre-PMF patients. We conducted a retrospective study of 168 pre-PMF patients defined by 2016 WHO diagnostic criteria in the First Affiliated Hospital of Zhejiang University School of Medicine and the Fourth Affiliated Hospital of Zhejiang University School of Medicine (ChiCTR2200061208). We retrospectively evaluated the clinical characteristics, prognosis of hemorrhage and thrombosis, and the evolution risk to overt-PMF and acute leukemia in 168 pre-PMF patients. We also assessed the risk factors for thrombosis events and validated the availability of IPSET-thrombosis scoring system in pre-PMF patients. Among 168 pre-PMF patients, 98 (58.3%) harbored JAK2V617F, 22 (13.1%) CALR and 2 (1.2%) MPL mutations; 46 (27.4%) patients were negative for all three mutations. Compared to JAK2V617F-mutated cases, TN pre-PMF patients were younger (P<0.001), had fewer constitutional symptoms (P=0.003), lower white blood cell count (WBC) (P=0.01), hemoglobin level (HB) (P=0.007), mean platelet volume (MPV) (P=0.03), hematocrit value (HCT) (P=0.045), red blood cell distribution width (RDW) (P<0.001) and lactic dehydrogenase (LDH) (P=0.021). They also had fewer thrombotic events (1, 2.2%) compared to JAK2V617F group (25, 26.3%) and CALR group (5, 22.7%) during the follow-up and at diagnosis (P<0.001) (Figure A). A proportion of 7.1% and 18.2% progressed to overt-PMF in JAK2V617F and CALR group, respectively, while no TN pre-PMF patients developed overt-PMF progression (0, 0.0%) (P=0.015) (Figure B). We also found, although not statistically significant, a lower incidence of hemorrhagic events (2% vs. 12%) and acute leukemia (2% vs. 5%) in TN pre-PMF groups compared with JAK2V617F pre-PMF groups (Figure C, Figure D). As for risk factors for thrombosis, univariable analysis found thrombosis was associated with constitutional symptoms (P=0.003), thrombosis history (P<0.001), smoking status (P=0.007) and JAK2V617F mutations (P=0.008). Patients with higher HB (P=0.013), MPV (P=0.042) and HCT (P=0.040) were also more likely to develop thrombotic events. In multivariable analysis, smoking history (P=0.036; HR 0.902, 95% CI 0.05-0.212), higher HB value (P=0.048; HR 1.336, 95% CI 1.001-1.156) and thrombosis history (P=0.001; HR 0.313, 95% CI 0.009-0.053) had negative impact on the development of thrombosis events (Figure E). When divided by HB value with cut-off point of 144.5, subjects with HB value <144.5 g/L were less likely to develop thrombosis events compared to ≥144.5 g/L (P=0.003) (Figure F). And subjects with no history of smoking were less likely to develop thrombosis events compared to subjects with a history of smoking (P=0.004) (Figure G). We also validated IPSET-thrombosis scoring system in pre-PMF patients, results showed significant statistical difference among very low-, low-, intermediate- and high-risk groups (P<0.001) (Figure H). In conclusion, TN pre-PMF patients were younger, had fewer constitutional symptoms, lower WBC, HB, MPV, HCT, RDW and LDH, and less likely to develop thrombotic events and overt-PMF compared to JAK2V617F and CALR groups. TN pre-PMF patients with smoking history, higher HB and thrombosis history had higher risks of thrombosis events. And IPSET-thrombosis scoring system was applicable in stratifying thrombosis risks of pre-PMF patients. Acknowledgment: The research was supported by the Key R&D Program of Zhejiang, No. 2022C03137; Public Technology Application Research Program of Zhejiang, China, No. LGF21H080003; the Key Project of Jinhua Science and Technology Plan, China, No. 2020-3-011; the 2019-2022 Key Medical Discipline (Hematology) Fund of Jinhua, China. Correspondence to: Dr Jian Huang, Department of Hematology, The First Affiliated Hospital of Zhejiang University School of Medicine. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Emergence of Chronic Myeloid Leukemia in Patients with Myeloproliferative Neoplasms: A Single-Institution Experience

Introduction: Chronic myeloid leukemia (CML) is characterized by the presence of the BCR-ABL1 translocation and is typically distinct from other myeloproliferative neoplasms (MPN) including polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis (MF). Concurrent diagnosis of CML and MF is uncommon and infrequently described in case reports.1 Moreover, very few cases have been described of CML occurring after an established diagnosis of MPN.2-3 Due to the rarity of concurrent CML and MPN, management strategies in this population are not defined. In addition, the latency time from the emergence of BCR-ABL1 positive clones to a clinical diagnosis of CML in this setting is largely unknown. To address these questions, we interrogated our institutional experience and retrospectively analyzed available specimens. Methods: We collected information on patients seen at Massachusetts General Hospital (MGH) from January 1 2012 to January 1 2022 for CML occurring after an established diagnosis of MPN. Available stored samples were retrospectively tested for BCR-ABL1 with a fusion transcript assay (MGH Center for Integrated Diagnostics, Boston, MA). Results: We identified four patients with CML preceded by MPN (Table 1). Three patients had prior MF, diagnosed 4.3-7.6 years before the onset of CML; two of these had post-ET MF. Patient #4 had ET diagnosed 4 years prior to onset of CML, then went on to develop MF 4.9 years after CML diagnosis. None of the patients were on JAK inhibitors, hydroxyurea or interferon at the time of CML diagnosis. All patients had a leukocytosis with characteristic features on the differential prompting evaluation for CML. The median white blood cell count was 45.83 x 10^9/L (range 27.49-121.02). All four patients had an elevated basophil count (median 2.99 x 10^9/L, range 1.1-22.99) and two had an elevated eosinophil count (median 1.11 x 10^9/L, range 0.55-44.17). Regarding MPN driver mutations, one patient had a JAK2 V617F, one patient had a CALR exon 9 deletion, and two patients had no identifiable mutations on extended next-generation sequencing. For CML treatment, three patients received dasatinib (two with 100 mg daily, one with 50 mg daily), whereas one received nilotinib (150 mg twice daily). Two achieved a major molecular response (MMR) (at 7 and 8 months); one had an early molecular response at 6 months (last follow-up); one (with e13-a3 transcript) could not be quantified on a standard PCR assay but achieved a complete cytogenetic response by 4 months. At the time of last follow-up, two patients were in MMR on tyrosine kinase inhibitors (TKI), whereas two underwent allogeneic hematopoietic cell transplantation (alloHCT), primarily for progression of MF. We retrospectively interrogated available samples for evidence of the BCR-ABL1 translocation prior to a clinical diagnosis of CML. Patient #2 was followed at MGH for MF and thus had available samples. We identified BCR-ABL1 transcripts in the peripheral blood three months prior to his clinical diagnosis of CML (unable to be quantified due to e13-a3 transcript); there were no available samples from earlier in his disease course for testing (Figure 1). Discussion: CML can emerge in patients with MPN. Herein, we describe four cases of this phenomenon. Signs of CML evolution included marked leukocytosis, as well as basophilia and eosinophilia. Despite underlying MF, three patients tolerated full-dose TKI and achieved molecular responses early in their disease course. However, MPN progression remained a significant challenge, with two patients ultimately requiring an alloHCT. Of note, we found evidence of BCR-ABL1 in the peripheral blood three months prior to clinically apparent CML in one patient. Experiments using single cell techniques in patients with concurrent MPN and CML would provide insights into the clonal architecture and are in planning stages.

AML-213 Unusual Presentation of Acute Myeloid Leukemia (AML-M2): A Case Report

Acute myeloid leukemia (AML) is rarely presented with thrombocytosis and marked thrombocytosis with a platelet count over 1.0×1012/L is an extremely rare phenomenon. A case of de novo AML with unusual presentation by extreme thrombocytosis. A case report. Hematology Unit at the Oncology Center Mansoura University, Egypt. A 37-year-old male patient with a history of sleeve gastrectomy in 2020, presented with oral mucositis, recurrent abscess, fever, and bilateral axillary lymphadenopathy. Initial complete blood count (CBC) showed a Hb level of 6 g/dL, a platelet count of 1.685×109/L, and a white blood cell (WBC) count of 19×109/L. Diagnosis of de novo AML (FAB-M2 AML) was confirmed by bone marrow aspiration, biopsy, and immunophenotyping. Cytogenetic study showed negative t(8;21)/inv 16/ t(9;22). A molecular study showed positive FLT-3 mutation and negative BCP/ABL1, JAK2, V617F, and CALR exon 9 mutations excluding blast transformation on top of myeloproliferative neoplasm (MPN) and myelodysplastic syndrome (MDS). The patient received induction chemotherapy including a course of 7-day-cytarabine along with 3-day-anthracycline on September 20th, 2021 but the patient was refractory as BMA showed blast cells 75%. The patient started salvage HAM (high-dose cytosine arabinoside and mitoxantrone) + Sorafenib on November 6th, 2021. Again, he could not achieve a response and received FLAG (high-dose cytosine arabinoside, fludarabine, and granulocyte colony-stimulating factors) + Sorafenib on December 18th, 2021, after recovery from COVID-19 infection. To shed light on fact that myeloid neoplasms as MPN, MDS, and de novo AML can share overlapping features. On January 25th, 2022, the last CBC showed a Hb level of 8.4 g/dL, a platelet Count of 395×109/L, and a WBC Count of 2.41×109/L with a differential Neutrophil count of 0.24×109/L. The patient lost follow-up since then. Only a few AML cases have been reported with thrombocytosis. Detailed molecular studies are mandatory to confirm the diagnosis of de novo AML patients with unusual presentation. Careful follow-up of those cases could help in establishing management guidelines for better outcomes as those patients usually have a poor prognosis.

Targeted sequencing of candidate gene regions for myelofibrosis in dogs.

BackgroundMyelofibrosis often lacks an identifiable cause in dogs. In humans, most primary myelofibrosis cases develop secondary to driver mutations in JAK2, CALR, or MPL.ObjectivesTo determine the prevalence of variants in JAK2, CALR, or MPL candidate regions in dogs with myelofibrosis and in healthy dogs.AnimalsTwenty‐six dogs with myelofibrosis that underwent bone marrow biopsy between 2010 and 2018 and 25 control dogs matched for age, sex, and breed.MethodsCross‐sectional study. Amplicon sequencing of JAK2 exons 12 and 14, CALR exon 9, and MPL exon 10 was performed on formalin‐fixed, decalcified, paraffin‐embedded bone marrow (myelofibrosis) or peripheral blood (control) DNA. Somatic variants were categorized as likely‐benign or possibly‐pathogenic based on predicted impact on protein function. Within the myelofibrosis group, hematologic variables and survival were compared by variant status (none, likely‐benign only, and ≥1 possibly‐pathogenic). The effect of age on variant count was analyzed using linear regression.ResultsEighteen of 26 (69%) myelofibrosis cases had somatic variants, including 9 classified as possibly‐pathogenic. No somatic variants were detected in controls. Within the myelofibrosis group, hematologic variables and survival did not differ by variant status. The number of somatic variants per myelofibrosis case increased with age (estimate, 0.69; SE, 0.29; P = .03).Conclusions and Clinical ImportanceSomatic variants might initiate or perpetuate myelofibrosis in dogs. Our findings suggest the occurrence of clonal hematopoiesis in dogs, with increasing incidence with age, as observed in humans.

Patients With Myeloproliferative Neoplasms Harbor High Frequencies of CD8 T Cell-Platelet Aggregates Associated With T Cell Suppression.

Myeloproliferative neoplasms (MPN) are chronic cancers of the hematopoietic stem cells in the bone marrow, and patients often harbor elevated numbers of circulating platelets (PLT). We investigated the frequencies of circulating PLT-lymphocyte aggregates in MPN patients and the effect of PLT-binding on CD8 T cell function. The phenotype of these aggregates was evaluated in 50 MPN patients and 24 controls, using flow cytometry. In vitro studies compared the proliferation, cytokine release, and cytoxicity of PLT-bound and PLT-free CD8 T cells. Frequencies of PLT-CD8 T cell aggregates, were significantly elevated in MPN patients. Advanced disease stage and CALR mutation associated with the highest aggregate frequencies with a predominance of PLT-binding to antigen-experienced CD8 T cells. PLT-bound CD8 T cells showed reduction in proliferation and cytotoxic capacity. Our data suggest that CD8 T cell responses are jeopardized in MPN patients. JAK2 and CALR exon 9 mutations – the two predominant driver mutations in MPN – are targets for natural T cell responses in MPN patients. Moreover, MPN patients have more infections compared to background. Thus, PLT binding to antigen experienced CD8 T cells could play a role in the inadequacy of the immune system to control MPN disease progression and prevent recurrent infections.

Mutational landscape of blast phase myeloproliferative neoplasms (MPN-BP) and antecedent MPN.

Myeloproliferative neoplasms (MPN) have an inherent tendency to evolve to the blast phase (BP), characterized by ≥20% myeloblasts in the blood or bone marrow. MPN-BP portends a dismal prognosis and currently, effective treatment modalities are scarce, except for allogeneic hematopoietic stem cell transplantation (allo-HSCT) in selected patients, particularly those who achieve complete/partial remission. The mutational landscape of MPN-BP differs from de novo acute myeloid leukemia (AML) in several key aspects, such as significantly lower frequencies of FLT3 and DNMT3A mutations, and higher incidence of IDH1/2 and TP53 in MPN-BP. Herein, we comprehensively review the impact of the three signaling driver mutations (JAK2 V617F, CALR exon 9 indels, MPL W515K/L) that constitutively activate the JAK/STAT pathway, and of the other somatic non-driver mutations (epigenetic, mRNA splicing, transcriptional regulators, and mutations in signal transduction genes) that cooperatively or independently promote MPN progression and leukemic transformation. The MPN subtype, harboring two or more high-molecular risk (HMR) mutations (epigenetic regulators and mRNA splicing factors) and "triple-negative" PMF are among the critical factors that increase risk of leukemic transformation and shorten survival. Primary myelofibrosis (PMF) is the most aggressive MPN; and polycythemia vera (PV) and essential thrombocythemia (ET) are relatively indolent subtypes. In PV and ET, mutations in splicing factor genes are associated with progression to myelofibrosis (MF), and in ET, TP53 mutations predict risk for leukemic transformation. The advent of targeted next-generation sequencing and improved prognostic scoring systems for PMF inform decisions regarding allo-HSCT. The emergence of treatments targeting mutant enzymes (e.g., IDH1/2 inhibitors) or epigenetic pathways (BET and LSD1 inhibitors) along with new insights into the mechanisms of leukemogenesis will hopefully lead the way to superior management strategies and outcomes of MPN-BP patients.

Clinical and Laboratory Features of JAK2 V617F, CALR, and MPL Mutations in Malaysian Patients with Classical Myeloproliferative Neoplasm (MPN).

Mutations of JAK2V617F, CALR, and MPL genes confirm the diagnosis of myeloproliferative neoplasm (MPN). This study aims to determine the genetic profile of JAK2V617F, CALR exon 9 Type 1 (52 bp deletion) and Type 2 (5 bp insertion), and MPL W515 L/K genes among Malaysian patients and correlate these mutations with clinical and hematologic parameters in MPN. Mutations of JAK2V617F, CALR, and MPL were analyzed in 159 Malaysian patients using allele-specific polymerase chain reaction, including 76 polycythemia vera (PV), 41 essential thrombocythemia (ET), and 42 primary myelofibrosis (PMF) mutations, and the demographics of the patients were retrieved. The result showed that 73.6% JAK2V617F, 5.66% CALR, and 27.7% were triple-negative mutations. No MPL W515L/K mutation was detected. In ET and PMF, the predominance type was the CALR Type 1 mutation. In JAK2V617F mutant patients, serum LDH was significantly higher in PMF compared to PV and ET. PV has a higher risk of evolving to post PV myelofibrosis compared to ET. A thrombotic event at initial diagnosis of 40.9% was high compared to global incidence. Only one PMF patient had a CALR mutation that transformed to acute myeloid leukemia. JAK2V617F and CALR mutations play an important role in diagnostics. Hence, every patient suspected of having a myeloproliferative neoplasm should be screened for these mutations.

Sotatercept (ACE-011) Alone and in Combination with Ruxolitinib in Patients (pts) with Myeloproliferative Neoplasm (MPN)-Associated Myelofibrosis (MF) and Anemia

Sotatercept (ACE-011) Alone and in Combination with Ruxolitinib in Patients (pts) with Myeloproliferative Neoplasm (MPN)-Associated Myelofibrosis (MF) and Anemia

Application of heteroduplex analysis for CALR mutation screening detection in patients with Ph-myeloproliferative neoplasms

Background. In accordance with the World health organization clinical guidelines, the analysis of somatic mutations in the CALR gene, as well as mutations in the JAK2 and MPL genes, are included in the list of criteria for the Ph-myeloproliferative neoplasms diagnosis.More than 50 different mutation variants have been found in the CALR gene, among which the most frequent are a 52 bp deletion (c.1092_1143del), also called type 1, and a 5 bp insertion (c.1154_1155insTTGTC), also called type 2 (88 %).The remaining 12 % are other type less frequent indels or combinations thereof.It is most convenient to use sequencing methods to identify all possible variants of CALR mutations. It is also important to develop inexpensive screening test that can detect any mutations in the analyzed DNA fragment of CALR gene. This method can be heteroduplex analysis followed by electrophoresis on polyacrylamide gel (PAGE).The objective: to develop and demonstrate the feasibility of using heteroduplex analysis with separation of the PCR product by electrophoresis on non-denaturing PAGE for the CALR exon 9 mutations detection as the screening test. Materials and methods. DNA samples of 13 CALR-positive patients with different phenotypic variants of Ph-myeloproliferative neoplasms were screened by heteroduplex analysis. For the most common variants of CALR mutations (c.1092_1143del and c.1154_1155insTTGTC), a threshold determination of the mutant allele presence was analyzed.Nucleotide sequence of exon 9 fragment was determined using Sanger sequencing. Also, all 13 samples were analyzed using the pyrosequencing method to assess the allelic burden level.Results. Heteroduplex analysis revealed mutations in exon 9 of the CALR gene in all 13 patients. The threshold determinations of the method in the case of the c.1154_1155insTTGTC and c.1092_1143del analysis are 6.25 % and 3.13 % of the mutant allele presence in the patient sample, respectively.Conclusion. The proposed variant of the heteroduplex analysis with separation of the PCR product by electrophoresis on non-denaturing PAGE can be recommended for use as the preliminary screening test which is carried out before the confirming sequencing methods for the different indels (or combinations thereof) CALR mutations determine.The presence of heteroduplexes indicates the presence of a mutation, even if the mutant product is not visualized (in case of small mutations).

MPL exon 10 mutations other than canonical MPL W515L/K mutations identified by in-house MPL exon 10 direct sequencing in essential thrombocythemia.

MPL exon 10 mutations are one of the driver mutations in essential thrombocythemia (ET) or myelofibrosis (MF). We have established an in-house MPL mutation analysis system, covering the entire region of MPL exon 10 by direct sequencing. Since 2009, MPL exon 10 mutation analysis has been performed for diagnosis of myeloproliferative neoplasms (MPN) without JAK2 V617F or CALR exon 9 mutations. So far, 11 cases of MPL exon 10 mutation have been found in 51 patients with suspected MPN. In patients with ET, we detected five non-canonical MPL mutations including one novel mutation, MPL R514_P518delinsK, and one canonical MPL W515L mutation. Notably, three ET patients without canonical MPL mutations had thrombotic events. Meanwhile, in primary or secondary MF, only canonical MPL W515L/K mutations were found. Further cases need to be examined to elucidate the full MPL mutation profile in MPN. However, our data indicate that analysis of the whole of MPL exon 10 is warranted for the diagnosis of MPL mutations, especially in ET, and that the use of Japanese commercial laboratory tests that only detect canonical MPL W515L/K mutations may miss a significant percentage of MPL exon 10 mutations, which could delay the administration of anti-thrombotic therapy.

Analysis of Common Driver Mutations in Philadelphia-Negative Myeloproliferative Neoplasms

BackgroundPhiladelphia-negative myeloproliferative neoplasms (MPNs) are a group of hematopoietic stem cell disorders that include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). This study examines the driver mutations among patients with MPNs in Kuwait. Patients and MethodsThis study was a retrospective review of 942 MPN cases with a driver mutation from July 2007 to June 2019 to examine their demographic, clinical, and laboratory attributes. ResultsThe annual incidence of MPNs is 1.6 per 100,000 persons, and ET is the most common subtype. The median age of our cohort was 55 years, and the patients were predominantly male. We found that the most frequent gene mutation of MPNs in our cohort was the JAK2V617F mutation, which was present in 90% of cases, followed by the CALR exon 9, MPLW515L/K, and JAK2 exon 12 mutations. In our cohort, thrombotic events were observed in 18.7% of cases. ConclusionAlthough Philadelphia-negative MPNs are rare hematologic malignancies, thrombosis is a relatively common initial presentation. The JAK2V617F mutation was the driver mutation in the majority of patients with MPN.

Inflammatory Microenvironment and Specific T Cells in Myeloproliferative Neoplasms: Immunopathogenesis and Novel Immunotherapies.

The Philadelphia-negative myeloproliferative neoplasms (MPNs) are malignancies of the hematopoietic stem cell (HSC) arising as a consequence of clonal proliferation driven by somatically acquired driver mutations in discrete genes (JAK2, CALR, MPL). In recent years, along with the advances in molecular characterization, the role of immune dysregulation has been achieving increasing relevance in the pathogenesis and evolution of MPNs. In particular, a growing number of studies have shown that MPNs are often associated with detrimental cytokine milieu, expansion of the monocyte/macrophage compartment and myeloid-derived suppressor cells, as well as altered functions of T cells, dendritic cells and NK cells. Moreover, akin to solid tumors and other hematological malignancies, MPNs are able to evade T cell immune surveillance by engaging the PD-1/PD-L1 axis, whose pharmacological blockade with checkpoint inhibitors can successfully restore effective antitumor responses. A further interesting cue is provided by the recent discovery of the high immunogenic potential of JAK2V617F and CALR exon 9 mutations, that could be harnessed as intriguing targets for innovative adoptive immunotherapies. This review focuses on the recent insights in the immunological dysfunctions contributing to the pathogenesis of MPNs and outlines the potential impact of related immunotherapeutic approaches.

Calreticulin – a multifaced protein

Summary Calreticulin (CALR) is a highly conserved multi-function protein that primarily localizes within the lumen of the endoplasmic reticulum (ER). It participates in various processes in the cells, including glycoprotein chaperoning, regulation of Ca2+ homeostasis, antigen processing and presentation for adaptive immune response, cell adhesion/migration, cell proliferation, immunogenic cell death, gene expression and RNA stability. The role of CALR in the assembly, retrieval and cell surface expression of MHC class I molecules is well known. A fraction of the total cellular CALR is localized in the cytosol, following its retro-translocation from the ER. In the cell stress conditions, CALR is also expressed on the cell surface via an interaction with phosphatidylserine localized on the inner leaflet of the plasma membrane. The abovementioned mechanism is relevant for the recognition of the cells, as well as immunogenicity and phagocytic uptake of proapoptotic and apoptotic cells. Lastly, the presence of CALR exon 9 gene mutations was confirmed in patients with myeloproliferative neoplasms. Their presence results in an abnormal CALR structure due to the loss of its ER-retention sequence, CALR extra-ER localisation, the formation of a complex with thrombopoietin receptor, and oncogenic transformation of hematopoietic stem cells. It is also known that CALR exon 9 mutants are highly immunogenic and induce T cell response. Despite this fact, CALR mutant positive hematopoietic cells emerge. The last phenomenon is probably the result of the inhibition of phagocytosis of the cancer cells exposing CALR mutant protein by dendritic cells.

JAK2 V617F Prevalence Study: Associations in the General Population and Vascular Disease Populations

Introduction Clonal hematopoiesis (CH) is defined as an expansion of blood stem cell clones that bear advantageous somatic mutations. JAK2V617F is not only the most common mutation association with MPN, it is the fifth most common lesion implicated in CH. While the JAK2V617F positive MPN are rare diseases, CH occurs at relatively common rates in the aging population. CH carriers, and in particular JAK2V617F CH carriers, similar to MPN patients, are at increased risk of developing blood cancers, and arterial and venous thromboses. While many large-scale general population studies have examined prevalence of JAK2V617F CH, a comprehensive assessment of associations to disease has not been performed. Methods We identified all published studies that evaluated the prevalence of the JAK2V617F and CALR exon 9 mutations in the general population and in vascular disease risk populations. We used PUBMED, EMBASE, and SCOPUS databases. Two reviewers independently performed study selection and extracted study characteristics. Studies were included if they met the following criteria : analysis of JAK2/CALR mutations was performed according commonly accepted criteria and study inclusion of at least 40 patients. Results We identified 5793 abstracts/manuscripts that met our search criteria. After title/abstract review, 104 manuscripts were included in the meta-analysis (Figure 1). Thirteen of these studies examined the prevalence of JAK2V617F and CALR exon 9 mutations in a general, unselected population. Among 546,504 participants included in these studies, 1,850 (0.34%) were positive for the JAK2V617F mutation. In contrast, 32 out of 19,958 (0.16%) were positive for CALR exon 9 mutations. In the general population, the JAK2V617F mutation was most commonly associated with male sex, increased age, and smoking. Three studies reported increased blood counts, predominantly platelets and white blood cells. Four studies noted an increased risk of MPN or hematologic cancer in those with the JAK2V617F mutation. The JAK2V617F mutation was associated with an increased risk of venous thrombosis in 3 studies and ischemic heart disease/MI in 2 studies. JAK2V617F variant allele fraction (VAF) when reported ranged 0.004% to 86%. The prevalence of the JAK2V617F in patients with venous or arterial vasculopathy was examined in 91 studies with a total of 35,982 participants. Of these, 26,279 individuals had a history of thrombotic disease and 8,944 were controls. Of the participants with a history of any type of vascular disease, 1,279 (4.9%) were noted to have the JAK2V617F mutation with VAF, when reported, ranging from 0.2-96.2%. On subgroup analysis, patients with splanchnic vein thrombosis had the highest prevalence of the JAK2V617F mutation (18.7%) followed by patients with ischemic stroke (8.5%) and cerebral vein thrombosis (6.0%) (Table 1). The prevalence of the JAK2V617F mutation in patients with DVT/PE and peripheral arterial disease (PAD) was 1.6% and 3.1% respectively. The prevalence of JAK2V617F among all of the vascular disease subsets was substantially higher than the control populations. Similarly, the prevalence of CALR was noted to be high in studies of splanchnic vein thrombosis and cerebral vein thrombosis. (Table 2). Conclusions JAK2V617F and CALR mutations are associated with alterations of blood counts and thrombosis. Even at very low VAF, JAK2V617F is a thrombosis risk factor in the general population and is over-represented in populations with thrombosis. In every thrombosis group, the prevalence in JAK2V617F in thrombosis patients was higher than that in the control population, suggesting the JAK2V617F mutation may contribute to both arterial and venous thrombotic risk, whether in the MPN or CH population context. Disclosures Naik: Elsevier: Consultancy; Rigel: Research Funding. Moliterno:Pharmessentia: Consultancy; MPNRF: Research Funding.