Clonal evolution process from essential thrombocythemia to acute myeloid leukemia in the original patient from whom the CALR-mutated Marimo cell line was established.

We previously reported the Marimo cell line, which was established from the bone marrow cells of a patient with essential thrombocythemia (ET) at the last stage after transformation to acute myeloid leukemia (AML). This cell line is widely used for the biological analysis of ET because it harbors CALR mutation. However, genetic processes during disease progression in the original patient were not analyzed. We sequentially analyzed the genetic status in the original patient samples during disease progression. The ET clone had already acquired CALR and MPL mutations, and TP53 and NRAS mutations affected the disease progression from ET to AML in this patient. Particularly, the variant allele frequency of the NRAS mutation increased along with the disease progression after transformation, and the NRAS-mutated clone selectively proliferated in vitro, resulting in the establishment of the Marimo cell line. Although CALR and MPL mutations co-existed, MPL was not expressed in Marimo cells or any clinical samples. Furthermore, mitogen-activated protein kinase (MAPK) but not the JAK2-STAT pathway was activated. These results collectively indicate that MAPK activation is mainly associated with the proliferation ability of Marimo cells.

NGS panel enhance precise diagnosis of myeloid neoplasms under WHO-HAEM5 and International Consensus Classification: An observational study.

This study aimed to assess hematological diseases next-generation sequencing (NGS) panel enhances the diagnosis and classification of myeloid neoplasms (MN) using the 5th edition of the WHO Classification of Hematolymphoid Tumors (WHO-HAEM5) and the International Consensus Classification (ICC) of Myeloid Tumors. A cohort of 112 patients diagnosed with MN according to the revised fourth edition of the WHO classification (WHO-HAEM4R) underwent testing with a 141-gene NGS panel for hematological diseases. Ancillary studies were also conducted, including bone marrow cytomorphology and routine cytogenetics. The cases were then reclassified according to WHO-HAEM5 and ICC to assess the practical impact of these 2 classifications. The mutation detection rates were 93% for acute myeloid leukemia (AML), 89% for myelodysplastic syndrome (MDS), 94% for myeloproliferative neoplasm (MPN), and 100% for myelodysplasia/myeloproliferative neoplasm (MDS/MPN) (WHO-HAEM4R). NGS provided subclassified information for 26 and 29 patients with WHO-HAEM5 and ICC, respectively. In MPN, NGS confirmed diagnoses in 16 cases by detecting JAK2, MPL, or CALR mutations, whereas 13 "triple-negative" MPN cases revealed at least 1 mutation. NGS panel testing for hematological diseases improves the diagnosis and classification of MN. When diagnosed with ICC, NGS produces more classification subtype information than WHO-HAEM5.

Lysosomal degradation targets mutant calreticulin and the thrombopoietin receptor in myeloproliferative neoplasms.

ABSTRACT: Somatic mutants of calreticulin (CRT) drive myeloproliferative neoplasms (MPNs) via binding to the thrombopoietin receptor (MPL) and aberrant activation of the JAK/STAT pathway. Compared with healthy donors, platelets from mutant CRT-expressing patients with MPN display low cell surface MPL. Additionally, coexpression of MPL with an MPN-linked CRT mutant (CRTDel52) reduces cell surface MPL, suggesting that CRTDel52 may induce MPL degradation. We show that lysosomal degradation is relevant to the turnover of CRTDel52 and MPL. Furthermore, CRTDel52 increases the lysosomal localization and degradation of MPL. Mammalian target of rapamycin (mTOR) inhibitors reduce cellular CRTDel52 and MPL, secreted CRTDel52 levels, and impair CRTDel52-mediated cell proliferation. mTOR inhibition also reduces colony formation and differentiation of CD34+ cells from patients with MPN but not from healthy donors. Together, these findings indicate that low-surface MPL is a biomarker of mutant CRT-mediated MPN and that induced degradation of CRTDel52 and MPL is an avenue for therapeutic intervention.

Thrombopoietin, the Primary Regulator of Platelet Production: From Mythos to Logos, a Thirty-Year Journey.

Thrombopoietin, the primary regulator of blood platelet production, was postulated to exist in 1958, but was only proven to exist when the cDNA for the hormone was cloned in 1994. Since its initial cloning and characterization, the hormone has revealed many surprises. For example, instead of acting as the postulated differentiation factor for platelet precursors, megakaryocytes, it is the most potent stimulator of megakaryocyte progenitor expansion known. Moreover, it also stimulates the survival, and in combination with stem cell factor leads to the expansion of hematopoietic stem cells. All of these growth-promoting activities have resulted in its clinical use in patients with thrombocytopenia and aplastic anemia, although the clinical development of the native molecule illustrated that "it's not wise to mess with mother nature", as a highly engineered version of the native hormone led to autoantibody formation and severe thrombocytopenia. Finally, another unexpected finding was the role of the thrombopoietin receptor in stem cell biology, including the development of myeloproliferative neoplasms, an important disorder of hematopoietic stem cells. Overall, the past 30 years of clinical and basic research has yielded many important insights, which are reviewed in this paper.

Essential thrombocythaemia: A contemporary approach with new drugs on the horizon.

Essential thrombocythaemia (ET) is a myeloproliferative neoplasm characterized by an increased risk of vascular complications and a tendency to progress to myelofibrosis and acute leukaemia. ET patients have traditionally been stratified into two thrombosis risk categories based on age older than 60 years and a history of thrombosis. More recently, the revised IPSET-thrombosis scoring system, which accounts for the increased risk linked to the JAK2 mutation, has been incorporated into most expert recommendations. However, there is increasing evidence that the term ET encompasses different genomic entities, each with a distinct clinical course and prognosis. Moreover, the effectiveness and toxicity of cytoreductive and anti-platelet treatments differ depending on the molecular genotype. While anti-platelets and conventional cytoreductive agents, mainly hydroxycarbamide (hydroxyurea), anagrelide and pegylated interferon, remain the cornerstone of treatment, recent research has shed light on the effectiveness of novel therapies that may help improve outcomes. This comprehensive review focuses on the evolving landscape of treatment strategies in ET, with an emphasis on the role of molecular profiling in guiding therapeutic decisions. Besides evidence-based management according to revised IPSET-thrombosis stratification, we also provide specific observations for those patients with CALR-, MPL-mutated and triple-negative ET, as well as cases with high-risk mutations.

Clinical laboratory characteristics and gene mutation spectrum of Ph-negative MPN patients with atypical variants of JAK2, MPL, or CALR.

OBJECTIVE: To evaluate the incidence, clinical laboratory characteristics, and gene mutation spectrum of Ph-negative MPN patients with atypical variants of JAK2, MPL, or CALR. METHODS: We collected a total of 359 Ph-negative MPN patients with classical mutations in driver genes JAK2, MPL, or CALR, and divided them into two groups based on whether they had additional atypical variants of driver genes JAK2, MPL, or CALR: 304 patients without atypical variants of driver genes and 55 patients with atypical variants of driver genes. We analyzed the relevant characteristics of these patients. RESULTS: This study included 359 patients with Ph-negative MPNs with JAK2, MPL, or CALR classical mutations and found that 55 (15%) patients had atypical variants of JAK2, MPL, or CALR. Among them, 28 cases (51%) were male, and 27 (49%) were female, with a median age of 64 years (range, 21-83). The age of ET patients with atypical variants was higher than that of ET patients without atypical variants [70 (28-80) vs. 61 (19-82), p = 0.03]. The incidence of classical MPL mutations in ET patients with atypical variants was higher than in ET patients without atypical variants [13.3% (2/15) vs. 0% (0/95), p = 0.02]. The number of gene mutations in patients with atypical variants of driver genes PV, ET, and Overt-PMF is more than in patients without atypical variants of PV, ET, and Overt-PMF [PV: 3 (2-6) vs. 2 (1-7), p < 0.001; ET: 4 (2-8) vs. 2 (1-7), p < 0.05; Overt-PMF: 5 (2-9) vs. 3 (1-8), p < 0.001]. The incidence of SH2B3 and ASXL1 mutations were higher in MPN patients with atypical variants than in those without atypical variants (SH2B3: 16% vs. 6%, p < 0.01; ASXL1: 24% vs. 13%, p < 0.05). CONCLUSION: These data indicate that classical mutations of JAK2, MPL, and CALR may not be completely mutually exclusive with atypical variants of JAK2, MPL, and CALR. In this study, 30 different atypical variants of JAK2, MPL, and CALR were identified, JAK2 G127D being the most common (42%, 23/55). Interestingly, JAK2 G127D only co-occurred with JAK2V617F mutation. The incidence of atypical variants of JAK2 in Ph-negative MPNs was much higher than that of the atypical variants of MPL and CALR. The significance of these atypical variants will be further studied in the future.

Differential in vivo roles of Mpl cytoplasmic tyrosine residues in murine hematopoiesis and myeloproliferative disease.

Thrombopoietin (Tpo), which binds to its specific receptor, the Mpl protein, is the major cytokine regulator of megakaryopoiesis and circulating platelet number. Tpo binding to Mpl triggers activation of Janus kinase 2 (Jak2) and phosphorylation of the receptor, as well as activation of several intracellular signalling cascades that mediate cellular responses. Three tyrosine (Y) residues in the C-terminal region of the Mpl intracellular domain have been implicated as sites of phosphorylation required for regulation of major Tpo-stimulated signalling pathways: Mpl-Y565, Mpl-Y599 and Mpl-Y604. Here, we have introduced mutations in the mouse germline and report a consistent physiological requirement for Mpl-Y599, mutation of which resulted in thrombocytopenia, deficient megakaryopoiesis, low hematopoietic stem cell (HSC) number and function, and attenuated responses to myelosuppression. We further show that in models of myeloproliferative neoplasms (MPN), where Mpl is required for pathogenesis, thrombocytosis was dependent on intact Mpl-Y599. In contrast, Mpl-Y565 was required for negative regulation of Tpo responses; mutation of this residue resulted in excess megakaryopoiesis at steady-state and in response to myelosuppression, and exacerbated thrombocytosis associated with MPN.

Deciphering the differential impact of thrombopoietin/MPL signaling on hematopoietic stem/progenitor cell function in bone marrow and spleen.

Thrombopoietin (TPO) and its receptor MPL play crucial roles in hematopoietic stem cell (HSC) function and platelet production. However, the precise effects of TPO/MPL signaling on HSC regulation in different hematopoietic niches remain unclear. Here, we investigated the effects of TPO/MPL ablation on marrow and splenic hematopoiesis in TPO-/- and MPL-/- mice during aging. Despite severe thrombocytopenia, TPO-/- and MPL-/- mice did not develop marrow failure during a 2-year follow-up. Marrow and splenic HSCs exhibited different responses to TPO/MPL ablation and exogenous TPO treatment. Splenic niche cells compensated for marrow HSC loss in TPO-/- and MPL-/- mice by upregulating CXCL12 levels. These findings provide new insights into the complex regulation of HSCs by TPO/MPL and reveal a previously unknown link between TPO and CXCL12, two key growth factors for HSC maintenance. Understanding the distinct regulatory mechanisms between marrow and spleen hematopoiesis will help to develop novel therapeutic approaches for hematopoietic disorders.

Germline MPL mutations may be a rare cause of "triple-negative" thrombocytosis.

Hereditary thrombocytosis (HT) is a rare inherited disorder with clinical features resembling those of sporadic essential thrombocythemia. This study included 933 patients with persistent isolated thrombocytosis for whom secondary reactive causes were excluded. Of 933 patients screened, 567 were JAK2-mutated, 255 CALR-mutated, 41 MPL-mutated, 2 double-mutated, and 68 were triple-negative. Two patients carried germline non-canonical mutations in exon 10: MPL W515* and MPL V501A. One triple-negative patient carried another germline non-canonical MPL mutation outside exon 10: MPL R102P. As germline MPL mutations may be underlying causes of HT, we recommend screening patients with triple-negative isolated thrombocytosis for non-canonical MPL mutations. Although clear evidence concerning HT treatment is still lacking, individuals with HT should probably be excluded from cytoreductive treatment. Thus, an accurate diagnosis is pivotal in avoiding unnecessary treatments.

Expanded molecular detection of MPL codon p.W515 and p.S505N mutations in myeloproliferative neoplasms.

BACKGROUND: Patients negative for the JAK2 p.V617F somatic variant are frequently reflexed to testing for MPL exon 10 variants. Detection of these variants via multiplexed allele-specific PCR followed by fragment analysis has been previously published. The present study builds on this concept by improving the detection of the p.W515A variant, adding a second allele-specific primer to detect the p.W515R variant, and incorporating an improved primer for p.S505N detection. METHODS: The W515 amplification employs 5'-labeled allele-specific forward primers to detect p.W515K, p.W515L, p.W515R, and p.W515A. The p.S505N amplification includes an allele-specific reverse primer with a tail extension. Fragments were subject to capillary electrophoresis on an ABI 3500 Genetic Analyzer and analyzed using GeneMapper 6.0 (Thermo Fisher Scientific). RESULTS: Thirty MPL-negative and 13 MPL-positive samples previously tested by a reference laboratory were tested with the MPL LDT. Results were 100% concordant. The MPL LDT has a limit of detection of at least 5% VAF for the p.W515 variants and 10% VAF for the p.S505N variant. CONCLUSION: Current MPL assays are predominantly focused on p.W515L/K and p.S505N mutations. We have engineered an MPL test for detecting p.W515A/L/K/R and p.S505N variants, thereby increasing the diagnostic yield with little additional expense or technician time.

JAK2, CALR, and MPL Mutation Profiles in Colombian patients with BCR-ABL Negative Myeloproliferative Neoplasms.

Background: Among the chronic myeloproliferative neoplasms (MPNs) not associated with BCR-ABL mutations are polycythemia vera, primary myelofibrosis, and essential thrombocythemia. These diseases are caused by mutations in genes, such as the JAK2, MPL, and CALR genes, which participate in regulating the JAK-STAT signaling pathway. Objective: This study aimed to establish the frequencies of mutations in the JAK2, MPL, and CALR genes in a group of Colombian patients with a negative clinical diagnosis of BCR-ABL chronic myeloproliferative neoplasms. Methods: The JAK2 V617F and MPL W515K mutations and deletions or insertions in exon 9 of the CALR gene were analyzed in 52 Colombian patients with polycythemia vera, primary myelofibrosis, and essential thrombocythemia. Results: The JAK2V617F mutation was carried by 51.9% of the patients, the CALR mutation by 23%, and the MPL mutation by 3.8%; 23% were triple-negative for the mutations analyzed. In these neoplasms, 6 mutation types in CALR were identified, one of which has not been previously reported. Additionally, one patient presented a double mutation in both the CALR and JAK2 genes. Regarding the hematological results for the mutations, significant differences were found in the hemoglobin level, hematocrit level, and platelet count among the three neoplasms. Conclusion: Thus, this study demonstrates the importance of the molecular characterization of the JAK2, CALR and MPL mutations in Colombian patients (the genetic context of which remains unclear in the abovementioned neoplasms) to achieve an accurate diagnosis, a good prognosis, adequate management, and patient survival.

Antecedentes: Entre las neoplasias mieloproliferativas crónicas no asociadas con mutaciones BCR-ABL se encuentran la policitemia vera, la mielofibrosis primaria y la trombocitemia esencial. Estas enfermedades están causadas por mutaciones en genes, como los genes JAK2, MPL y CALR, que participan en la regulación de la vía de señalización JAK-STAT. Objetivo: Establecer las frecuencias de mutaciones en los genes JAK2, MPL y CALR en un grupo de pacientes colombianos con diagnóstico clínico negativo de NMP BCR-ABL. Metodos: Se analizaron las mutaciones y deleciones o inserciones JAK2 V617F y MPL W515K en el exón 9 del gen CALR en 52 pacientes colombianos con policitemia vera, mielofibrosis primaria y trombocitemia esencial. Resultados: La mutación JAK2V617F la portaban el 51.9% de los pacientes, la mutación CALR el 23.0% y la mutación MPL el 3.8%; El 23.0% fueron triple negativos para las mutaciones analizadas. En estas neoplasias se identificaron seis tipos de mutación en CALR, uno de los cuales no ha sido reportado previamente. Además, un paciente presentó una doble mutación tanto en el gen CALR como en el JAK2. En cuanto a los resultados hematológicos para las mutaciones, se encontraron diferencias significativas en el nivel de hemoglobina, el nivel de hematocrito y el recuento de plaquetas entre las tres neoplasias. Conclusiones: Así, este estudio demuestra la importancia de la caracterización molecular de las mutaciones JAK2, CALR y MPL en pacientes colombianos (cuyo contexto genético aún no está claro en las neoplasias antes mencionadas) para lograr un diagnóstico certero, un buen pronóstico, un manejo adecuado y una mejoría del paciente. supervivencia.

A novel mutation in MECOM affects MPL regulation in vitro and results in thrombocytopenia and bone marrow failure.

MECOM-associated syndrome (MECOM-AS) is a rare disease characterized by amegakaryocytic thrombocytopenia, progressive bone marrow failure, pancytopenia and radioulnar synostosis with high penetrance. The clinical phenotype may also include finger malformations, cardiac and renal alterations, hearing loss, B-cell deficiency and predisposition to infections. The syndrome, usually diagnosed in the neonatal period because of severe thrombocytopenia, is caused by mutations in the MECOM gene, encoding for the transcription factor EVI1. The mechanism linking the alteration of EVI1 function and thrombocytopenia is poorly understood. In a paediatric patient affected by severe thrombocytopenia, we identified a novel variant of the MECOM gene (p.P634L), whose effect was tested on pAP-1 enhancer element and promoters of targeted genes showing that the mutation impairs the repressive activity of the transcription factor. Moreover, we demonstrated that EVI1 controls the transcriptional regulation of MPL, a gene whose mutations are responsible for congenital amegakaryocytic thrombocytopenia (CAMT), potentially explaining the partial overlap between MECOM-AS and CAMT.

Structure of the thrombopoietin-MPL receptor complex is a blueprint for biasing hematopoiesis.

Thrombopoietin (THPO or TPO) is an essential cytokine for hematopoietic stem cell (HSC) maintenance and megakaryocyte differentiation. Here, we report the 3.4 Å resolution cryoelectron microscopy structure of the extracellular TPO-TPO receptor (TpoR or MPL) signaling complex, revealing the basis for homodimeric MPL activation and providing a structural rationalization for genetic loss-of-function thrombocytopenia mutations. The structure guided the engineering of TPO variants (TPOmod) with a spectrum of signaling activities, from neutral antagonists to partial- and super-agonists. Partial agonist TPOmod decoupled JAK/STAT from ERK/AKT/CREB activation, driving a bias for megakaryopoiesis and platelet production without causing significant HSC expansion in mice and showing superior maintenance of human HSCs in vitro. These data demonstrate the functional uncoupling of the two primary roles of TPO, highlighting the potential utility of TPOmod in hematology research and clinical HSC transplantation.

Genomic and computational analysis of four novel variants of MPL gene in Congenital Amegakaryocytic Thrombocytopenia.

Congenital amegakaryocytic thrombocytopenia (CAMT) is a rare, genetic, autosomal recessive disorder characterized by severe thrombocytopenia, due to inefficient bone marrow megakaryopoiesis eventually leading to aplasia. Majority of the cases are due to homozygous or compound heterozygous mutations in MPL gene encoding for thrombopoietin (THPO) receptor protein. CAMT can be diagnosed at early phase of life, with major complication of transfusion dependency and hematopoietic transplantation as only curative treatment. We have investigated the sequence variations in MPL gene of 7 bone marrow failure (BMF) subjects, who presented with clinically diverse phenotypes, through next generation sequencing (NGS). Plasma THPO levels were estimated using ELISA. Insilico sequence and structure-based analyses were performed to understand the structural and functional implications of mutations, identified through NGS. We studied 7 CAMT subjects suspected of BMF, who presented with severe thrombocytopenia followed by pancytopenia, bleeding manifestation and physical anomalies. The plasma THPO levels were significantly elevated (p<0.05) in all the cases. Molecular analysis by NGS identified 9 genomic mutations in MPL gene. These included 7 non-synonymous substitution, 1 nonsense substitution and 1 in-del mutations, of which 4 are novel mutations. Insilico analysis predicted damaging effects on THPO-R and its reduced affinity for THPO for all the identified mutations. CAMT is a rare disorder with diverse clinical phenotypes and diagnosis is challenging. The elevated plasma THPO levels should be considered for the primary diagnosis and prognosis of the disease. However, molecular analysis of MPL gene is important for the diagnosis and management of the disease through genetic counselling. Though the cytokines, THPO-R agonist are used for the treatment of CAMT, HSCT is the only curative therapy.

MPL gene mutation is a possible risk factor for thrombosis in patients with essential thrombocythemia in Japan.

OBJECTIVES: Since MPL mutation is a rare driver gene mutation found in a small number of essential thrombocythemia (ET) patients, the clinical characteristics of patients with MPL mutations and their association with thrombotic events have not yet been elucidated in Japan. METHODS: We enrolled 579 Japanese ET patients based on the diagnostic criteria of the WHO classification 2017 and compared clinical characteristics of MPL-mutated patients (n = 22; 3.8%) to JAK2V617F-mutated (n = 299; 51.6%), CALR-mutated (n = 144; 24.9%), and triple-negative (TN) (n = 114; 19.7%) patients. RESULTS: Thrombosis during follow up was observed in 4 out of 22 (18.2%) in the MPL-mutated group, which was the highest among all driver gene mutation groups (JAK2V617F-mutated, 8.7%; CALR-mutated, 3.5%; TN,1.8%). The MPL- and JAK2V617F-mutated groups had worse thrombosis-free survival (TFS) than the CALR-mutated (p = 0.043) and TN groups (p = 0.006). Univariable analysis revealed that a history of thrombosis was a possible risk factor for thrombosis among MPL-mutated patients (hazard ratio: 9.572, p = 0.032). CONCLUSIONS: MPL-mutated ET patients should require more intensive management to prevent recurrence of thrombosis.

Constitutive activation and oncogenicity are mediated by loss of helical structure at the cytosolic boundary of thrombopoietin receptor mutant dimers.

Dimerization of the thrombopoietin receptor (TpoR) is necessary for receptor activation and downstream signaling through activated Janus kinase 2. We have shown previously that different orientations of the transmembrane (TM) helices within a receptor dimer can lead to different signaling outputs. Here we addressed the structural basis of activation for receptor mutations S505N and W515K that induce myeloproliferative neoplasms. We show using in vivo bone marrow reconstitution experiments that ligand-independent activation of TpoR by TM asparagine (Asn) substitutions is proportional to the proximity of the Asn mutation to the intracellular membrane surface. Solid-state NMR experiments on TM peptides indicate a progressive loss of helical structure in the juxtamembrane (JM) R/KWQFP motif with proximity of Asn substitutions to the cytosolic boundary. Mutational studies in the TpoR cytosolic JM region show that loss of the helical structure in the JM motif by itself can induce activation, but only when localized to a maximum of six amino acids downstream of W515, the helicity of the remaining region until Box 1 being required for receptor function. The constitutive activation of TpoR mutants S505N and W515K can be inhibited by rotation of TM helices within the TpoR dimer, which also restores helicity around W515. Together, these data allow us to develop a general model for activation of TpoR and explain the critical role of the JM W515 residue in the regulation of the activity of the receptor.

Genetic and Clinical Characteristics of Patients with Philadelphia-Negative Myeloproliferative Neoplasm Carrying Concurrent Mutations in JAK2V617F, CALR, and MPL.

Simultaneous mutations in Janus kinase 2 (JAK2), calreticulin, and myeloproliferative leukemia (MPL) genes are generally not considered for characterizing Philadelphia-negative myeloproliferative neoplasms (MPNs), leading to misdiagnosis. Sanger sequencing and quantitative polymerase chain reaction were used to detect gene mutations in patients with MPN. We retrospectively screened the data of patients with double mutations in our center and from the PubMed database. Two patients tested positive for both JAK2V617F and CALR mutations (2/352 0.57%) in our center, while data of 35 patients from the PubMed database, including 26 patients with essential thrombocythemia (ET), 6 with primary myelofibrosis (PMF), 2 with unexplained thrombosis, and 1 with polycythemia vera were screened for double mutations. Among these mutations, co-mutation of JAKV617F-CALR constituted the majority (80.0%), when compared with JAKV617F-MPL (17.1%) and CALR-MPL (2.9%) mutations. Moreover, patients with concurrent mutational myeloproliferative neoplasm (MPN) were relatively older (P = .010) with significantly higher platelet counts than their counterparts with single gene mutations (P < .001). The occurrence of palpable splenomegaly (P < .001) and leukocyte count (P = .041) were also significantly different between patients with single and simultaneous gene mutations. These 4 risk factors also showed significant test effectiveness in the ET and PMF cohorts (P < .05). In terms of clinical characteristics of patients with ET, those with JAK2V617F-CALR mutation had higher but normal hemoglobin levels (P = .0151) than those carrying JAK2V617F-MPL mutation. From a clinical perspective, patients with multiple mutational MPN are different from those with single gene mutations. The poor treatment response by patients in our center and unfavorable indicators for patients with co-mutations in published literature indicate that customized treatment may be the best choice for patients with MPN carrying co-mutations.

Identification of putative noncanonical driver mutations in patients with essential thrombocythemia.

Essential thrombocythemia (ET) cases without canonical JAK2, CALR, or MPL mutations, that is, triple-negative (TN) ET, have been found in 10%-20% of ET cases. Owing to the limited number of TN ET cases, its clinical significance remains unclear. This study evaluated TN ET's clinical characteristics and identified novel driver mutations. Among 119 patients with ET, 20 (16.8%) had no canonical JAK2/CALR/MPL mutations. Patients with TN ET tended to be younger and had lower white blood cell counts and lactate dehydrogenase values. We identified putative driver mutations in 7 (35%): MPL S204P, MPL L265F, JAK2 R683G, and JAK2 T875N were previously reported as candidate driver mutations in ET. Moreover, we identified a THPO splicing site mutation, MPL*636Wext*12, and MPL E237K. Four of the seven identified driver mutations were germline. Functional studies on MPL*636Wext*12 and MPL E237K revealed that they are gain-of-function mutants that increase MPL signaling and confer thrombopoietin hypersensitivity with very low efficiency. Patients with TN ET tended to be younger, although this was thought to be due to the inclusion of germline mutations, hereditary thrombocytosis. Accumulating the genetic and clinical characteristics of noncanonical mutations may help future clinical interventions in TN ET and hereditary thrombocytosis.

Genomics of clonal evolution in a rare essential thrombocythemia with coexisting Type 2 CALR and MPL S204P mutations.

Essential thrombocythemia (ET) with double driver mutations is a rare disease. ET patients with both MPL and Type 1 CALR mutations have been reported. Here, we report the first case of an ET patient with both MPL S204P and Type 2 CALR mutations and a summary of our literature review findings. In the patient whose case is reported here, the disease progressed to an accelerated phase 3.5 months after diagnosis. CALR mutation disappeared and new mutations emerged as the disease progressed, such as ASXL1, CBL, ETV6, and PTPN11 mutations. This case highlights that screening for additional mutations using NGS should be considered in patients with ET to assess the prognosis, especially as the disease progresses.

Distinctive Attributes of Indian Patients With Classical BCR::ABL1 Negative Myeloproliferative Neoplasms: Unified Clinical and Laboratory Data.

INTRODUCTION: We report one of the largest single center data from a mixed referral setting in India describing baseline characteristics and outcomes of patients with classical BCR::ABL1 negative myeloproliferative neoplasms (MPNs). MATERIALS AND METHODS: Patients diagnosed from June 2019 to 2022 were included. Workup and treatment was as per current guidelines. RESULTS: Diagnosis comprised polycythemia vera (PV) in 51(49%), ET in 33(31.7%) and prefibrotic primary myelofibrosis (MF) pre fibrotic myelofibrosis (prePMF) and myelofibrosis in 10(9.6%) patients each. Median age at diagnosis was 52 years for PV and ET, 65.5 for MF and 79 years for prePMF. Diagnosis was incidental in 63(56.7%) and after thrombosis in 8(7.2%) patients. Baseline next generation sequencing (NGS) was available for 63(60.5%) patients. Driver mutations in PV: JAK2 in 80.3%; in ET: JAK2 in 41%, CALR in 26%, MPL in 2.9%; in prePMF JAK2 in 70%, CALR in 20%, MPL in 10%, and in MF: JAK2 in 10%, MPL in 30% and CALR in 40%. Seven novel mutations were detected of which 5 were potentially pathogenic on computational analysis. After median follow up of 30 months, 2 patients had disease transformation and none had new episodes of thrombosis. Ten patients died, most commonly with cardiovascular events(n = 5,50%). Median overall survival was not reached. Mean OS time was 10.19 years(95%CI, 8.6 to 11.74) and mean time to transformation was 12.2 years(95% CI,11.8 to 12.6). CONCLUSION: Our data indicates comparatively indolent presentation of MPNs in India with younger age and lower risk of thrombosis. Further follow up will enable correlation with molecular data and guide modification of age based risk stratification models.