Background: Diagnostic and treatment response criteria for the JAK2/CALR/MPL mutation-related myeloproliferative neoplasms (MPNs) are largely based on bone marrow (BM) biopsy results. However, these biopsies have several limitations, such as the risk of sampling error. Also, the prognostic impact of BM abnormalities is largely unclear. Although not currently used in clinical practice, imaging techniques might offer additional information. In this review, we investigated the value of BM, liver, and spleen imaging for diagnosis, prognostication, and response monitoring of the JAK2/CALR/MPL mutation-related MPNs (i.e. essential thrombocythemia (ET), polycythemia vera (PV), and myelofibrosis (MF)).

Methods: A systematic literature search was performed via PubMed, Embase and the Cochrane Library up to 2020 March 26th. Of 5505 identified records, 55 publications met the eligibility criteria (i.e. containing original data on the imaging appearance of BM, spleen, or liver in adult ET, PV, or MF patients, published in a peer-reviewed journal, written in English).

Results: Many explorative studies described imaging features, sometimes with comparisons to clinical characteristics. Studies reporting measures of diagnostic accuracy included 1) splenic transient elastography to predict BM fibrosis grade in MF, 2) dynamic contrast-enhanced MRI to discern MF patients from ET patients and healthy controls, and 3) 18-fluorodeoxyglucose PET to detect residual disease after stem cell transplantation in MF. The diagnostic accuracies of radiography and ^99mTc-colloid scintigraphy were derived from several other articles. Except for the study on 18-fluorodeoxyglucose PET, we established substantial concerns regarding risk of bias and applicability across these studies, using the QUADAS-2 tool. Three publications described a correlation between imaging results and prognosis, of which one quantified the effect.

Conclusions: Based on current data, MRI (T1-weighted/STIR, Dixon) seems especially promising for the evaluation of BM fat content - and indirectly cellularity/fibrosis - in MF, and possibly for estimating BM cellularity in ET/PV. 18-fluorodeoxyglucose and 18-fluorothymidine PET/CT might be useful for evaluating BM fibrosis, with good reported accuracy of the former for the diagnosis of residual disease. Further research on these and other techniques is warranted to determine their exact value. Future researchers should improve methodology and focus on evaluation of diagnostic accuracy and prognostic implications of results.

Keywords: Computed tomography; Diagnostic accuracy; Dual-energy X-ray absorptiometry; Magnetic resonance imaging; Myelofibrosis; Myeloproliferative neoplasms; PET/CT; Radiography; Ultrasound.

BACKGROUND

We developed and evaluated the feasibility of peptide nucleic acid (PNA)-based fluorescence melting curve analysis (FMCA) to detect common mutations in myeloproliferative neoplasms (MPNs).

METHODS

We have set up two separate reactions of PNA-based FMCA: JAK2 V617F &CALR p.Leu367fs*46 (set A) and MPL W515L/K &CALR p.Lys385fs*47 (set B). Clinical usefulness was validated with allele-specific real-time PCR, fragment analysis, Sanger sequencing in 57 BCR-ABL1-negative MPNs.

RESULTS

The limit of detection (LOD) of PNA-based FMCA was approximately 10% for each mutation and interference reactions using mixtures of different mutations were not observed. Non-specific amplification was not observed in normal control. PNA-based FMCA was able to detect all JAK2 V617F (n=20), CALR p.Leu367fs*46 (n=10) and p.Lys385fs*47 (n=8). Three of six MPL mutations were detected except three samples with low mutant concentration in out of LOD. JAK2 exon 12 mutations (n=7) were negative without influencing V617F results. Among six variant CALR exon 9 mutations, two were detected by this method owing to invading of probe binding site.

CONCLUSIONS

PNA-based FMCA for detecting common JAK2, MPL, and CALR mutations is a rapid, simple, and sensitive technique in BCR-ABL1-negative MPNs with >10% mutant allele at the time of initial diagnosis.

The frequency of the polymorphism rs11801197 of calcitonin receptor gene (CALCR) was studied by real-time PCR in 422 Moscow women and children, including pregnant women (n=96), lactating (n=29) and non-pregnant women (n=28) and children (n=269) of preschool (2-6 years, n=76) and school age (7-16 years, n=193) with different levels of bone strength (BS) as determined by ultrasound densitometry. It was found that the decrease in the value of the BS (Z-score<-1) was observed in 60% of women, 54% of preschool children and 48% of school children. In the cohort studied the predominant genotype of rs11801197 polymorphism of CALCR gene was CT (38%), the frequency of the genotypes CC and TT - 31%, C and T allele - 50%. There was statistically significant association of BS reduction risk with a C allele of rs11801197 polymorphism of CALCR gene in all examined women (QR=2.034, p=0.02). A positive but not statistically significant association of BS reduction risk with C allele of polymorphism in non-pregnant and pregnant women was found (OR=6.905, p=0.09 and OR=1.902, p=0.09 respectively). The same tendency was observed in preschool children (OR=1.880, p=0.104). In school-aged children C allele was not associated with the risk of BS reduction (OR=0.866, p=0.595). Thus, the allele C is the risk allele of BS reduction. The frequency of CC genotype in Moscow women is much higher than that in women in Europe and in women of North-West region of Russian Federation. Women in the Moscow region - the carriers of rs11801197 polymorphism of CALR - gene need in personalized support of their bone health.

Primary myelofibrosis has the worst outcome among classical chronic myeloproliferative neoplasms. The past decade has brought numerous discoveries elucidating the role of proliferative mutations in disease pathogenesis. Mutations of the genes JAK2, MPL and CALR are present in about 90 percent of all primary myelofibrosis cases. The prognosis of myelofibrosis is considered heterogeneous, the expected survival of patients may range from one year to more than a decade based on several prognostic factors. Estimated survival can be assessed based on clinical prognostic scores. The aim of treatment is to reduce mortality and to alleviate the main aspects of disease-associated morbidity, e.g. anemia, splenomegalia and systemic symptoms. The effect of conventionally used cytoreductive agent hydroxyurea is usually transient. Use of allogeneic hematopoietic stem cell transplantation is limited by significant procedure-associated mortality. JAK2 tyrosine kinase inhibitors are the first treatment modality with evidence of improved overall survival, however, even these molecularly targeted therapies have failed to bring complete and permanent remission for the majority of myelofibrosis patients. Further improvement in overall survival for myelofibrosis can be expected from better understanding of the underlying molecular pathology and novel molecular therapeutic targets.

In the poultry, only less than 5% primordial follicles in the ovary can develop into the prehierarchical follicles (PHFs) leading to progressive development, ovulation, and egg formation. This low rate of recruitment indicates a huge potential for improvement of the laying performance. A great reduction in egg production is caused by aging with extensive follicular atresia. In this study, age-related changes in the laying performance and ovarian status were compared between the peak-lay (D280) and aged (D580) chickens. Subsequently, a cross coculture of PHFs and granulosa cells (GCs) from D280 or D580 hens was adopted to reveal the mechanism of declined follicle development. Results showed that persistent endoplasmic reticulum (ER) stress in GCs of the aged hens was accompanied with intensified apoptosis. Bone morphogenetic protein 4 (BMP4) secreted by GCs of PHFs in D280 hens was capable of relieving ER stress and improving follicular dominance for selection in D580 hens. During this action, BMP4 reduced free calreticulin (CALR, an ER marker) content and attenuated cell apoptosis in PHFs of D580 hens via the PERK-CHOP-BCL2/caspase3 or CALR-Ca²⁺-BCL2-caspase12 pathway. Furthermore, BMP4 prevented follicular atresia by promoting production of steroid hormones to improve survival of GCs in PHFs from the aged hens. In conclusion, intensified ER stress and apoptosis occurred in GCs of PHFs in aged chickens, while BMP4 secreted by GCs was capable of improving follicular viability by alleviating ER stress to promote follicular development.

Vitellogenesis is a principal process during ovarian maturation in crustaceans. This process is negatively regulated by gonad-inhibiting hormone (GIH), a neuronal peptide hormone from eyestalks. However, the detailed mechanism through which GIH regulates Vg expression is still ambiguous. In this study, suppression subtractive hybridization (SSH) under specific GIH-knockdown condition was utilized to determine the expression of genes in the ovary that may act downstream of GIH to control vitellogenin synthesis in Penaeus monodon. The total of 102 and 82 positive clones of up-regulated and down-regulated genes in GIH- knockdown shrimp were identified from the forward and reverse SSH libraries, respectively. Determination of the expression profiles of these reproduction-related genes during ovarian development revealed that the expression of calreticulin (CALR) was significantly reduced in vitellogenic ovary suggesting its role in vitellogenesis. Suppression of CALR by specific dsRNA showed elevated vitellogenin (Vg) transcript level in the ovary at day 7 post-dsRNA injection. Since CALR can bind to steroid hormone receptors and prevents the binding of the receptor to its responsive element to regulate gene expression, it is possible that CALR is an inhibitory mediator of vitellogenin synthesis via steroidal pathway. Our results posted a possible novel pathway of GIH signaling that might interfere the steroid signaling cascade to mediate Vg synthesis in the shrimp.

The identification of somatic calreticulin (CALR) mutations can be used to confirm the diagnosis of a myeloproliferative disorder in Philadelphia chromosome-negative, JAK2 and MPL wild type patients with thrombocytosis. All pathogenic CALR mutations result in an identical C-terminal protein and therefore may be identifiable by immunohistochemistry. We sought to test the sensitivity and specificity of mutation specific immunohistochemistry for pathogenic CALR mutations using a commercially available mouse monoclonal antibody (clone CAL2). Immunohistochemistry for mutant calreticulin was performed on the most recent bone marrow trephine from a cohort of patients enriched for CALR mutations and compared to mutation testing performed by polymerase chain reaction (PCR) amplification followed by fragment length analysis. Twenty-nine patients underwent both immunohistochemistry and molecular testing. Eleven patients had CALR mutation, and immunohistochemistry was positive in nine (82%). One discrepant case appeared to represent genuine false negative immunohistochemistry. The other may be attributable to a 12 year delay between the bone marrow trephine and the specimen which underwent molecular testing, particularly because a liver biopsy performed at the same time as molecular testing demonstrated positive staining in megakaryocytes in extramedullary haematopoiesis. All 18 cases which lacked CALR mutation demonstrated negative staining. In this population enriched for CALR mutations, the specificity was 100%; sensitivity 82-91%, positive predictive value 100% and negative predictive value 90-95%. We conclude that mutation specific immunohistochemistry is highly specific for the presence of CALR mutations. Whilst it may not identify all mutations, it may be very valuable in routine clinical care.

The term "idiopathic erythrocytosis (IE)" is applied to those cases where a causal clinical or pathological event cannot be elucidated and likely reflects a spectrum of underlying medical and molecular abnormalities. The clinical course of a patient with IE is described manifesting as a persistent erythrocytosis with a low serum erythropoietin level, mild eosinophilia, and with evidence of a thrombotic event. The patient subsequently developed a myelodysplasic syndrome (MDS) and acute myeloid leukemia (AML), an event not observed in erythrocytosis patients other than those with polycythemia vera (PV). Application of a next-generation sequencing (NGS) approach targeted for myeloid malignancies confirmed wild-type JAK2 exons 12-15 and identified a common SH2B3 W262R single-nucleotide polymorphism associated with the development of hematological features of myeloproliferative neoplasms (MPNs). Further NGS analysis detected a CBL L380P mutated clone expanding in parallel with the development of MDS and subsequent AML. Despite the absence of JAK2, MPL exon 10, or CALR exon 9 mutations, a similarity with the disease course of PV/MPN was evident. A clonal link between the erythrocytosis and AML could be neither confirmed nor excluded. Future molecular identification of the mechanisms underlying IE is likely to provide a more refined therapeutic approach.

BIOLOGICAL ASPECTS OF JAK/STAT SIGNALING IN BCR-ABL-NEGATIVE MYELOPROLIFERATIVE NEOPLASMS: Myeloproliferative disorders more recently named Myeloproliferative neoplasms (MPN) display several clinical entities: chronic myeloid leukemia (CML), the classical MPN including polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PMF) and atypical and unclassifiable NMP. The term MPN is mostly used for classical BCR-ABL-negative (myeloproliferative disorder) (ET, PV, PMF). These are clonal diseases resulting from the transformation of an hematopoietic stem cell and leading to an abnormal production of myeloid cells. The genetic defects responsible for the myeloproliferative abnormalities are called « driver » mutations and all result in deregulation of the cytokine receptor / JAK2 / STAT axis. Among them, JAK2, the thrombopoietin receptor (MPL) and calreticulin (CALR) mutations are found in around 90% of the cases. These driver MPN mutations can be associated with other driver mutations also found in other hematological malignancies, especially in PMFs. These are chronic diseases with major risks being thrombosis, hemorrhage and cytopenias for PMF and the long-term progression to myelofibrosis and the transformation to leukemia. Most recent therapeutic have focused on targeting the JAK2 signaling pathway directly by inhibitors of JAK2 or indirectly. Interferon a allows in some cases hematologic and molecular remission patients.

The molecular landscape of patients with myelofibrosis (MF) includes "phenotypic driver" and "subclonal" mutations. The three driver (JAK2, MPL and CALR)-mutated genes currently represent major diagnostic criteria, unlike subclonal mutations that are not specific for the disease and occur in other myeloid neoplasms. Recent data indicate that selected mutations deserve prognostic significance allowing to identify categories of patients with different survival and risk of leukemia. This review focuses on current knowledge regarding genotype-prognostic correlates in MF, however, with the understanding that this is a rapid moving field and no definite recommendations for the clinicians can be done yet.

Hemorrhage is a well-known complication of essential thrombocythemia (ET) and polycythemia vera (PV), but evidence-based data on its management and prevention are lacking to help inform clinicians. In this review, appropriate published data from the past 15 years regarding bleeding epidemiology, classification, location, and risk factors are presented and discussed. Research was conducted using the Medline database. The bleeding classifications were heterogeneous among the collected studies. The median incidences of bleeding and major bleeding were 4.6 and 0.79% patients/year, in ET patients and 6.5 and 1.05% patients/year in PV patients, respectively. The most frequent location was the gastrointestinal tract. Bleeding accounted for up to 13.7% of deaths, and cerebral bleeding was the main cause of lethal hemorrhage. Thirty-nine potential risk factors were analyzed at least once, but the results were discrepant. Among them, age >60 years, bleeding history, splenomegaly, myeloproliferative neoplasm subtype, and platelet count should deserve more attention in future studies. Among the treatments, aspirin seemed to be problematic for young patients with ET (especially CALR-mutated ET patients) and anagrelide was also identified as a bleeding inducer, especially when associated with aspirin. Future studies should analyze bleeding risk factors in more homogeneous populations and with common bleeding classifications. More tools are needed to help clinicians manage the increased risk of potentially lethal bleeding events in these diseases.

Activation of JAK-STAT signaling is one of the hallmarks of myelofibrosis, a myeloproliferative neoplasm that leads to inflammation, progressive bone marrow failure, and a risk of leukemic transformation. Around 90% of patients with myelofibrosis have a mutation in JAK2, MPL, or CALR: so-called 'driver' mutations that lead to activation of JAK2. Ruxolitinib, and other JAK2 inhibitors in clinical use, provide clinical benefit but do not have a major impact on the abnormal hematopoietic clone. This phenomenon is termed 'persistence', in contrast to usual patterns of resistance. Multiple groups have shown that type 1 inhibitors of JAK2, which bind the active conformation of the enzyme, lead to JAK2 becoming resistant to degradation with consequent accumulation of phospho-JAK2. In turn, this can lead to exacerbation of inflammatory manifestations when the JAK inhibitor is discontinued, and it may also contribute to disease persistence. The ways in which JAK2 V617F and CALR mutations lead to activation of JAK-STAT signaling are incompletely understood. We summarize what is known about pathological JAK-STAT activation in myelofibrosis and how this might lead to future novel therapies for myelofibrosis with greater disease-modifying potential.

Janus kinase 2 (JAK2) and signal transducer and activator of transcription-5 (STAT5) play a key role in the pathogenesis of myeloproliferative neoplasms (MPN). In most patients, JAK2 V617F or CALR mutations are found and lead to activation of various downstream signaling cascades and molecules, including STAT5. We examined the presence and distribution of phosphorylated (p) STAT5 in neoplastic cells in patients with MPN, including polycythemia vera (PV, n = 10), essential thrombocythemia (ET, n = 15) and primary myelofibrosis (PMF, n = 9), and in the JAK2 V617F-positive cell lines HEL and SET-2. As assessed by immunohistochemistry, MPN cells displayed pSTAT5 in all patients examined. Phosphorylated STAT5 was also detected in putative CD34⁺/CD38^- MPN stem cells (MPN-SC) by flow cytometry. Immunostaining experiments and Western blotting demonstrated pSTAT5 expression in both the cytoplasmic and nuclear compartment of MPN cells. Confirming previous studies, we also found that JAK2-targeting drugs counteract the expression of pSTAT5 and growth in HEL and SET-2 cells. Growth-inhibition of MPN cells was also induced by the STAT5-targeting drugs piceatannol, pimozide, AC-3-019 and AC-4-130. Together, we show that CD34⁺/CD38^- MPN-SC express pSTAT5 and that pSTAT5 is expressed in the nuclear and cytoplasmic compartment of MPN cells. Whether direct targeting of pSTAT5 in MPN-SC is efficacious in MPN patients remains unknown.

Essential thrombocythemia (ET) is a blood cancer defined by a strong increase of platelet numbers. A quarter of patients suffering from ET show mutations in the last exon of calreticulin (CALR) gene. Two variants named type 1 and type 2 represent 85% of these patients. However, a large number of other variants have been determined. In this study, we have compiled variants taken from COSMIC database and literature leading to 155 different variants. This large number of variants allowed redefining 5 new classes extending the classification of type 1-like and type 2-like to a finer description. These analyses showed that last class, named E, corresponding to more than 10% of CALR variants seemed not attached to ET. Structural properties analyzed showed that CALR variants associated to ET have common features. All the compiled and refined information had been included into a freely dedicated database CALR-ETdb (https://www.dsimb.inserm.fr/CALR-ET).

Keywords: Classification; MPN; database; protein structural models; sequence analyses.

Philadelphia-negative chronic myeloproliferative neoplasms (MPNs) have been traditionally considered as indistinctly slowly progressing conditions; recent evidence proves that a subset of cases have a rapid evolution, so that MPNs' prognosis needs to be personalized. We identified a new morphological parameter, defined as Megakaryocytic Activation (M-ACT) based on the coexistence of megakaryocytic emperipolesis, megakaryocytes (MK) clusters formation and evidence of arrangement of collagen fibers around the perimeter of MK. We retrospectively analyzed the bone marrow biopsy of two MPNs cohorts of patients with polycythemia (PV) (n=64) and non-PV patients [including essential thrombocythemia (ET), and early/prefibrotic primary myelofibrosis (PMF)] (n=222). M-ACT showed a significant correlation with splenomegaly, white blood cell (WBC) count, and LDH serum levels in both groups, with JAK2 V617F allele burden in PV patients, and with CALR mutations, and platelet count in non-PV patients. Progression-free survival, defined as PV-to-secondary MF progression and non-PV-to-overt PMF, was worse in both PV and early/prefibrotic PMF patients with M-ACT in comparison to those without M-ACT (P<.0001). Interestingly, M-ACT was not found in the subgroup of ET patients. In conclusion, M-ACT can be helpful in the differential diagnosis of MPNs and can represent a new morphologic parameter with a predictive value for progression of MPNs.

Background: Primary myelofibrosis (PMF) is a myeloproliferative neoplasm (MPN) characterized by recurrent mutations in the JAK2, CALR, and MPL genes. The CALR and MPL co-mutation is very rare. To our knowledge, no more than five cases have been reported. Here, we report a case of PMF in which a CALR and MPL co-mutation was detected by next-generation sequencing (NGS) technology, and a literature review was performed.

Case summary: A 73-year-old woman was admitted to our hospital in 2018 due to abdominal distension. The patient had splenomegaly, lymphadenopathy, leukopenia, anemia, and immature granulocytes in peripheral blood. There were dacrocytes and atypical megakaryocytes in bone marrow, and megakaryocytic proliferation was very active, accompanied by reticulin fibrosis grade 2. By NGS analysis of the bone marrow sample, we detected mutations in CALR, MPL, and PIK3RI, while JAK2 V617F and BCR-ABL were negative. Therefore, the patient was diagnosed with PMF and received oral ruxolitinib. However, the spleen and hematologic responses were poor. We review the literature, analyze previous reports of the mutation sites in our patient and differences between our patient and other reported cases of co-mutated CALR and MPL genes, and discuss the reason why the CALR and MPL co-mutations are rare and possible mechanisms and their impact on the prognosis of patients.

Conclusion: CALR and MPL mutations can be concurrent in MPN, but they are rare. The use of NGS may help to identify more patients with co-mutated CALR and MPL genes. This will help to further explore the mechanism and its impact on these patients to develop appropriate treatment strategies.

Keywords: CALR; Case report; Co-mutation; MPL; Next-generation sequencing; Primary myelofibrosis.

Essential thrombocythemia (ET) is a clonal bone marrow disease, characterized by increased production of platelets along with other clinical and bone marrow findings. Most patients with ET will have a somatic mutation in one of the known gene locations of JAK2, CALR, or MPL that can upregulate the JAK-STAT pathway. MPL mutation is present in 5% of cases with the most common mutations being W515L and W515K. In this report we describe 2 cases of patients with clinical and laboratory picture of ET. One patient carried MPLY252H mutation which is previously unreported in the adult population but has been shown to be a gain-of-function mutation. The other patient carried MPL F126fs mutation which is not known to be of clinical importance and has not been previously reported.

Philadelphia-negative (Ph-) classical myeloproliferative neoplasms (MPNs) include polycythemia vera, essential thrombocythemia (ET), and primary myelofibrosis. Somatic driver mutations in the JAK2, CALR, and MPL genes serve as major diagnostic criteria of the Ph- MPNs and these mutations occur in a mutually exclusive manner. In this report, we describe the first case of ET harboring double mutations in JAK2 V617F and MPL. For MPL, the patient had multiple clones of MPL mutations: c.1543_1546delinsAGGG (p.Trp515_Gln516delinsArgGlu) and c.1546C>G (p.Gln516Glu). The JAK2 V617F allele burden in our patient is very low (4%) compared to the relatively high (17%-78%) allele frequency of MPL mutations. The low JAK2 mutant burden might be explained by preexisting clonal hematopoiesis before overt signs of MPNs, followed by the acquisition of a second oncogenic mutation of CALR or MPL leading to the MPN phenotype. This highlights that screening for a second driver mutation should be considered in patients with a low JAK2 mutant burden by reporting a 57-year-old Korean man with ET.

Keywords: Essential Thrombocythemia; JAK2; MPL; Mutation.

Besides histopathological findings, there are no indicators of increased risk for fibrotic progression in myeloproliferative neoplasms (MPNs). Age-related clonal hematopoiesis (ARCH) or clonal hematopoiesis of indetermined potential (CHIP) are frequent findings in the elderly and combinations with MPN driver mutations (JAK2, MPL, and CALR) have been described. To determine the impact of ARCH/CHIP-related mutations for the development of fibrosis in primary myelofibrosis (PMF), the mutational status of cases with fibrotic progression from grade 0 to grade 2/3 (n = 77) as evidenced by follow-up bone marrow biopsies (median 6.2 years) was compared to prefibrotic PMF samples without the development of fibrosis (n = 27; median follow-up 7.3 years). Frequent ARCH/CHIP-associated mutations (TET2, ASXL1, DNMT3A) demonstrable at presentation were not connected with fibrotic progression. However, mutations that are rarely found in ARCH/CHIP (SRSF2, U2AF1, SF3B1, IDH1/2, and EZH2) were present in 24.7% of cases with later development of fibrosis and not detectable in cases staying free from fibrosis (P = 0.0028). Determination of tumor mutational burden (TMB) in a subgroup of cases (n = 32) did not show significant differences (7.68 mutations/MB vs. 6.85 mutations/MB). We conclude that mutations rarely found in ARCH/CHIP provide an independent risk factor for rapid fibrotic progression (median 2.0 years) when already manifest at first presentation.

Keywords: Age-related clonal hematopoiesis; Hematopoietic mutations; Myelodysplasia; Primary myelofibrosis; Risk of fibrotic progression.

The epidemiology, genetics, and thrombosis risk of MPNs among Arabs are largely unknown. This may be attributed to scarce epidemiological data, particularly from our region. Our study included 381 Kuwaiti nationals with Ph-negative MPNs and a confirmed driver mutation involving JAK2 (exon 12 14), CALR, or MPL. This first regional study examines the demographics, clinical parameters, and thrombosis-related attributes of the participants. This study reported a median age of 58 years, with females and males representing 54.9% and 45.1%, respectively. ET was the most frequent subtype of Ph-negative MPNs in our population, accounting for 52.0% of the cases, followed by PV, found in 34.6% of the participants, and PMF, found in 8.4% of participants. The crude annual cumulative incidence of Ph-negative MPNs in Kuwait ranged from 0.674 to 3.177 per 100,000 population across the study period. The most common driver mutation was JAK2V617F, with a frequency of 89.5%. At diagnosis, 19.2% of the patients presented with unexplained thrombosis, and almost half were of arterial origins. Males were more likely to present with arterial thrombosis than females (61.5% vs. 35.3%), whereas venous thrombotic events were more common in females than in males (47.1% vs. 17.9%; p-value = 0.025). Ph-negative MPNs in Kuwait are rare; however, thrombosis is a frequent complication, being documented in up to 19.2% of cases at presentation, more commonly at arterial sites. These findings call for thorough evaluation of patients with unexplained derangements in their hematological parameters during follow-ups.

Keywords: Myeloproliferative neoplasms; driver mutations; epidemiology.

Myeloproliferative neoplasms are characterized by mutations in JAK2, MPL and CALR genes. Commonly in diagnostics and previous studies mainly sequencing and common PCR techniques under conventional detection limits are used.Splanchnic vein thromboses are rare, but often appear associated with myeloproliferative neoplasms and represent serious complications.Herein, blood from patients with abdominal vein thromboses in Mecklenburg-West Pomerania (federal district of northern Germany), included in an ongoing prospective prevalence study, was analyzed by next generation sequencing representing the complete protein coding regions of JAK2, MPL and CALR genes with a coverage of > 2000 reads, therefore an ultradeep targeting approach.JAK2 V617F mutations were detected in 11/44 patients. In four of these cases allele frequencies ranged below the conventional cut off of 2%. MPL W515R was detected in 3/44 cases in low frequencies.Very low allele frequencies of JAK2 and MPL variants in patients with abdominal vein thromboses may indicate early manifestations of myeloproliferative neoplasms.

Keywords: Blood coagulation; JAK2; Low variant allele frequencies; MPL; Molecular genetics; Myeloproliferative neoplasms; Next generation sequencing; Splanchnic vein thrombosis; Ultradeep targeted sequencing.