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Cell-autonomous megakaryopoiesis associated with polyclonal hematopoiesis in triple-negative essential thrombocythemia.

ABSTRACT: A subset of essential thrombocythemia (ET) cases are negative for disease-defining mutations on JAK2, MPL, and CALR and defined as triple negative (TN). The lack of recurrent mutations in TN-ET patients makes its pathogenesis ambiguous. Here, we screened 483 patients with suspected ET in a single institution, centrally reviewed bone marrow specimens, and identified 23 TN-ET patients. Analysis of clinical records revealed that TN-ET patients were mostly young female, without a history of thrombosis or progression to secondary myelofibrosis and leukemia. Sequencing analysis and human androgen receptor assays revealed that the majority of TN-ET patients exhibited polyclonal hematopoiesis, suggesting a possibility of reactive thrombocytosis in TN-ET. However, the serum levels of thrombopoietin (TPO) and interleukin-6 in TN-ET patients were not significantly different from those in ET patients with canonical mutations and healthy individuals. Rather, CD34-positive cells from TN-ET patients showed a capacity to form megakaryocytic colonies, even in the absence of TPO. No signs of thrombocytosis were observed before TN-ET development, denying the possibility of hereditary thrombocytosis in TN-ET. Overall, these findings indicate that TN-ET is a distinctive disease entity associated with polyclonal hematopoiesis and is paradoxically caused by hematopoietic stem cells harboring a capacity for cell-autonomous megakaryopoiesis.

SUBMITTER: Inano T

PROVIDER: S-EPMC8421373 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Lack of demonstrable mutations affecting JAK2, CALR, or MPL driver genes within the spectrum of BCR-ABL1-negative myeloproliferative neoplasms (MPNs) is currently referred to as a triple-negative genotype, which is found in about 10% of patients with essential thrombocythemia (ET) and 5-10% of those with primary myelofibrosis (PMF). Very few papers are presently available on triple-negative ET, which is basically described as an indolent disease, differently from triple-negative PMF, which is an aggressive myeloid neoplasm, with a significantly higher risk of leukemic evolution. The aim of the present study was to evaluate the bone marrow morphology and the clinical-laboratory parameters of triple-negative ET patients, as well as to determine their molecular profile using next-generation sequencing (NGS) to identify any potential clonal biomarkers. We evaluated a single-center series of 40 triple-negative ET patients, diagnosed according to the 2017 WHO classification criteria and regularly followed up at the Hematology Unit of our Institution, between January 1983 and January 2019. In all patients, NGS was performed using the Illumina Ampliseq Myeloid Panel; morphological and immunohistochemical features of the bone marrow trephine biopsies were also thoroughly reviewed. Nucleotide variants were detected in 35 out of 40 patients. In detail, 29 subjects harbored one or two variants and six cases showed three or more concomitant nucleotide changes. The most frequent sequence variants involved the TET2 gene (55.0%), followed by KIT (27.5%). Histologically, most of the cases displayed a classical ET morphology. Interestingly, prevalent megakaryocytes morphology was more frequently polymorphic with a mixture of giant megakaryocytes with hyperlobulated nuclei, normal and small sized maturing elements, and naked nuclei. Finally, in five cases a mild degree of reticulin fibrosis (MF-1) was evident together with an increase in the micro-vessel density. By means of NGS we were able to identify nucleotide variants in most cases, thus we suggest that a sizeable proportion of triple-negative ET patients do have a clonal disease. In analogy with driver genes-mutated MPNs, these observations may prevent issues arising concerning triple-negative ET treatment, especially when a cytoreductive therapy may be warranted.

Project description:In order to explore the gene expression signature in essential thrombocythemia (ET) patients in relation to JAK2V617F mutational status, expression profiling in circulating granulocytes was performed. Twenty ET were studied by microarray analysis and the results were confirmed by real-time quantitative RT-PCR in 40 ET patients, not receiving cytoreductive treatment. A heterogeneous molecular signature characterized by two main gene expression patterns was found: one with an up-regulation of inflammatory genes related to neutrophil activation and thrombosis, and the other one with significantly lower expression of these genes. Supervised clustering analysis showed 30 genes differentially expressed between JAK2V617F-negative and JAK2V617F-positive ET patients. Among the JAK2V617F-negative, a set of 14 genes (CISH, C13orf18, CCL3, PIM1, MAFF, SOCS3, ID2, GADD45B, KLF5, TNF, LAMB3, HRH4, TAGAP and TRIB1) showed an abnormal expression pattern. In this group of patients CISH, SOCS2, SOCS3 and PIM1 genes, all involved in JAK-STAT signaling pathway, presented a lower expression,. A two-gene predictor model was built comprising FOSB and CISH genes, which were the best discriminators of JAK2V617F status. In conclusion, JAK2V617F-negative ET patients present a characteristic gene expression profile, different from JAK2V617F-positive patients. Other pathways besides JAK-STAT might be implicated in the pathophysiology of JAK2V617F-negative ET patients. Experiment Overall Design: Twenty ET were studied by microarray analysis and the results were confirmed by real-time quantitative RT-PCR in 40 ET patients. Microarray expression profiles were obtained using Whole Human Genome oligonucleotide microarrays (G4112A, Agilent Technologies, Palo Alto, CA). In each microarray experiment, RNA obtained from granulocytes from a single ET patient was compared with a pool of granulocyte RNAs from 10 healthy individuals. Duplicate hybridizations were performed for each comparison with dye-swapping to control.

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Cell-autonomous megakaryopoiesis associated with polyclonal hematopoiesis in triple-negative essential thrombocythemia.

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