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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. [ABSTRACT FROM AUTHOR]

Simple Summary: Philadelphia-chromosome-negative myeloproliferative neoplasms (MPNs) are a group of clonal hematopoietic stem cell disorders characterized by the excessive production of differentiated myeloid cells. Other than the well-known propensity for leukemia transformation, MPN patients are also prone to developing second cancers (SCs) that arise from different embryonic dermal origins. This brings up an intriguing question: what is the molecular background leading to the development of SCs in MPN patients? To explore further, we used whole exome sequencing to characterize the genomic landscapes in 26 paired MPN samples stratified by the presence or absence of SCs. We found that mutated genes in MPN–SC samples were enriched in some critical immune-related pathways and inflammatory networks, an observation further supported by the increased plasma levels of cytokines. Our work provides the genomic landscape that profiles the genetic basis for SC tumorigenesis in MPN patients, and also demonstrates that inflammation could be indispensable in MPN–SC pathogenesis. Patients with myeloproliferative neoplasms (MPNs) are characterized by systemic inflammation. With the indolent nature of the diseases, second cancers (SCs) have emerged as a challenging issue in afflicted patients. Epidemiological studies have confirmed the excessive risk of SCs in MPNs, but little is known about their molecular basis. To explore further, we used whole exome sequencing to explore the genetic changes in the granulocytes of 26 paired MPN patients with or without SC. We noticed that MPN–SC patients harbor genomic variants of distinct genes, among which a unique pattern of co-occurrence or mutual exclusiveness could be identified. We also found that mutated genes in MPN–SC samples were enriched in immune-related pathways and inflammatory networks, an observation further supported by their increased plasma levels of TGF-β and IL-23. Noteworthily, variants of KRT6A, a gene capable of mediating tumor-associate macrophage activity, were more commonly detected in MPN–SC patients. Analysis through OncodriveCLUST disclosed that KRT6A replaces JAK2V617F as the more prominent disease driver in MPN–SC, whereas a major mutation in this gene (KRT6A c.745T>C) in our patients is linked to human carcinoma and predicted to be pathogenic in COSMIC database. Overall, we demonstrate that inflammation could be indispensable in MPN–SC pathogenesis. [ABSTRACT FROM AUTHOR]

Hepatitis C virus-associated immune thrombocytopenia (HCV-ITP) has been assumed to be one of secondary ITP and associated with antiplatelet antibodies. This study was to clarify the antibody profile in HCV-ITP compared with primary ITP. We enrolled 55 HCV-ITP, 30 primary ITP, 11 Helicobacter pylori-ITP, 21 HCV control, and 16 healthy volunteers. We reviewed their blood cell counts, autoimmune markers, and spleen size. We used enzyme-linked immunosorbent assay kit to detect the specific antibody to glycoproteins IIb/IIIa, Ia/IIa, Ib/IX, IV, and human leukocyte antigen (HLA) class I. Compared with primary ITP patients, HCV-ITP patients had an older age, lower white blood cell (WBC) count and fewer presented with severe thrombocytopenia. The rate of positive antibody detection was 63.6% for the HCV-ITP group higher than the rate of 40% for the primary ITP. In the HCV control, antiplatelet antibodies were detected in 38.1% patients and no one had more than two types of antibodies. The antiplatelet antibodies correlated to severer thrombocytopenia. An HLA class I antibody was associated with lower WBCs and larger spleen. In conclusion, HCV-ITP patients had a high rate of positive antiplatelet antibody. The antibodies were associated with not only lower platelets but also leukopenia and splenomegaly. [ABSTRACT FROM AUTHOR]

Summary: Significant phenotypic heterogeneity exists in patients with all subtypes of myeloproliferative neoplasms (MPN), including essential thrombocythaemia (ET). Single‐cell RNA sequencing (scRNA‐Seq) holds the promise of unravelling the biology of MPN at an unprecedented level of resolution. Herein we employed this approach to dissect the transcriptomes in the CD34+ cells from the peripheral blood of seven previously untreated ET patients and one healthy adult. The mutational profiles in these patients were as follows: JAK2 V617F in two, CALR in three (one type I and two type II) and triple‐negative (TN) in two. Our results reveal substantial heterogeneity within this enrolled cohort of patients. Activation of JAK/STAT signalling was recognized in discrepant progenitor lineages among different samples. Significantly disparate molecular profiling was identified in the comparison between ET patients and the control, between patients with different driver mutations (JAK2 V617F and CALR exon 9 indel), and even between patients harbouring the same driver. Intra‐individual clonal diversity was also found in the CD34+ progenitor population of a patient, possibly indicating the presence of multiple clones in this case. Estimation of subpopulation size based on cellular immunophenotyping suggested differentiation bias in all analysed samples. Furthermore, combining the transcriptomic information with data from targeted sequencing enabled us to unravel key somatic mutations that are molecularly relevant. To conclude, we demonstrated that scRNA‐Seq extended our knowledge of clonal diversity and inter‐individual heterogeneity in patients with ET. The obtained information could potentially leapfrog our efforts in the elucidation of the pathogenesis of the disease. [ABSTRACT FROM AUTHOR]