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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.

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.

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

Background/purpose Ropeginterferon alpha-2b (Ropeg) is a novel pegylated interferon-alpha recently approved for the treatment of polycythemia vera (PV) in Europe. However, other than data from clinical trials, little is known about this agent in real world practice. Methods A compassionate use program employing Ropeg for treating patients with unmet medical need was initiated in Taiwan in 2017. Herein, we collected clinical data and assessed the safety as well as efficacy of Ropeg in nine patients treated in this program. Results Collectively, among evaluable patients, both the molecular response and complete blood count remission rates were 62.5%. Most therapy-related side effects were mild, and there was no treatment discontinuation attributable to intolerable adverse events. The agent also showed efficacy in symptom amelioration and spleen size reduction. Although no specific patterns of cytokine level alteration could be identified, significantly attenuated plasma levels of inflammation markers were observed in one particular patient who happened to have normalized spleen size and most remarkable reduction in JAK2 mutant allele burden, indicating all-around improvement in every aspect of this case. Furthermore, plasma hepcidin levels increased in two-thirds of PV patients, illustrating the potential of Ropeg to restore normal regulation of erythropoiesis. Using RNA sequencing on pre- and post-treatment samples from one patient, we demonstrated altered expression of genes participating in IFN response, inflammation, apoptosis, and cellular differentiation. Conclusion Conclusively, observed signs of efficacy and safety in our real-world experience prove Ropeg as a promising option for the treatment of MPN.

Background Aberrant MYC and BCL2 expression, cell of origin (COO), and National Comprehensive Cancer Network international prognostic index (NCCN-IPI) are commonly used for risk assessment and treatment decision in patients with diffuse large B-cell lymphoma (DLBCL). Although obesity has been shown to be of predictive value in DLBCL patients, it remains unclear whether it retains its prognostic relevance after those aforementioned novel factors being taken into consideration. Methods Patients with DLBCL were identified retrospectively in a single institute and data were collected through electronic databases and pharmacy records. Results Fifteen (17.6%) out of the 85 patients with DLBCL in our cohort were categorized as obese. They had lower platelet counts, were younger and more likely to harbor either BCL2- or MYC-overexpressing tumors. The NCCN-IPI scores, COO, and other clinical parameters were not significantly different between obese and non-obese patients. In spite that obesity adversely affected the treatment response to immunochemotherapy, multivariate analysis showed that only NCCN-IPI risk categories [hazard ratio (HR) 2.83 for high-intermediate or high-risk, versus low-intermediate or low-risk, P=0.034] and BCL2/MYC expressional status (HR 4.12 for BCL2high and/or MYChigh, versus both low expressors, P=0.004) independently predicted progression-free survival (PFS) outcome, whereas obesity lost its prognostic value in this regard (HR 1.81 for obese patients, P=0.242). Similarly, high-intermediate to high NCCN-IPI risk (HR 3.11, P=0.034) and increased expression in either BCL2 or MYC (HR 5.63, P=0.001) both portended an inferior overall survival (OS), but the presence of obesity did not affect the outcome (HR 1.65, P=0.352). Conclusions Our study has demonstrated that, for the first time, obesity increases the frequency of BCL2- or MYC-overexpressing tumors in patients with DLBCL.

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.

High mobility group AT-hook 2 (HMGA2) is an architectural transcription factor that is negatively regulated by let-7 microRNA through binding to it’s 3′-untranslated region. Transgenic mice expressing Hmga2 with a truncation of its 3′-untranslated region has been shown to exhibit a myeloproliferative phenotype. To decipher the let-7-HMGA2 axis in myeloproliferative neoplasms, we employed an in vitro model supplemented with clinical correlation. Ba/F3 cells with inducible JAK2V617F expression (Ton.JAK2.V617F cells) showed upregulation of HMGA2 with concurrent let-7a repression. Ton.JAK2.V617F cells treated with a let-7a inhibitor exhibited further escalation of Hmga2 expression, while a let-7a mimic diminished the Hmga2 transcript level. Hmga2 overexpression conferred JAK2-mutated cells with a survival advantage through inhibited apoptosis. A pan-JAK inhibitor, INC424, increased the expression of let-7a, downregulated the level of Hmga2, and led to increased apoptosis in Ton.JAK2.V617F cells in a dose-dependent manner. In samples from 151 patients with myeloproliferative neoplasms, there was a modest inverse correlation between the expression levels of let-7a and HMGA2. Overexpression of HMGA2 was detected in 29 (19.2%) of the cases, and it was more commonly seen in patients with essential thrombocythemia than in those with polycythemia vera (26.9% vs. 12.7%, P=0.044). Patients with upregulated HMGA2 showed an increased propensity for developing major thrombotic events, and they were more likely to harbor one of the 3 driver myeloproliferative neoplasm mutations in JAK2, MPL and CALR. Our findings suggest that, in a subset of myeloproliferative neoplasm patients, the let-7-HMGA2 axis plays a prominent role in the pathogenesis of the disease that leads to unique clinical phenotypes.

Background: Somatic mutations of Janus kinase 2 (JAK2V617F), calreticulin (CALR), and myeloproliferative leukemia virus oncogene (MPL) are the major clonal molecules that drive the pathogenesis of myeloproliferative neoplasms (MPN). It is well recognized that MPN patients carry an excessive risk of thrombohemorrhagic complications. However, little is known about the prevalence of these clonal markers in patients with cerebral vascular disease. Methods: To address this issue, 153 consecutive stroke patients in Taiwan were enrolled in the study. Allele-specific PCR (AS-PCR), real-time AS-PCR, and Illumina paired-end sequencing were employed to detect the presence of MPL, JAK2V617F, and CALR exon 9 mutations, respectively. Results: JAK2V617F mutation was detectable in 13 samples (8.5%), but the allele burdens (AB) were greater than 1% in only six (3.9%) of them. Compared to JAK2-unmutated patients, those with JAK2V617F AB > 1% had significantly higher white blood count (p = 0.01), although four of the six did not exhibit MPN phenotypes. Two patients had a heterozygous CALR exon9 mutation locating outside the coding region and did not alter the amino acid sequence of this protein. On the other hand, there were no patients carrying the MPL mutations. Using patient age, baseline hemogram, and stroke-relevant risk factors, we developed a predictive model that could successfully identify stroke patients at risk of carrying clonal JAK2V617F mutation. Conclusions: The prevalence of JAK2V617F mutation in stroke patients was higher than that seen in general population. Based on our newly developed probability stratification model, genotyping of JAK2V617F mutation in selected patients with stroke might be warranted.

Background Myeloproliferative neoplasm (MPN) is a heterogeneous group of clonal disorders. The underlying mechanisms of pathogenesis, especially in subtype specification and stochastic malignant transformation, are still largely unknown. Single-cell RNA sequencing (scRNA-seq) is a novel tool that can be used to identify the transcriptomic signature of individual cells. In the current study, we aimed to employ scRNA-seq to analyze genetic profiling of individual cells at different hematopoietic hierarchy in essential thrombocythemia (ET) patients. Methods We enrolled 7 ET patients and one healthy adult. Individual CD34+ progenitor cells were enriched from peripheral blood (PB). Harvested viable cells were barcoded and sequenced with the Illumina HiSeq 4000. Data was visualized with 10x Genomics Loupe software. We performed t-distributed stochastic neighbor embedding (t-SNE) plotting to dissect the scRNA-seq data and cluster cells with transcriptional similarity. Cellular sub-populations were stratified by the surface markers, including hematopoietic stem cells (HSC, CD34+CD38-Lin-), common myeloid progenitors (CMP, CD34+KIT+FLT3+IL3RAlowLin-), megakaryocyte-erythroid progenitor (MEP, CD34+CD38+PTPRC-) and granulocyte-macrophage progenitor (GMP, CD34+CD38+PTPRC+). Differentially expressed genes were subjected to gene ontology analysis. Gene Set Enrichment Analysis (GSEA) and the Reactome analysis were also employed. To clarify the distinct genetic background of these patients, targeted deep sequencing of PB granulocytes was also performed. Results Among the 7 ET patients, two carry JAK2 mutation [one heterozygous (h-JAK2)and one homozygous (H-JAK2)] and three carry CALR mutation (1 type I, 2 type II). The remaining two cases are triple-negative (TN). Integrative analysis showed significant activation of JAK-STAT signaling in ET patients. Compared with control, the t-SNE analysis revealed disparate expression profiling in ET patients across various hematopoietic lineages. The discrepancy grew wider as the hematopoiesis became more lineage-restricted. Significant heterogeneity existed even among different ET patients, suggesting the high diversity of the disease. In the two JAK2-mutated patients, the t-SNE analysis demonstrated divergent transcriptomic profiling which scarcely overlapped. In the H-JAK2 sample, the HSCs exhibited a distinct profile different from the rest of hematopoietic progenitors. The CMPs were more closely related to MEP, which possibly suggested skewed differentiation and resultant ET phenotype. The phenomenon, however, was not similarly seen in the h-JAK2 sample. Using GSEA, we identified a subset of miR-21-targeted genes that were down-regulated in the h-JAK2 sample. Furthermore, there was apparent aberrant signaling activity of TGF-β, widely considered a regulator of miR-21, in this ET sample as compared with the H-JAK2 sample. Therefore, it was probably not a coincidence that, two months after blood sampling, the h-JAK2 patient suffered from disease transformation to secondary myelofibrosis. Among the three CALR-mutated patients, the expression patterns and the mutational profiles were also significantly discrepant. In the patient with type I CALR mutation, the CD34+ cells exhibited aberrant activity in epigenetic regulators. Coupled with the identified somatic mutations in some epigenetic modifiers from the targeted sequencing results, it is speculated that these mutations occur in a very early hematopoietic stage and contribute to ET pathogenesis through abnormal epigenetic regulation in this patient. Lastly, principal component analysis showed that the pathognomonic molecular events initiated at different hierarchical level of hematopoiesis in the 2 TN ET patients. Reactome analysis also disclosed one patient had altered DNA repair activity, and targeted sequencing confirmed the presence of TP53 mutation. Clinically, this patient exhibited highly aggressive, treatment-refractory disease. Conclusion We demonstrate that scRNA-seq extends our knowledge of clonal diversity and inter-individual heterogeneity in patients with ET. Combined with the results from targeted sequencing, we were able to uncover unique transcriptomic pattern in samples carrying specific somatic mutations. The obtained information could potentially leapfrog our effort in the elucidation of the pathogenesis of ET. Disclosures No relevant conflicts of interest to declare.

Mutually exclusive mutations in JAK2 (JAK2V617F and exon 12), MPL, and calreticulin (CALR) constitute the driving force of Philadelphia-negative myeloproliferative neoplasms (MPN), as they have been shown to promote disease propagation through dysregulated JAK-STAT signaling pathways. However, none of these mutations have been proved to be specific to disease subtype, and they cannot be used in the molecular sub-classification of MPNs. It remains a major mystery why the same driver mutation could lead to discrepant phenotypes. High mobility group AT-hook 2 (HMGA2) is an architectural transcriptional factor that is negatively regulated by Let-7 microRNA through binding to its 3’-untranslated region and is known to promote cell proliferation and survival. Transgenic mice expressing HMGA2 with a truncation of its 3’-UTR has been leading to increased megakaryopoiesis as well as MPN phenotypes in animal models. To decipher the Let-7-HMGA2 axis in myeloproliferative neoplasms (MPN), we employed an in vitro model supplemented with clinical correlation. Ba/F3 cells with inducible JAK2V617F expression (Ton.JAK2.V617F cells) showed up-regulation of HMGA2 with concurrent let-7a repression. Ton.JAK2.V617F cells treated with a let-7a inhibitor exhibited further escalation of HMGA2 expression, while a let-7a mimic diminished the HMGA2 transcript level. HMGA2 overexpression conferred JAK2-mutated cells a survival advantage through inhibited apoptosis. Pan-JAK inhibitor INC424 increased the expression of let-7a, down-regulated the level of HMGA2, and led to increased apoptosis in Ton.JAK2.V617F cells in a dose-dependent manner. Furthermore, up-regulation of HMGA2 was significantly associated with MPN patients carrying the JAK2V617F mutation. In vitro studies showed that Ba/F3 cells carried JAK2V617F (Ba/F3-JAK2V617F) had decreased let-7a and up-regulated HMGA2. Silencing of HMGA2 in Ba/F3-JAK2V617F cells resulted in growth inhibition coupled with a significant increase in apoptosis. In our model cells, HMGA2 up-regulation is seen in both JAK2-mutated and CALR-mutated cells, indicating its profound participation in the disease patterning and a phenotype modifier in MPN. Hence, we studied a cohort of 151 MPN patients. Overexpressed HMGA2 was detected in about one-fifth of the cases, and it was more commonly seen in ET (26.9%, vs. 12.7% in PV, p=0.044). Compared to their counterparts, HMGA2-overexpressing patients had higher platelet counts, increased thromboembolic risk, and inferior thrombosis-free survival. Fluorescence in situ hybridization analysis showed that chromosomal translocation was not a major cause of HMGA2 overexpression in MPN patients, yet there was an inverse correlation between the expression levels of let-7a and HMGA2. Our findings suggest that, in a subset of MPN patients, Let-7-HMGA2 axis plays a prominent role in the pathogenesis of the disease that leads to unique clinical phenotypes in JAK2 and CALR mutations. Citation Format: Chia-Chen Hsu, Jie-Yu You, Cih-En Huang, Yi-Yang Chen, Hsing-Ying Ho, Chian-Pei Li, Chang-Hsien Lu, Kuan-Der Lee, Jyh-Pyng Gau, Yu-Wei Leu, Chih-Cheng Chen. Driver mutation and collaborating signaling in phenotype patterning in human myeloproliferative neoplasms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-043. doi:10.1158/1538-7445.AM2017-LB-043

High mobility group AT-hook 2 (HMGA2) is an architectural transcriptional factor that is negatively regulated by Let-7 microRNA. Dysregulated HMGA2 has been shown to exhibit a myeloproliferative phenotype in engineered mice. To decipher the Let-7-HMGA2 axis in myeloproliferative neoplasms (MPN), we studied a cohort of 151 MPN patients. Overexpressed HMGA2 was detected in about one-fifth of the cases, and it was more commonly seen in ET (26.9%, vs. 12.7% in PV, p=0.044). Compared to their counterparts, HMGA2-overexpressing patients had higher platelet counts, increased thromboembolic risk, and inferior thrombosis-free survival. Fluorescence in situ hybridizationanalysis showed that chromosomal translocation was not a major cause of HMGA2 overexpression in MPN patients, yet there was an inverse correlation between the expression levels of let-7a and HMGA2. Furthermore, up-regulation of HMGA2 was significantly associated with MPN patients carrying JAK2V617F mutation. In vitro studies showed that Ba/F3 cells carried JAK2V617F (Ba/F3-JAK2V617F) had decreased let-7a and up-regulated HMGA2. Silencing of HMGA2 in Ba/F3-JAK2V617F cells resulted in growth inhibition coupled with a significant increase in apoptosis. Our findings suggest that, in a subset of JAK2-mutated MPN patients, Let-7-HMGA2 axis plays a prominent role in the pathogenesis of the disease that leads to unique clinical phenotypes. Disclosures Hsu: Ministry of Science and Technology (Taiwan): Research Funding; Chang-Gung Memorial Hospital: Employment, Research Funding. Chen:Ministry of Science and Technology (Taiwan): Research Funding; Chang Gung Memorial Hospital, Chiayi branch: Research Funding. Gau:Taipei Veterans General Hospital, Taipei, Taiwan: Employment. Huang:Chang-Gung Memorial Hospital: Employment, Research Funding. You:Lotung Poh-Ai Hospital, Yilan, Taiwan: Employment. Lung:Chang-Gung Memorial Hospital: Employment, Research Funding. Chen:Chang-Gung Memorial Hospital: Employment. Ho:Chang-Gung Memorial Hospital: Employment, Research Funding. Li:Chang-Gung Memorial Hospital: Employment, Research Funding. Lu:Chang Gung Memorial Hospital, Chiayi, Taiwan: Employment. Lee:Chang-Gung Memorial Hospital: Employment, Research Funding.