Activated mesenchymal stem/stromal cells promote myeloid cell differentiation via CCL2/CCR2 signaling.

Myeloid cells, which originate from hematopoietic stem/progenitor cells (HSPCs), play a crucial role in mitigating infections. This study aimed to explore the impact of mesenchymal stem/stromal cells (MSCs) on the differentiation of HSPCs and progenitors through the C-C motif chemokine CCL2/CCR2 signaling pathway. Murine MSCs, identified as PDGFRα+Sca-1+ cells (PαS cells), were found to secrete CCL2, particularly in response to lipopolysaccharide stimulation. MSC-secreted CCL2 promoted the differentiation of granulocyte/macrophage progenitors into the myeloid lineage. MSC-derived CCL2 plays an important role in the early phase of myeloid cell differentiation in vivo. Single-cell RNA sequencing analysis confirmed that CCL2-mediated cell fate determination was also observed in human bone marrow cells. These findings provide valuable insights for investigating the in vivo effects of MSC transplantation.

Impact of non-driver gene mutations on thrombo-haemorrhagic events in ET patients.

Risk-adapted therapy is recommended to prevent major clinical complications, such as thrombo-haemorrhagic events, in patients with essential thrombocythaemia (ET). In this study, we analysed the association between non-driver gene mutations and thrombo-haemorrhagic events in 579 patients with ET. ASXL1 and TP53 mutations were frequently identified in patients with ET complicated by thrombosis (22.7% and 23.1%, respectively), and the DNMT3A mutation was frequently identified in patients who experienced haemorrhage (15.2%). Multivariate analyses of thrombosis-free survival (TFS) revealed that ASXL1 and TP53 mutations are associated with thrombosis (hazard ratio [HR] = 3.140 and 3.752 respectively). Patients harbouring the ASXL1 or TP53 mutation had significantly worse TFS rates than those without mutation (p = 0.002 and p < 0.001 respectively). Furthermore, JAK2V617F-mutated patients with accompanying ASXL1 mutations showed significantly shorter TFS compared with those without ASXL1 mutations (p = 0.003). Multivariate analyses of haemorrhage-free survival (HFS) revealed that the DNMT3A mutation (HR = 2.784) is associated with haemorrhage. DNMT3A-mutated patients showed significantly shorter HFS than those without the mutation (p = 0.026). Non-driver gene mutations should be considered in treatment strategies and may provide important information for personalised treatment approaches.

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.

Establishment of isogenic induced pluripotent stem cells with or without pathogenic mutation for understanding the pathogenesis of myeloproliferative neoplasms.

Identification and functional characterization of disease-associated genetic traits are crucial for understanding the pathogenesis of hematologic malignancies. Various in vitro and in vivo models, including cell lines, primary cells, and animal models, have been established to examine these genetic alterations. However, their nonphysiologic conditions, diverse genetic backgrounds, and species-specific differences often limit data interpretation. To evaluate somatic mutations in myeloproliferative neoplasms (MPNs), we used CRISPR/Cas9 combined with the piggyBac transposon system to establish isogenic induced pluripotent stem (iPS) cell lines with or without JAK2V617F mutation, a driver mutation of MPNs. We induced hematopoietic stem/progenitor cells (HSPCs) from these iPS cells and observed phenotypic differences during hematopoiesis using fluorescence-activated cell sorting analysis. HSPCs with pathogenic mutations exhibited cell-autonomous erythropoiesis and megakaryopoiesis, which are hallmarks in the bone marrow of patients with MPNs. Furthermore, we used these HSPCs as a model to validate therapeutic compounds and showed that interferon alpha selectively inhibited erythropoiesis and megakaryopoiesis in mutant HSPCs. These results demonstrate that genome editing is feasible for establishing isogenic iPS cells, studying genetic elements to understand the pathogenesis of MPNs, and evaluating therapeutic compounds against MPNs.

Secreted mutant calreticulins as rogue cytokines in myeloproliferative neoplasms.

Mutant calreticulin (CALR) proteins resulting from a -1/+2 frameshifting mutation of the CALR exon 9 carry a novel C-terminal amino acid sequence and drive the development of myeloproliferative neoplasms (MPNs). Mutant CALRs were shown to interact with and activate the thrombopoietin receptor (TpoR/MPL) in the same cell. We report that mutant CALR proteins are secreted and can be found in patient plasma at levels up to 160 ng/mL, with a mean of 25.64 ng/mL. Plasma mutant CALR is found in complex with soluble transferrin receptor 1 (sTFR1) that acts as a carrier protein and increases mutant CALR half-life. Recombinant mutant CALR proteins bound and activated the TpoR in cell lines and primary megakaryocytic progenitors from patients with mutated CALR in which they drive thrombopoietin-independent colony formation. Importantly, the CALR-sTFR1 complex remains functional for TpoR activation. By bioluminescence resonance energy transfer assay, we show that mutant CALR proteins produced in 1 cell can specifically interact in trans with the TpoR on a target cell. In comparison with cells that only carry TpoR, cells that carry both TpoR and mutant CALR are hypersensitive to exogenous mutant CALR proteins and respond to levels of mutant CALR proteins similar to those in patient plasma. This is consistent with CALR-mutated cells that expose TpoR carrying immature N-linked sugars at the cell surface. Thus, secreted mutant CALR proteins will act more specifically on the MPN clone. In conclusion, a chaperone, CALR, can turn into a rogue cytokine through somatic mutation of its encoding gene.

Reevaluation of cardiovascular risk factors for thrombotic events in 580 Japanese patients with essential thrombocythemia.

Risk-adapted therapy is recommended to prevent thrombosis in essential thrombocythemia (ET) patients. An advanced age, a history of thrombosis, and the presence of the JAK2V617F mutation are well-defined risk factors for thrombosis in ET; however, the impact of cardiovascular risk (CVR) factors on thrombosis in ET remains elusive. Therefore, we herein investigated the impact of CVR factors on thrombosis in 580 ET patients who met the 2017 World Health Organization Classification diagnostic criteria. A univariate analysis identified hypertriglyceridemia and multiple CVR factors as strong risk factors for thrombosis (hazard ratio [HR] 3.530, 95% confidence interval [CI] 1.630-7.643, P = 0.001 and HR 3.368, 95% CI 1.284-8.833, P = 0.014, respectively) and hyper-LDL cholesterolemia as a potential risk factor (HR 2.191, 95% CI 0.966-4.971, P = 0.061). A multivariate analysis revealed that hypertriglyceridemia was an independent risk factor for thrombosis (HR 3.364, 95% CI 1.541-7.346, P = 0.002). Furthermore, poor thrombosis-free survival was observed in patients with a serum triglyceride level ≥ 1.2 mmol/L (HR = 2.592, P = 0.026 vs. < 1.2 mmol/L) or two or more CVR factors (P = 0.011 vs. no CVR factors and P = 0.005 vs. one CVR factor). These results revealed the impact of CVR factors on thrombosis in ET. Since CVR factors are manageable, lifestyle interventions, such as the control of serum triglyceride levels, may effectively prevent thrombosis in ET patients.

Clinical features of acquired erythrocytosis: Low levels of serum erythropoietin in a subset of non-neoplastic erythrocytosis patients.

BACKGROUND: Acquired erythrocytosis can be classified into polycythemia vera (PV) and non-neoplastic erythrocytosis (NNE). The vast majority of PV patients harbor JAK2 mutations, but differentiating JAK2 mutation-negative PV from NNE is challenging due to a lack of definitive molecular markers. METHODS: We studied the clinical features of 121 patients with erythrocytosis of which 47 (38.8%) were JAK2 mutation-positive and also fulfilled the diagnostic criteria for PV, and 67 (55.4%) JAK2 mutation-negative erythrocytosis patients who were diagnosed as NNE. Diagnosis was strictly based on driver mutation analysis and central pathology review. RESULTS: No JAK2 mutation-negative PV patients were found in our cohort. The NNE group showed significantly younger (p < 0.01) age with higher frequency of smoking (p < 0.001), alcohol consumption (p < 0.001), and diabetes mellitus (p < 0.05), whereas the PV group (n = 47) showed significantly higher white blood cell count, platelet count, and lactate dehydrogenase (p < 0.001). Although serum erythropoietin (EPO) levels were significantly higher in NNE compared to PV (p < 0.001), approximately 40% of the NNE patients had EPO levels below the lower range of normal, fulfilling a minor diagnostic criterion of PV and raising the possibility of PV misdiagnosis. CONCLUSION: Low EPO levels in JAK2 mutation-negative erythrocytosis may not be a reliable diagnostic criterion for distinguishing PV from NNE.

Phase 1 Clinical Trial of PPMX-T003, a Novel Human Monoclonal Antibody Specific for Transferrin Receptor 1, to Evaluate Its Safety, Pharmacokinetics, and Pharmacodynamics.

This study aimed to evaluate the safety, pharmacokinetics, and pharmacodynamics of PPMX-T003, a novel human monoclonal antibody for transferrin receptor 1 (TFR1), in healthy individuals. Forty participants were enrolled and randomized to PPMX-T003 dose groups (n = 6/group) and the placebo group (n = 10). The safety and pharmacokinetics profiles were assessed according to the sequential, ascending single-dose intravenous infusions of PPMX-T003 from 0.008 mg/kg to 0.25 mg/kg. Adverse events (AEs) after PPMX-T003 administration occurred in 16 of 30 participants. Any severe AE and AE incidence were not reported, but they tended to increase depending on the dose. Laboratory tests, vital signs, and standard 12-lead electrocardiogram showed no clinically relevant changes. Five participants experienced an infusion-related reaction but recovered on days 5-10. Regarding pharmacokinetics, PPMX-T003 has a nonlinear elimination pattern. PPMX-T003 in the 0.25 mg/kg group showed apparent (>50%) decreased serum levels of reticulocytes from day 3 and sustained moderate (<10%) fall of hematocrit and hemoglobin counts from day 7. In conclusion, the antibody-mediated blockade of TFR1 elicited the expected fall in blood cell levels and showed an acceptable safety profile, supporting the continuing development of PPMX-T003 as a new candidate for polycythemia vera treatment.

Non-driver gene mutation analysis in a large cohort of polycythemia vera and essential thrombocythemia.

OBJECTIVES: A proportion of patients with polycythemia vera (PV) and essential thrombocythemia (ET) harbor non-driver mutations associated with poor prognosis. In this study, we analyzed the frequency of non-driver mutations in a large Japanese PV and ET cohort. Furthermore, we studied the relationship of these mutations and prognosis in Japanese patients. METHODS: We enrolled 843 Japanese patients with PV or ET. Non-driver mutations were analyzed by target resequencing using next-generation sequencing. The association of the mutations with the prognosis was estimated using multivariable logistic regression analysis and log-rank test. RESULTS: Non-driver mutations were detected in 31.1% and 24.5% patients with PV and ET, respectively. Among them, ASXL1 mutations were identified as a risk factor for leukemic/myelofibrotic transformation in PV and ET patients (hazard ratio: 4.68, p = .006). The higher-risk groups of the mutation-enhanced international prognostic system (MIPSS)-PV and MIPSS-ET incorporating non-driver mutations exhibited significantly shorter overall survival compared with the low-risk group (p < .001). CONCLUSIONS: These results implicate the importance of studying non-driver mutations for predicting the prognosis and survival of Japanese PV and ET patients.

Causes of Thrombocytosis: A Single-center Retrospective Study of 1,202 Patients.

Objective Thrombocytosis can occur as a primary event accompanying hematological diseases or as a secondary event. Since the publication of the World Health Organization classification in 2008, thrombocytosis is now generally defined as a platelet count above 450×109/L. Furthermore, the discovery of driver-gene mutations in myeloproliferative neoplasms (MPNs) has simplified the diagnostic approach for thrombocytosis. To identify the causes of thrombocytosis using this new definition, we conducted a retrospective study. Methods We identified outpatients and inpatients aged 20 years or older with platelet counts >450×109/L in a half-year period at a single institute and analyzed the causes of thrombocytosis and associated clinical characteristics. Results Among 1,202 patients with thrombocytosis, 150 (12.5%) had primary and 999 (83.1%) had secondary thrombocytosis. Of these patients with primary thrombocytosis, 129 (86%) had at least 1 molecular marker indicative of MPNs. The major causes of secondary thrombocytosis were tissue injury (32.2%), infection (17.1%), chronic inflammatory disorders (11.7%) and iron deficiency anemia (11.1%). The median platelet count and the incidence of thrombosis were significantly higher in patients with primary thrombocytosis than in those with secondary thrombocytosis. Conclusion Thrombocytosis mainly occurs as a secondary event; however, it is important to determine the cause of and prevent thrombosis, particularly in cases of primary thrombocytosis.

A marine sponge-derived lectin reveals hidden pathway for thrombopoietin receptor activation.

N-glycan-mediated activation of the thrombopoietin receptor (MPL) under pathological conditions has been implicated in myeloproliferative neoplasms induced by mutant calreticulin, which forms an endogenous receptor-agonist complex that traffics to the cell surface and constitutively activates the receptor. However, the molecular basis for this mechanism is elusive because oncogenic activation occurs only in the cell-intrinsic complex and is thus cannot be replicated with external agonists. Here, we describe the structure and function of a marine sponge-derived MPL agonist, thrombocorticin (ThC), a homodimerized lectin with calcium-dependent fucose-binding properties. In-depth characterization of lectin-induced activation showed that, similar to oncogenic activation, sugar chain-mediated activation persists due to limited receptor internalization. The strong synergy between ThC and thrombopoietin suggests that ThC catalyzes the formation of receptor dimers on the cell surface. Overall, the existence of sugar-mediated MPL activation, in which the mode of activation is different from the original ligand, suggests that receptor activation is unpredictably diverse in living organisms.

Clinical and biological relevance of CREB3L1 in Philadelphia chromosome-negative myeloproliferative neoplasms.

Cyclic AMP-response element-binding protein 3-like 1 (CREB3L1) is a gene involved in the unfolded protein response (UPR). Recently, we demonstrated that CREB3L1 is specifically overexpressed in the platelets of patients with Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs). In this study, we aimed to show the clinical and biological relevance of CREB3L1 in these hematological diseases. Overexpression of CREB3L1 was specific to platelets in MPNs and associated with a higher risk of thrombosis and fibrotic transformation in essential thrombocythemia (ET) and polycythemia vera (PV) cases, respectively. Furthermore, we found that UPR genes were downregulated in platelets of patients with ET and PV, which were more pronounced in patients harboring the JAK2 V617F mutation. However, CREB3L1 overexpression does not alter UPR gene expression or cell proliferation in UT-7/TPO/CALRm cells exogenously expressing mutated calreticulin and HEL cells harboring endogenous JAK2 V617F. Furthermore, CREB3L1 overexpression did not modulate sensitivity to endoplasmic reticulum stress in these cell lines. Taken together, our data show 1) a potential role of CREB3L1 expression in platelets as a new marker of high-risk MPNs and 2) an association between CREB3L1 overexpression and UPR gene downregulation in these patients' platelets, with CREB3L1 not altering UPR in our in vitro models and possibly further in vivo mechanisms being involved.

Eliminating chronic myeloid leukemia stem cells by IRAK1/4 inhibitors.

Leukemia stem cells (LSCs) in chronic myeloid leukemia (CML) are quiescent, insensitive to BCR-ABL1 tyrosine kinase inhibitors (TKIs) and responsible for CML relapse. Therefore, eradicating quiescent CML LSCs is a major goal in CML therapy. Here, using a G0 marker (G0M), we narrow down CML LSCs as G0M- and CD27- double positive cells among the conventional CML LSCs. Whole transcriptome analysis reveals NF-κB activation via inflammatory signals in imatinib-insensitive quiescent CML LSCs. Blocking NF-κB signals by inhibitors of interleukin-1 receptor-associated kinase 1/4 (IRAK1/4 inhibitors) together with imatinib eliminates mouse and human CML LSCs. Intriguingly, IRAK1/4 inhibitors attenuate PD-L1 expression on CML LSCs, and blocking PD-L1 together with imatinib also effectively eliminates CML LSCs in the presence of T cell immunity. Thus, IRAK1/4 inhibitors can eliminate CML LSCs through inhibiting NF-κB activity and reducing PD-L1 expression. Collectively, the combination of TKIs and IRAK1/4 inhibitors is an attractive strategy to achieve a radical cure of CML.

Validation and reliability of current guidelines for the treatment of essential thrombocythemia under real-world clinical settings in Japan.

OBJECTIVE: Current guidelines for essential thrombocythemia (ET) patients recommend different treatment approaches based on thrombosis risk stratification models. However, these recommendations may not be applicable to some patients under real clinical settings. Therefore, we carried out a retrospective real-world validation study. METHODS: Thrombosis-free survival (TFS) was compared between treatment naïve ET patients receiving different treatment approaches. ET patients were stratified by three representative risk models, the conventional, the International Prognostic Score for thrombosis in ET (IPSET-thrombosis), and revised IPSET-thrombosis. Treatment decisions were largely made by individual physicians, taking into account patient preferences and backgrounds. RESULTS: A total of 179 ET patients were included, and thrombotic events were observed in 26 patients. TFS was significantly longer in high-risk patients of all risk models receiving a combination of cytoreductive therapy (CRT) and antiplatelet therapy (APT) compared to CRT alone. Similar results were seen in intermediate-risk patients stratified by IPSET-thrombosis. In contrast, in very low- and low-risk patients of all risk models, TFS was not affected by addition of CRT, indicating that observation or APT alone is an appropriate treatment approach for these patients. CONCLUSION: We demonstrate that current guidelines provide optimal treatment approaches for Japanese ET patients under real-world clinical settings.

In silico-labeled ghost cytometry.

Characterization and isolation of a large population of cells are indispensable procedures in biological sciences. Flow cytometry is one of the standards that offers a method to characterize and isolate cells at high throughput. When performing flow cytometry, cells are molecularly stained with fluorescent labels to adopt biomolecular specificity which is essential for characterizing cells. However, molecular staining is costly and its chemical toxicity can cause side effects to the cells which becomes a critical issue when the cells are used downstream as medical products or for further analysis. Here, we introduce a high-throughput stain-free flow cytometry called in silico-labeled ghost cytometry which characterizes and sorts cells using machine-predicted labels. Instead of detecting molecular stains, we use machine learning to derive the molecular labels from compressive data obtained with diffractive and scattering imaging methods. By directly using the compressive 'imaging' data, our system can accurately assign the designated label to each cell in real time and perform sorting based on this judgment. With this method, we were able to distinguish different cell states, cell types derived from human induced pluripotent stem (iPS) cells, and subtypes of peripheral white blood cells using only stain-free modalities. Our method will find applications in cell manufacturing for regenerative medicine as well as in cell-based medical diagnostic assays in which fluorescence labeling of the cells is undesirable.

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

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.

MPL overexpression induces a high level of mutant-CALR/MPL complex: a novel mechanism of ruxolitinib resistance in myeloproliferative neoplasms with CALR mutations.

Ruxolitinib (RUX), a JAK1/2-inhibitor, is effective for myeloproliferative neoplasm (MPN) with both JAK2V617 F and calreticulin (CALR) mutations. However, many MPN patients develop resistance to RUX. Although mechanisms of RUX-resistance in cells with JAK2V617 F have already been characterized, those in cells with CALR mutations remain to be elucidated. In this study, we established RUX-resistant human cell lines with CALR mutations and characterized mechanisms of RUX-resistance. Here, we found that RUX-resistant cells had high levels of MPL transcripts, overexpression of both MPL and JAK2, and increased phosphorylation of JAK2 and STAT5. We also found that mature MPL proteins were more stable in RUX-resistant cells. Knockdown of MPL in RUX-resistant cells by shRNAs decreased JAK/STAT signaling. Immunoprecipitation assays showed that binding of mutant CALR to MPL was increased in RUX-resistant cells. Reduction of mutated CALR decreased proliferation of the resistant cells. When resistant cells were cultured in the absence of RUX, the RUX-resistance was reversed, with reduction of the mutant-CALR/MPL complex. In conclusion, MPL overexpression induces higher levels of a mutant-CALR/MPL complex, which may cause RUX-resistance in cells with CALR mutations. This mechanism may be a new therapeutic target to overcome RUX-resistance.

Clinical impacts of the mutational spectrum in Japanese patients with primary myelofibrosis.

Patients with primary myelofibrosis (PMF) have a poorer prognosis than those with other subtypes of myeloproliferative neoplasms (MPNs). To investigate the relationship between gene mutations and the prognosis of Japanese PMF patients, we analyzed mutations in 72 regions located in 14 MPN-relevant genes (CSF3R, MPL, JAK2, CALR, DNMT3A, TET2, EZH2, ASXL1, IDH1/2, SRSF2, SF3B1, U2AF1, and TP53) utilizing a target resequencing platform. In our cohort, ASXL1 mutations were more frequently detected in both overt and prefibrotic PMF patients than other mutations. The frequency of ASXL1 mutations was slightly higher among overt PMF patients than among prefibrotic PMF patients (44.6% vs 25.0%, FDR = 0.472). Decision tree classification algorithms revealed that ASXL1, EZH2, and SRSF2 mutations were associated with a poor prognosis for overt PMF. Overall survival was significantly shorter in patients harboring ASXL1, EZH2, or SRSF2 mutations than in those without these mutations (p = 0.03). These results suggest that, as reported in Western countries, MIPSS70 is applicable to Japanese PMF patients and ASXL1, EZH2, and SRSF2 mutations may be utilized as surrogate markers of a poor prognosis.

CREB3L1 overexpression as a potential diagnostic marker of Philadelphia chromosome-negative myeloproliferative neoplasms.

Discrimination of Philadelphia-negative myeloproliferative neoplasms (Ph-MPNs) from reactive hypercytosis and myelofibrosis requires a constellation of testing including driver mutation analysis and bone marrow biopsies. We searched for a biomarker that can more easily distinguish Ph-MPNs from reactive hypercytosis and myelofibrosis by using RNA-seq analysis utilizing platelet-rich plasma (PRP)-derived RNAs from patients with essential thrombocythemia (ET) and reactive thrombocytosis, and CREB3L1 was found to have an extremely high impact in discriminating the two disorders. To validate and further explore the result, expression levels of CREB3L1 in PRP were quantified by reverse-transcription quantitative PCR and compared among patients with ET, other Ph-MPNs, chronic myeloid leukemia (CML), and reactive hypercytosis and myelofibrosis. A CREB3L1 expression cutoff value determined based on PRP of 18 healthy volunteers accurately discriminated 150 driver mutation-positive Ph-MPNs from other entities (71 reactive hypercytosis and myelofibrosis, 6 CML, and 18 healthy volunteers) and showed both sensitivity and specificity of 1.0000. Importantly, CREB3L1 expression levels were significantly higher in ET compared with reactive thrombocytosis (P < .0001), and polycythemia vera compared with reactive erythrocytosis (P < .0001). Pathology-affirmed triple-negative ET (TN-ET) patients were divided into a high- and low-CREB3L1-expression group, and some patients in the low-expression group achieved a spontaneous remission during the clinical course. In conclusion, CREB3L1 analysis has the potential to single-handedly discriminate driver mutation-positive Ph-MPNs from reactive hypercytosis and myelofibrosis, and also may identify a subgroup within TN-ET showing distinct clinical features including spontaneous remission.