Anagrelide Research Articles

Anagrelide and idarubicin combination induces GSDME-mediated pyroptosis as a potential therapy for high-PDE3A acute myeloid leukemia.

Acute myeloid leukemia (AML) is an invasive hematopoietic malignancy requiring novel treatment strategies. In this study, we identified phosphodiesterase 3 A (PDE3A) as a potential new target for drug repositioning in AML. PDE3A was preferentially overexpressed in AML cells than in normal cells, and high expression of PDE3A was correlated with lower event-free survival (EFS) in de novo AML patients. The PDE3A inhibitor anagrelide (ANA) profoundly suppresses the proliferation of high PDE3A-expressing AML cells while exhibiting minimal impact on those with low PDE3A expression. Moreover, synergistic effect of ANA with other chemotherapeutic drugs in high PDE3A expression AML cells was observed. The ANA-idarubicin (IDA) combination showed the most remarkable synergistic effect among all ANA-chemotherapeutic drugs commonly used in AML cell line models. Mechanistically, the synergy between ANA and IDA inhibited the survival of PDE3Ahigh AML cell lines through pyroptosis. This mechanism was initiated by GSDME cleavage triggered by caspase-3 activation. In vivo combination treatment of leukemic animals with high PDE3A expression significantly reduced leukemia burden and prolonged survival time compared with single-drug and vehicle control treatments. Our findings suggest that combined ANA and IDA treatment is an innovative and promising therapeutic strategy for AML patients with high PDE3A expression.

Initial Low-Dose Hydroxyurea and Anagrelide Combination in Essential Thrombocythemia: Comparable Response with Lower Toxicity.

Background and Objectives: Essential thrombocythemia (ET) is a myeloproliferative neoplasm that overproduces platelets and is associated with life-threatening thrombosis. Medical cytoreduction either with hydroxyurea (HU) or anagrelide (AG) is widely used, but drug intolerance or resistance are major concerns. Low-dose combination of HU and AG as an alternative strategy has been explored in various studies. It showed comparable response with acceptable toxicity in second-line settings for patients who experienced side effects from prior monotherapy. In this study, we evaluated the efficacy and safety of upfront combination for ET patients. Materials and Methods: From January 2018 to June 2022, a total of 241 ET patients with intermediate to high risk were enrolled. We identified 21 patients with initial drug combinations and compared treatment outcomes and adverse events (AEs) between combination and monotherapy groups. Results: The median age was 62 years old (range, 26-87) and median platelet count was 912 × 109/L (range, 520-1720). Overall treatment response did not exhibit significant differences between the groups, although there was a trend towards a lower response rate in patients treated with AG alone at 3 months post-treatment (AG + HU, 85.7% vs. AG alone, 75.4%, p = 0.068). AEs of any grade occurred in 52.3% of the combination group, 44.3% of the HU monotherapy group, and 43.4% of the AG single group, respectively. Of note was that the HU plus AG combination group suffered a lower incidence of grade 3-4 AEs compared to the other two groups, with statistical significance (p = 0.008 for HU monotherapy vs. combination therapy and p < 0.01 for AG monotherapy vs. combination therapy). Conclusions: Our findings demonstrated that the upfront low-dose combination approach showed feasible clinical outcomes with significantly lower severe AEs compared to conventional monotherapy. These results may offer valuable insights to clinicians for future prospective investigations.

ROP-ET: a prospective phase III trial investigating the efficacy and safety of ropeginterferon alfa-2b in essential thrombocythemia patients with limited treatment options

Interferon-based therapies, such as ropeginterferon alfa-2b have emerged as promising disease-modifying agents for myeloproliferative neoplasms (MPNs), including essential thrombocythemia (ET). Current ET treatments aim to normalize hematological parameters and reduce the thrombotic risk, but they do not modify the natural history of the disease and hence, have no impact on disease progression. Ropeginterferon alfa-2b (trade name BESREMi®), a novel, monopegylated interferon alfa-2b with an extended administration interval, has demonstrated a robust and sustained efficacy in polycythemia vera (PV) patients. Given the similarities in disease pathophysiology and treatment goals, ropeginterferon alfa-2b holds promise as a treatment option for ET. The ROP-ET trial is a prospective, multicenter, single-arm phase III study that includes patients with ET who are intolerant or resistant to, and/or are ineligible for current therapies, such as hydroxyurea (HU), anagrelide (ANA), busulfan (BUS) and pipobroman, leaving these patients with limited treatment options. The primary endpoint is a composite response of hematologic parameters and disease-related symptoms, according to modified European LeukemiaNet (ELN) criteria. Secondary endpoints include improvements in symptoms and quality of life, molecular response and the safety profile of ropeginterferon alfa-2b. Over a 3-year period the trial assesses longer term outcomes, particularly the effects on allele burden and clinical outcomes, such as disease-related symptoms, vascular events and disease progression. No prospective clinical trial data exist for ropeginterferon alfa-2b in the planned ET study population and this study will provide new findings that may contribute to advancing the treatment landscape for ET patients with limited alternatives.Trial registrationEU Clinical Trials Register; EudraCT, 2023-505160-12-00; Registered on October 30, 2023.

Phosphodiesterase 3A Acts As a Tumor Activator and Its Inhibitor Anagrelide Combined with Idarubicin Represents a Potential Therapeutic Approach for PDE3A High Expressed Acute Myeloid Leukemia through Pyroptosis By Triggering Cleavage of Gasdermin E

Acute myeloid leukemia (AML) is a clinically aggressive and heterogeneous hematologic neoplasm with a poor overall outcome. Platelets can interact with malignant cells including leukemic blasts and play a crucial role in cancer progression, metastasis and angiogenesis, budding of platelet microparticles can transfer a plethora of bioactive molecules to cancer cells upon internalization and in AML this can induce chemoresistance. Anti-platelet therapy using Phosphodiesterase 3A (PDE3A) inhibitors, such as anagrelide (ANA), holds promise in tumor elimination. Additionally, upregulation of PDE3A in solid tumors is associated with a poor prognosis in patients and activates inflammatory pathways, impacting cancer cell stemness. However, the role of PDE3A in AML remains unclear. This study aims to investigate the correlation between PDE3A expression and AML prognosis, as well as to characterize the feasibility and mechanism of ANA as a potential anti-leukemia therapy and its synergistic effect to chemotherapeutic drugs. To explore the correlation between PDE3A expression and AML prognosis, we initially analyzed its expression in AML patients and healthy donors from the Children's Hospital of Soochow University. The results demonstrated significantly higher levels of PDE3A in bone marrow mononuclear cells (BMMNCs) from AML patients, both at the protein and mRNA levels, compared to BMMNCs from healthy donors. Subsequently, we investigated PDE3A expression and survival data of 152 AML patients from our center, revealing that patients with high PDE3A expression exhibited significantly lower overall survival (OS), relapse-free survival (RFS), and event-free survival (EFS) compared to those with low PDE3A expression. These findings were further corroborated by data from other databases (R2 and GEPIA), confirming the significant role of PDE3A in AML. Among AML cell lines, HEL and MOLM-16 cells exhibited notably higher PDE3A expression at both the protein and mRNA levels, which were chosen for further study. ANA treatment specifically inhibited the proliferation of PDE3A-high-expressing AML cells but not low expression cells in vitro, which was further confirmed in the HEL-mediated xenograft leukemia model. Notably, the RNA sequencing data revealed ANA treatment significantly downregulated several chemotherapy-resistant genes, including PAK1, HMGCS1, p-SRC, DUSP16 and EGR1, indicating ANA has the potential to synergize with chemotherapeutic drugs for AML therapy. Therefore, we combine ANA and chemotherapy drugs to investigate the synergetic inhibitory effect. IDA with ANA simultaneously exerted the strongest synergistic effect among the six anti-leukemia drugs depend on the judgement of CI value and had the synergistic effect in HEL, MOLM-16 and primary cells from PDE3A high expressed AML patients. The GSEA analysis of RNA sequencing data showed that the IDA-ANA combination treatment led to a significant enrichment of the inflammatory response. Moreover, at the protein level, pyroptosis-related proteins including NLRP3, Cleaved-CASP3, Cleaved-CASP8 and GSDME-N were notably upregulated in IDA-ANA combination group compared to IDA or ANA group. Finally, we established an AML xenograft mouse model using the HEL cell line and administered different treatments: vehicle (control), IDA (0.5 mg/kg), ANA (5 mg/kg), or the indicated combination of IDA and ANA. The results demonstrated that the IDA-ANA combination treatment significantly extended the survival time of the mice and reduced the leukemia burden when compared to the individual treatments with either IDA or ANA. In second HEL xenograft mice study, separate cohorts of vehicle and drug treated mice were sacrificed and leukemic livers as well as spleens were harvested and weighed, the IDA-ANA combination treated mice exhibited smaller and lighter livers and spleens than the other groups and controls. HE results explained the histological decrease of HEL cells infiltration of IDA-ANA combination treatment in the livers, spleens and bone marrow compared with the treatment of IDA or ANA. Congruently, the analysis of IHC confirmed the same result. In summary, high expression of PDE3A related to poor prognosis in AML and PDE3A inhibitor ANA combined with IDA exerted anti-leukemia effect through pyroptosis on PDE3A high expressed AML. Our findings represent a potential therapeutic regimen for AML.

Impact of Cytoreductive Drugs upon Outcomes in a Contemporary Cohort of Adolescent and Young Adults with Essential Thrombocythemia and Polycythemia Vera

Introduction Essential thrombocythemia (ET) and polycythemia vera (PV) are well described in adolescent and young adults (AYA) patients (pts); the most prevalent complications are thrombotic events, but progression to myelofibrosis (MF) is also associated with significant morbidity and mortality and this risk is time-dependent. Here we investigated the impact of cytoreductive drugs on outcomes in a well-defined cohort of AYA pts. Method and objectives Patients with a diagnosis (Dx) of ET or PV established at less than 25 years (yrs) of age and having known driver mutation status were included from a collaborative group of centers within the EHA MPN special working group. Patients were classified by the first cytoreductive treatment they received or no cytoreduction (NoCYTO). Main endpoints were thrombosis free survival (TFS) and, for those who received a minimum of 2 years of treatment, myelofibrosis free survival (MFS). Kaplan-Meier and log-rank tests were used to compare groups. Results Overall we included 348 pts (278 ET, 70 PV) with median age 20 yrs at Dx (IQR: 18-23). Median age at Dx for ET was 21 yrs (18-23) and for PV 20 yrs (16-23). 249 pts were female (216 ET, 33 PV). Median follow-up was 9 yrs (4-15). Overall, 237/348 (68%) started a cytoreductive therapy. First treatment was hydroxycarbamide (HU) in 126 pts (100 ET, 26 PV), IFN in 55 pts (33 ET, 22 PV), anagrelide (ANA) in 52 pts (51 ET, 1 PV) and other in 4 pts (1 ET, 3 PV). 111 pts (32 %) did not receive cytoreduction (93 ET, 18 PV) deemed NoCYTO. According to ELN risk group at Dx, 39/43 pts (26 ET, 13 PV) receiving a cytoreductive drug were high-risk and 197/304 pts (159 ET, 38 PV) were low-risk. Reason for treatment initiation was thrombotic event in 32 pts (24 ET, 8 PV), platelets &amp;gt;1000x10 9/L in 96 pts (87 ET, 9 PV), MPN-related symptoms 12 pts (7 ET, 5 PV), and other/unknown for 97 pts (67 ET, 30 PV). Ten year TFS was 87% (95%CI: 82-91%) overall, 87% (82-92%) for ET and 86% (77-96%) for PV. In multivariate analysis, elevated WBC (&amp;gt;11x10 9/L) was associated with higher thrombosis risk (HR: 2.8, 95%CI: 1.2-6.2, p=0.01) and splenomegaly with a lower risk (HR: 0.2, 95%CI: 0.1-0.9, p=0.04). In the ELN low-risk group, 10yrs TFS was 82% (73-90%) for HU, 84% (70-98%) for IFN, 93% (84-100%) for ANA and 94% (88-100%) for noCYTO, suggesting no benefit for cytoreduction, p=0.04. For high-risk patients, 10yrs TFS was 78% (56-100%) for HU, 67% (35-98%) for IFN, and 86% (59-100%) for ANA, p=0.689 (Fig 1A). Ten year MFS was 95% (92-98%) overall, 95% (91-98%) for ET, 93% (85-100%) for PV. On multivariate analysis, CALR mutation (HR: 6.0, 95%CI: 2.3-16.1, p&amp;lt;0.05) and interestingly thrombotic history at Dx (HR 3.8, 95%CI: 1.3-11, p=0.02) were associated with increased risk of MF progression overall. For ET: CALR mutation (HR 4.1, 95%CI: 1.6-10.5, p &amp;lt;0.05) and splenomegaly (HR: 2.9, 95% CI: 1.1-7.7, p=0.03) were associated with MF progression, but only CALR mutation was significant on multivariate analysis (HR: 4.2, 95% CI: 1.6-11.6, p&amp;lt;0.05). For PV, we did not identify risk factors associated with MF progression. Finally, we investigated the impact of cytoreductive therapy on MF progression. Overall, 10yrs and 20yrs MFS for IFN was 100%, for HU 10yrs and 20yrs MFS were 93% (86-99%) and 74% (57-92%), for ANA 10yrs and 20yrs MFS were 92% (82-100%) and 73% (40-100%), and for NoCYTO patients 10yrs and 20yrs MFS were 94% (88-100%) and 74% (47-100%), respectively (Fig 1b). Log-rank test comparing IFN vs. other management (HU, ANA or NoCYTO) was significantly in favour of IFN (p=0.046). Finally, during the follow-up there was only one MDS-progression and four deaths (1 of GVHD following HSCT 34 yrs after Dx, 1 of CMV disease 15 yrs after Dx, 2 of unknown cause, 4 yrs and 41 yrs after Dx). Discussion This study is the largest of its kind in contemporary young ET and PV patients focusing on specifically upon the impact of treatment. We demonstrate that early initiation of cytoreduction in low-risk patients does not impact TFS and that the choice of drug does not impact TFS in high-risk patients. In addition, importantly our data demonstrate that IFN specifically compared to other cytoreductive agents yields significantly better MFS compared to other treatments. These results support the use of IFN as a currently available disease-modifying agent to improve long-term MFS and also warrant reconsideration of earlier treatment in patients with ET and PV with IFN to improve MFS potentially as a primary aim.

EXCEED-ET: A single-arm multicenter study to assess the efficacy, safety, and tolerability of ropeginterferon alfa-2b-njft (P1101) in North American adults with essential thrombocythemia.

TPS7088 Background: Essential thrombocythemia (ET) is a myeloproliferative neoplasm (MPN) characterized by a high platelet count due to an overacting JAK-STAT pathway as a result of the most common mutations in JAK2, CALR and MPL genes. There is a high, unmet clinical need for safe and effective treatment options for patients with ET who require cytoreduction, both naïve and previously exposed to hydroxyurea (HU) or anagrelide (ANA). In addition to blood count control and thrombohemorrhagic risk reduction, treatment for ET should also halt/prevent disease progression to post-ET myelofibrosis. Reduction in variant allele frequency and changes in marrow morphology may lead to the elimination of the malignant clone, and treatment with an interferon may achieve this outcome. Interferons have been used in the treatment of MPNs for over 30 years with well-established improvements in hematological and molecular rates, but interferons are not approved by a regulatory agency for the treatment of ET. The objective of this study is to assess the efficacy of ropeginterferon alfa-2b-njft, a new generation interferon alfa, in adults with ET who live in the US or Canada. Methods: This single-arm, open-label study includes a 28-day screening period, a 12-month treatment phase, and a 28-day follow-up period, for a total trial duration of 14 months. An extension phase will supply ropeginterferon alfa-2b-njft to patients deriving benefit for a total of 3 years of treatment. Adults diagnosed with ET according to the WHO 2016 criteria with a platelet count at screening &gt; 450 x 109/L, cytoreductive treatment naïve, or with prior use or currently on HU and/or ANA may participate. Prior treatment with an interferon is an exclusion factor. Patients with a history or presence of clinically relevant depression, risk of suicide, or autoimmune disease may not participate. Ropeginterferon alfa-2b-njft will be administered subcutaneously every 2 weeks at the starting dose of 250 mcg (Week 0), 350 mcg (Week 2), and target dose of 500 mcg (Week 4) and remain fixed for the 12-month treatment period. Subjects currently on HU or ANA will follow a pre-specified dose-tapering schedule. All subjects will receive low-dose aspirin unless contraindicated. The primary endpoint is a durable response defined as the proportion of subjects who achieve simultaneous peripheral blood count remission (platelets ≤400 x109/L and WBC &lt; 10 x 109/L) for at least 80% of bi-weekly measurements consecutively between weeks 32-52, a durable resolution of disease-related symptoms according to the MPN Symptom Assessment Form Total Symptom Score (MPN-SAF TSS), and absence of bleeding or thrombotic events. This study is being sponsored by PharmaEssentia USA. Clinical trial information: NCT05482971 .

Identification and enrichment of a UV-induced degradant of Anagrelide drug substance

Anagrelide (ANG) is a widely used drug for the treatment of essential thrombocytosis and myeloproliferative neoplasms. Recently, a new oxidative degradant was identified when the drug product capsule underwent stress testing. Full structural characterization of this previously unidentified degradant was conducted. Preliminary LC-MS analysis indicated the targeted degradant as a mono-oxygenated product of ANG. For the purpose of facile isolation and purification, various forced degradation conditions were screened to enrich the desired degradant, among which, pyridinium chlorochromate (PCC)-treatment effectively afforded a yield of 55 % unknown degradant. Following isolation by prep-HPLC, 1D and 2D NMR studies and HRMS characterization assigned the products as a pair of 5-hydroxy-Anagrelide (5-OH-ANG) enantiomers. A plausible mechanism of formation is proposed.

Anagrelide Represents a Potential Therapeutic Therapy for Acute Myeloid Leukemia Cells Based on Phosphodiesterase 3A Expression

Acute myeloid leukemia (AML) is a molecularly and clinically heterogeneous disease. PDE3A is a well-characterized cyclic nucleotide phosphodiesterase (PDE) that hydrolyzes cAMP, cGMP, and cUMP. Recent studies have demonstrated that PDE3A was upregulated in several solid tumors of patients including breast cancer, ovarian carcinoma, melanoma, which correlated with poor prognosis. PDE3A activates inflammatory pathways that mediate cancer cell stemness by suppressing the cAMP/PKA pathway. Targeting PDE3A with chemicals suppressed cancer cells by inducing cell apoptosis and reducing metastasis. PDE3A inhibitor Anagrelide (ANA) is an antithrombotic and platelet reducing agent used to treat the thrombocythemia associated with myeloproliferative diseases. This study aims to explore the relationship between PDE3A expression and AML prognosis, and to investigate the feasibility and mechanism of PDE3A as a therapeutic target for AML. To explore the relationship between PDE3A expression and the survival time of patients, a total of 141 pediatric AML patients were recruited in this retrospective study from October 2013 to September 2021 in the Children's Hospital of Soochow University. The follow-up period was until March 2022. The transcriptomic expression level of PDE3A in newly diagnosed bone marrow samples was obtained according to the results of RNA sequencing. Patients were divided into two groups based on median PDE3A expression: PDE3Ahigh group (n= 70) and PDE3Alow group(n=71). Results showed AML patients in PDE3Ahigh group had significantly lower 5-year overall survival (OS) (71.4% vs 92.9%, P = 0.0010), event free survival (52.1% vs 84.6%, P = 0.0150) and relapse free survival (73.2% vs 91.4%, P = 0.0325) compared to patients in PDE3Alow group. Next, to evaluate PDE3A expression level in AML cell lines, we used RT-PCR and Western Blot to compare the difference of PDE3A expression in HEL, Kasumi-1, MV4-11, NB4, K562, HL60 and MEG-01. Among these cell lines, HEL has significantly higher PDE3A expression compared to others at both transcriptional and protein levels. We found that ANA specifically inhibited HEL growth in a concentration dependent manner after 72 hours of ANA treatment. ANA reached half of the inhibitory concentration on HEL at 0.9μM. Interestingly, ANA had no inhibitory effects on other tested AML cell lines which expressed low level of PDE3A. Thus, we choose HEL as PDE3Ahigh AML cell model and treatment with 0.9μM ANA for 72 hours for further experiments. By flow cytometry, we found ANA interrupted cell cycle by G1 arrest in HEL, but did not induce cell apoptosis. In consistent with these results, the expression of cell cycle-related proteins including CDK2, CDK4, CDK6, CDK9 and C-MYC were significantly down-regulated in a time-dependent manner after ANA treatment in HEL compared to control group. To explore the effect of ANA on PDE3A high expression AML in vivo, we established the preclinical model of AML by injecting 2 million luciferase overexpressed HEL cells into NSG mice via tail vein. Fourteen days later, the mice were treated with vehicle or 5mg/kg ANA treatment by gavage daily for 15 days as the therapeutic model. More interestingly, in vivo imaging assay indicated that ANA treatment reduced leukemia burden compared with the vehicle group. The anti-leukemia effects of ANA significantly prolonged survival time compared to vehicle (median survival time, 32.5 vs 24.5 days, P = 0.0091). In conclusion, our clinical data suggested that high expression of PDE3A in newly diagnosed pediatric AML patients was associated with poor clinical survival. Besides, our in vitro and in vivo data indicated that ANA targeting PDE3A suppressed progression of AML by interrupting cell cycle, providing a promising approach to eradicate AML with high level of PDE3A expression.

Clinicohematologic and Molecular Response of Essential Thrombocythemia Patients Treated with Pegylated Interferon-Alpha: A Multicenter Study on Behalf of the German Study Group-MPN (GSG-MPN)

Cytoreductive treatment with hydroxyurea (HU) or anagrelide (ANA) aims at reducing the risk of vascular complications in essential thrombocythemia (ET), while (pegylated) Interferon-α (pegIFN) may also prevent progression to secondary myelofibrosis (SMF) or acute myeloid leukemia (AML). In contrast to polycythemia vera (PV), the accessibility to pegIFN is limited in ET due to lack of approval. To investigate the clinical benefits of pegIFN in ET, we identified within the German Study Group-Myeloproliferative Neoplasms (GSG-MPN) registry a total of 127 WHO-defined patients (pts) treated at seven University centres. In this study, we analysed clinically relevant parameters before and during pegIFN treatment and assessed the JAK2 V617F variant allele frequency (VAF) in DNA from granulocytes by droplet digital PCR (sensitivity 10-3) in JAK2 mutated pts with available biosamples. Baseline characteristics of the pts were: median age at diagnosis 37.0 years (yrs, range 8.2-77.4), median platelet (PLT) count 780 /nl (124-2,776), median white blood cell (WBC) count 8.2 /nl (3.0-17.3); 67% were female; 53% were JAK2 V617F-, 33% CALR-, and 5% MPL-mutated. According to the International Prognostic Score of thrombosis in ET (IPSET), 37% were low-, 31% intermediate-, and 30% high-risk (2% unknown). Median time from diagnosis to pegIFN start was 1.9 yrs (0.0-37.2); in 59% pegIFN was used as first-line drug, 20% were pre-treated with HU, 9% with ANA, and 11% with both. Main reasons for pegIFN start were high PLT count (39%), prior thrombosis (31%), ET-associated symptoms (9%), age >60 yrs (7%), and pregnancy (7%). Since 31 pts (24%) had ≥2 lines of treatment (LOT) with pegIFN, a total number of 161 LOT was recorded. PegIFN 2a was used in most LOT (54%), followed by pegIFN 2b (35%) and ropegIFN 2b (11%). Median total pegIFN treatment duration per pt was 2.3 yrs (0.1-18.1); 30/127 (24%) and 10/127 (8%) were treated for ≥5 yrs and ≥10 yrs, respectively. At last follow-up, 60% of pts were still on pegIFN. The discontinuation rate was highest in the first yr of treatment (14.3%). Adverse events (16%) and market withdrawal of pegIFN 2b (15%) were the two most frequent reasons for discontinuing a LOT. According to European LeukemiaNET response criteria (Barosi et al. Blood 2009), 89% of pts achieved clinicohematologic response at any time which was complete in 54% and partial in 35%. Median PLT and WBC counts at last follow-up (380 /nl and 5.5 /nl, respectively) were significantly lower compared to baseline (780 /nl and 8.2 /nl) (Figure). Only one pt progressed to SMF during pegIFN treatment, while no pt transformed to secondary AML. Vascular complications were rare with three arterial and five venous events in 469 pegIFN treatment yrs, resulting in an incidence of 0.6% and 1.1% per pt and yr, respectively. Flu-like symptoms were recorded in 41%, abnormal liver tests in 16%, and depression in 14% of LOT (grading and time points unknown). Quantitative assessment of the JAK2 V617F VAF on 70 samples from 14 pts with available DNA before pegIFN start and ≥2x thereafter (each at least one yr apart) revealed a reduction from median 30.5% (7.2-48.1) at baseline to 8.3% and 18.1% after 4 and 8 yrs of pegIFN treatment, respectively (n.s.); 5/14 pts (36%) achieved a ≥50% reduction (partial molecular response) and one pt showed a VAF <1%. In 10/127 pts (8%), pegIFN 2b was stopped due to market withdrawal and not immediately switched to an alternative drug as they had been treated for a median of 9.5 yrs (6-18): 3/10 pts maintained normalized blood cell counts until data cut-off (1.5-4.5 yrs after discontinuation); of these 3, one had 0.1% JAK2 V617F at the time of discontinuation, while the other two were JAK2 V617F negative (baseline VAF not available); 2/3 pts underwent bone marrow biopsy and showed histomorphological remission of ET. Analyses of a large series of ET pts treated with pegIFN confirm a high efficacy of the drugs in achieving clinicohematologic response. Our data, including long-term treated pts with low rates of vascular complications, disease progressions, and adverse events underscore the medical benefit of pegIFN in ET. Although reduction of the JAK2 V617F VAF was not significant, molecular responses <1% and histomorphological remissions were observed in single pts. For treatment discontinuation, molecular thresholds for JAK2 V617F and CALR mutations need to be defined in the future. FS and LLT contributed equally to this study. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Patients with Essential Thrombocythemia Have a Gut Microbiota That Differs from Healthy Controls

Background:Essential thrombocythemia (ET), along with polycythemia vera (PV) and primary myelofibrosis (PMF), belongs to the Philadelphia chromosome negative myeloproliferative neoplasms (MPNs). Chronic inflammation is considered a hallmark in the pathogenesis and progression of the MPNs. The human gut microbiota helps regulate and mature the immune system, digest food, protect against pathogens and induce hematopoiesis. As our immune system constantly interacts with our gut microbiota any change to the microbiota could have significant impact on the overall health of the patient. This influence of the gut microbiota on the patient has been described as being involved in the pathogenesis of several diseases, such as obesity and diabetes mellitus. We recently added to this list by showing that the gut microbiota in patients with PV differs significantly from that of healthy controls (HCs), however the composition of the gut microbiota in patients with ET remains unknown. Thus, we hypothesized that patients with ET have a distinct gut microbiota signature compared with that of HCs. This study is the first to explore the gut microbiota in patients with ET specifically and compared with that of HCs. Methods: We included patients with ET, diagnosed according to the 2016 WHO classification. Included patients had not received antibiotics within the last 2 months and no change in cytoreductive treatment had occurred within the last 3 months. Comorbidities were assessed using the Charlson Comorbidity Index score (CCI). The gut microbiota was characterized using amplicon-based next generation sequencing of the V3-V4 region of the 16S ribosomal unit while dada2 was used for taxonomic classification. Alpha diversity, beta diversity and linear discriminant analysis effect size (LEfSe), were used to assess the microbiota between and within the patient groups. Alpha diversity describes the richness and evenness within a sample and beta diversity the difference in composition between the samples, while LEfSe is used for differential abundance analysis. Results: In total, 54 patients with ET (median age 68, range 38-83) and 42 HCs (median age 71, range 66-74), fulfilled the inclusion criteria. Patients with ET had a higher CCI score (p=0.038) compared with the HCs. Thirty-six patients had the JAK2V617F mutation. Seven were positive for calreticulin (CALR), three for myeloproliferative leukemia virus gene (MPL), two for CALR+JAK2V617F mutations, and 6 were triple negative. The treatments were: no cytoreductive treatment (n=15), PEG-interferon-α2 (n=11), anagrelide (ANG)(n=3), hydroxyurea (HU)(n=23) and HU+ANG(n=2). Patients with ET had a significantly higher observed richness (number of different bacteria present) compared with HCs (median, 283.5 vs 191.5; p<0.001), however, no significant difference was found in alpha diversity. The bacterial composition differed significantly between ET and HCs (p=0.004 and R=0.063). Eight taxa, with a relative abundance >1%, differed between ET and HCs. Patients with ET had a lower relative abundance of the phylum Firmicutes (p=0.03), class Clostridia (p=0.001), order Oscillospirales (p<0.01), and family Ruminococcaceae (p<0.001) compared with HCs. On genus level, patients with ET had a lower relative abundance of Faecalibacterium (p <0.001). While all mutations were associated with a significantly different microbiota in ET compared with HCs, ET patients who only had the JAK2V617F differed to a larger degree. Conclusion: The gut microbiota in patients with ET differed significantly from that of HCs, both in regard to richness and bacterial composition. The relative abundance of several taxa within the phylum Firmicutes differed significantly, with ET patients having a lower relative abundance in general. These differences appear to be more pronounced in patients who have the JAK2V617F mutation. Thus, it needs to be elucidated if the change of the microbiota is the cause or the consequence of the disease. Is it the increased inflammatory response which changes the microbiota or is it the change in microbiota which aggravates the inflammatory effects of the JAK2V617F mutation?

MPN-546 A Single-Arm Multicenter Study to Assess the Efficacy, Safety, and Tolerability of Ropeginterferon alfa-2b-njft (P1101) in North American Adults With Essential Thrombocythemia.

There is an unmet need for treatment options for patients with essential thrombocythemia (ET) who require cytoreduction who may or may not have been treated with hydroxyurea (HU) or anagrelide (ANA), which is commonly used outside of North America. In addition to the reduction of thrombotic risk, treatment options for ET should reduce all blood cell types, prevent disease progression, and modify the disease. To assess the efficacy of ropeginterferon alfa-2b-njft (P1101) in adults with ET who live in the US or Canada. Single-arm, open-label study includes a 28-day screening period, a 12-month treatment phase, and a 28-day follow-up period, for a total trial duration of 14 months. P1101 will be administered every 2 weeks during the treatment phase at each study visit, and once a maintenance dose is identified for a patient, bi-weekly minor assessment visits may be replaced with phone visits. A subsequent extension phase will supply P1101 to patients deriving benefit for a total of 3 years of treatment. Study results will impact the general management of patients with ET. Adults diagnosed with ET according to the WHO 2016 criteria with a platelet count at screening >4 50 × 109/L, cytoreductive treatment naïve or with prior HU and/or ANA may participate. Patients with a history or presence of clinically relevant depression, risk of suicide, or autoimmune disease may not participate. P1101 will be administered subcutaneously every 2 weeks at the starting dose of 250 mcg (Week 0), 350 mcg (Week 2), and target optimal dose of 500 mcg (Week 4) and remain fixed for the 12-month treatment period. Subjects currently on HU or ANA will follow a pre-specified dose-tapering schedule. All subjects will receive low-dose aspirin unless contraindicated. The primary endpoint is a durable response defined as the proportion of subjects who achieve simultaneous peripheral blood count remission (platelets ≤400×109/L and WBC <10 × 109/L) for at least 80% of bi-weekly measurements consecutively between weeks 32-52. This study is being sponsored by PharmaEssentia USA.

P997: PROTEOMIC ANALYSIS ON PLATELETS OF ESSENTIAL THROMBOCYTHEMIA PATIENTS UNDERSCORES THE ROLE OF MITOCHONDRIA IN JAK2 V617F PLATELET REACTIVITY AND FUNCTION

Background: Essential thrombocythemia (ET) is a heterogeneous disease subdivided into five genetic groups according to WHO, based on the common MPN driver mutations (JAK2 V617F, MPL W515K/L, and CALR Type I&II). Current treatment strategies include anti-platelet (e.g. acetylsalicylic acid (ASA)) or cytoreductive agents (e.g. hydrea (HU)) and target mainly complications related to platelet dysfunction (i.e. hemorrhage/thrombosis). Platelet activation is an energy demanding process fueled mainly by a dynamic equilibrium between mitochondria oxidative phosphorylation and glycolysis1. Importantly, altering the platelet catabolic response to activation has been shown to prevent thrombus formation2. JAK2 V617F platelets have been reported to be more activated than CALR mutated platelets3, as well as JAK2 mutated patients have a greater thrombotic risk in comparison to other ET groups. However, the cause of this phenotype is not completely understood. Aims: In this study we aimed to analyze the proteome of ET platelets and to characterize their functional properties according to the mutational background and treatment regimens. Methods: 22 healthy donors (HD) and 67 ET patients have been included in the study. Most of the patients were treated with low dose aspirin (ASA), anagrelide (ANA) or hydrea (HU) or a combination of these. Specifically, 35 out of the 67 ET platelet samples and 8 out of 22 HD were subjected to label-free quantitation mass spectrometry (LFQ-MS). All HD and ET platelets were also analyzed for surface marker expression, degranulation and aggregation capacity by flow cytometry. Results: Hierarchical clustering of the mass spectrometry data revealed different proteomic profiles between HD and ET mutational groups and specifically between JAK2 V617F and CALR I and CALR Type II treated and/or untreated platelets. In general, HD platelets were more enriched for proteins related to platelet activation, and degranulation as compared to ET platelets and clustered next to JAK2 V617F ASA-treated platelet samples (Figure 1). In particular, the JAK2 V617F ASA-treated platelets presented significant enrichment in platelet activation metabolic and mitochondrial proteins in contrast to the CALR Type I and Type II ET platelets. Of note, no peptides corresponding to the mutant CALR were detected using label-free MS methods. Surface marker expression was variable among ET patients. However, CD49B and CD36 surface markers were the most affected among the JAK2V617F and CALR Type I and II platelets and their levels (mean fluorescence intensity-MFI-) were inversely correlated. With regards to platelet aggregation, JAK2 V617F platelets presented similar or lower levels when compared to HD platelets, in line with their proteomic profiles. Differences in aggregation and degranulation levels were also observed between CALR Type I and Type II platelets specifically in the untreated condition. Image:Summary/Conclusion: ET platelets are functional but they show different capacities to respond upon various stimuli and treatment regimens. JAK2 V61F platelets are more activated than any other ET group according to their proteome profile, which is not fully reflected by the functional assays. Mitochondrial activity arises as an important factor in the control of platelet reactivity and it could be critical in disease management and treatment strategies, specifically for the JAK2 V617F patients.

Anagrelide for platelet-directed cytoreduction in polycythemia vera: Insights into utility and safety outcomes from a large multi-center database

Anagrelide (ANA) is a platelet-specific cytoreductive agent utilized in the guideline-directed management of high-risk essential thrombocythemia. In the context of polycythemia vera (PV), ANA is occasionally employed in clinical practice, although data has not consistently demonstrated a benefit to targeting a platelet goal as a therapeutic endpoint. The aim of the current study was to delineate the patterns of ANA use in PV, and to describe outcomes and toxicities. Within a multi-center cohort of 527 patients with PV, 48 received ANA (9 excluded for absent data). 27 (69.2%) had high-risk PV, 10 (25.6%) had prior thrombosis, and none had extreme thrombocytosis, acquired von Willebrand disease, and/or documented resistance to hydroxyurea. While ANA effectively lowered median platelet count, 43.5% of patients had an unresolved thrombocytosis at time of ANA discontinuation. Treatment-emergent adverse events—including headaches, cardiac palpitations and arrhythmias, nausea, vomiting and/or diarrhea—led to ANA discontinuation in 76.9% of patients. Further, three patients experienced arterial thromboses during a median duration of 27.5 months of ANA therapy. In conclusion, this study highlights ANA’s restrictive tolerability profile which, compounded by the absence of clear advantage to strict platelet control in PV, suggests the use of ANA should be limited in this setting.

Interaction of Anagrelide drug molecule on pristine and doped boron nitride nanocages: a DFT, RDG, PCM and QTAIM investigation

Nowadays, a nanostructure-based drug delivery system is one of the most noticeable topics to be studied, and in this regard, boron nitride nanoclusters are promising drug carriers for targeted drug delivery systems. In this article, the interaction mechanism of Anagrelide (AG) drug with B12N12 and Al- and Ga-doped B12N12 nanocages have been investigated using DFT with B3LYP/6-31 G (d, p) method in both gas and water media. All our studied complexes are thermodynamically stable, and doped nanocage complexes have higher negative adsorption energy (EAd.) and negative solvation energy than AG/B12N12 complexes which correspond to the stability of these systems in both media. The negative highest EAd value is 64.98 kcal/mol (63.17 kcal/mol) and 65.69 kcal/mol (65.11 kcal/mol) in gas (water) media for complex F (AG/AlB11N12) and complex I (AG/GaB11N12) respectively, which refers to the highest stability of these systems. The enhanced values of dipole moment (from 12.40 (12.65) Debye to 17.21 (17.69) Debye in complex F (complex I)) also confirm their stability. The QTAIM and RDG analysis endorse the strong adsorption nature of the AG drug onto the AlB11N12, and GaB11N12 nanocages, which is consistent with the adsorption energy as chemisorption occurs for these complexes. According to the electronic properties, doped nanocages show high sensitivity that infers their promising nature for drug delivery purposes. Thus, complex F and complex I are promising drug delivery systems, and doped nanocages (AlB11N12 and GaB11N12) are better carriers than pristine nanocages for the AG drug delivery system. Communicated by Ramaswamy H. Sarma

A Development and Validation of RP-HPLC Method for the Determination of Degradation Impurities in Anagrelide Dosage Form

A simple and selective method for liquid chromatography has been developed to determine the degradation impurities in solid oral dosage forms of Anagrelide (Antithrombocythemic agent). An excellent resolution between Anagrelide (ANG) and its related substances was achieved with Symmetry C8, 250 mm × 4.6 mm, 3.0 μm column. and mobile phase A (phosphate buffer, pH 4.1) and mobile phase B (the mixture of acetonitrile, methanol, and buffer in the ratio of 40:40:20 v/v/v). The column oven temperature was maintained at 40°C. The flow rate was fixed at 0.8 mL/min, and elution was monitored at 254 nm using a UV detector. The method was validated for precision, accuracy, specificity, linearity, and sensitivity. The validation studies demonstrated that the current HPLC method is reproducible, specific and accurate for its intended purpose. Linearity was observed for ANG, and its impurities are more than (R2 > 0.995). The limit of detection and limit of quantitation (LOQ) was found to be adequate (< 0.05 %) for the estimation of impurities. The RSD for intra- day and inter-day precision was found to be between 0.5 % to 2.5 %. The percentage of recovery was found between 95 % to 105 %. Forced degradation studies were performed on ANG under different conditions such as acidic, basic, oxidation, photostability, and thermal degradation. Based on the results, this is found to be simple, specific, precise, accurate, linear, and stability-indicating method. Hence it can be used to determine the Anagrelide-related impurities in the capsule dosage form. It is economically feasible and user-friendly method. The proposed method was successfully applied in quality control labs for stability analysis.

Possible Role Of Treatment On Secondary Malignancies In Patients Affected By Essential Thrombocytemia

IntroductionSurvival of Essential Thrombocytemia (ET) patients in the first 10 years of disease follow-up is similar to the general population one. While it is well known that alkylating agents (given alone or sequentially) increase the risk for acute myeloid leukemia, the specific role of different therapies on the development of secondary malignancies (SM) in ET patients, is still under investigation. AimsTo retrospectively evaluate the role of different treatments on the development of SM in a large population of ET patients. Patients and methodsWe collected a series of 1026 ET patients (diagnosis period 1980-2010), followed in 11 Hematological Centers (universities 5, general hospitals 6) from the region Lazio in Central Italy. Median age at ET diagnosis is 62.5 years (range 17-93); 392 males, 634 females; median follow-up of the entire population is 6.2 years (range 0.1 – 31.9). Seventy/1026 patients (6.8%) developed 71 SM during follow-up. Observed SM were grouped as follows: genito-urinary 15, breast 14, non-melanoma skin cancer 12, lung 11, gastro-intestinal 8, hematologic 5, thyroid 3, central nervous system 1, soft tissue 1 and unknown 1.Taking in consideration the different treatment approach, we divided our population in 5 different groups: group 0, no treated patients; group 1, patients treated either with Hydroxiurea (HU) alone or in combination/sequentially with Interpheron (IFN)/Anagrelide (ANA); group 2, patients treated either with alkylating agents (ALK) alone or in combination/sequentially with IFN/ANA; group 3, patients treated with ALK + HU sequentially; group 4, patients treated with ANA and/or IFN only. Characteristics of the patients are shown in the table. Results and conclusionsTaking into account every single treatment group, although we observed an increased rate in the ALK one (4/26, 15.4%), we could not find a statistically significant difference in the risk of SM development. Instead, combining different groups we obtained the subsequent results: group 0 + 4 versus group 1, no statistically significant difference (p= 0.174); group 0 + 4 versus 2 + 3 statistically significant difference (p= 0.031). Compared to anagrelide, interferon or no treatment, alkylating agents alone or given sequentially with hydroxiurea, seem to increase the risk of SM development, while HU treatment alone do not. Disclosures:No relevant conflicts of interest to declare.

Treatment of Essential Thrombocythemia with Anagrelide Is Associated with an Increased Risk of Worsened Kidney Function

Background and Purpose: When choosing a cytoreduction method for patients suffering from essential thrombocythemia (ET), it is important to know the safety profile of the medicine used. Few articles have been published about the effects of hydroxycarbamide (hydroxyurea, HU) and anagrelide (ANA) on renal function in ET patients. This study is the largest analysis of nephrotoxicity of cytoreductive drugs used in ET therapy so far, which additionally includes risk factors for the progression of kidney disease and coexisting genetic mutation. Experimental Approach: The retrospective study included 310 patients diagnosed with ET. Demographic data, comorbidities, Cr, and estimated glomerular filtration rate (eGFR) were all taken into account prior to diagnosis and after 6 months of HU and ANA treatment. Key Results: A statistically significant difference was found between Cr and eGFR levels at baseline and after 6 months of treatment (p < 0.001). The applied treatment (HU and ANA) had the greatest impact on kidney function. ANA significantly increased the risk of worsening renal function in contrary to hydroxycarbamide after 6 months of treatment (eGFR change: median +1 mL/min/1.73 m² [interquartile range (IQR) (−4)–(+7)] in the HU group vss. median −13 mL/min/1.73 m² [IQR (−18)–(−6)] in the ANA group, odds ratio [OR] 7.92 95% confidence interval [95% CI] [4.17–15.08], p < 0.001). Lowering of eGFR <60 mL/min/1.73 m² occurred in 31 patients (31.0%) from the ANA group and 10 people (4.8%) treated with HU (p = 0.000). In 1 patient from the ANA group, >50% decrease in eGFR was observed. The chance for an increase in Cr levels was higher in people with pre-existing arterial hypertension (OR 1.92 CI = 95% [1.21–3.05], p = 0.006). Sex, type of mutation found (JAK2 V617F or CALR), and previous renal impairment did not affect renal function after 6 months of treatment. In addition, there was no difference in the efficacy of ET treatment between HU and ANA (p = 0.998). Conclusions and Implications: The observations indicate that ANA should be used in patients with ET with great caution and taking into account the risk of worsened kidney function.

Anagrelide Modulates Proplatelet Formation Resulting in Decreased Number and Increased Size of Platelets.

We retrospectively evaluated 48 essential thrombocythemia (ET) patients who were treated in our institute (male/female, 14/34, median age, 61.5 years). In 14 patients treated with anagrelide (ANA), the degree of platelet count reduction (median, -56.6%) was strongly correlated with increase of mean platelet volume (MPV) (median, +11.7%) (R = 0.777). This correlation was not observed in ET patients treated with hydroxycarbamide alone (R = 0.245). The change in size of platelets strongly suggested that ANA affected the final process of platelet production. Thus, we hypothesized that ANA modifies the process by which platelets are released from proplatelets. To verify the association in an in vitro setting, we compared MEG-01 cells treated with PMA ± ANA. The number of platelet-like particles (PLPs) was decreased (P < 0.05) and the size of PLPs estimated by using flow cytometry was significantly increased when MEG-01 cells were treated with PMA + ANA (P < 0.05 vs PMA alone), recapitulating the clinical findings. The cytoplasmic protrusions extending from MEG-01 cells were shorter and thicker and the number of proplatelets was decreased when MEG-01 cells were treated with PMA + ANA (P < 0.01 vs PMA alone). Western blotting analysis showed that ANA treatment resulted in increased phosphorylation of MLC2 and reduced phosphorylation of focal adhesion kinase (FAK). The morphological change of proplatelets were reversed by blebbistatin, a specific inhibitor of myosin II. These findings indicated that ANA modulates the FAK-RhoA-ROCK-MLC2-myosine IIA pathway and suppresses proplatelet maturation, leading to a decrease in platelet count and increase in MPV.

PF682 MULTICENTER RETROSPECTIVE STUDY EVALUATING FIBROSIS RISK IN PATIENTS WITH ET DEPENDING ON THE TREATMENT SCENARIO

Background:The main objective of the treatment of essential thrombocythemia (ET) is to reduce thrombohemorrhagic events. According to last European LeukemiaNet (ELN) guidelines, hydroxycarbamide (HU) and interferon (IFN) are recommended as a first line therapy and anagrelide (ANA) is a second‐line therapy option in high‐risk patients with ET. According to the literature, ANA is not inferior to HU, but in some studies there was observed an association of ANA with a higher incidence of arterial thrombosis, bleeding complications and fibrotic progression. Thus, the panel of the ELN did not reached a consensus on recommending the ANA in first line therapy.Aims:to analyse the outcome of ET patients according to received therapy (ANA vs HU vs ANA plus HU vs no treatment)Methods:We performed the multicentre, retrospective study. The study was conducted in accordance with the Declaration of Helsinki. We analysed 993 ET patients diagnosed and treated between 1985 and 2017. Clinical and analytical data were collected at diagnosis (age, sex, WBC, Hgb, Plt, LDH, mutation status, karyotype, splenomegaly, IPSET, thrombosis previous at diagnosis). Patients with suspicious of prefibrotic fase of myelofibrosis (pre‐PMF), were excluded from the analysis. Patients were classified according to the therapy they received (HU, ANA, HU plus ANA, no therapy). The statistical analysis due the lack of normal distribution of variables was based on analysis of variance (ANOVA). The survival analysis was prepared using Kaplan‐Meier estimator.Results:There was 542 (54.6%) patients on HU, 117 (11.8%) on ANA, 75 (7.5%) on ANA plus HU, 33 (3.3%) with other therapy and 226 (22.8%) with no treatment. We did not find significant differences in clinical and analytical data at diagnosis between 4 groups of patients. The median follow‐up was 99 (&gt; 12–354) months. Respect to the frequency of transformation to myelofibrosis, there was no significant differences between patients on ANA vs on HU (p = 0,848). However, we observed increased frequency of progression to myelofibrosis in patients on HU plus ANA vs no treatment (9.33 vs 3.54%; p = 0,046). There was no significant differences between patients on ANA vs no treatment and patients on HU vs no treatment. The overall survival free from fibrotic transformation (HU vs ANA vs HU plus ANA vs no treatment) is presented on a figure. Patients who presented fibrotic transformation vs no transformation had significantly higher LDH level at diagnosis (284.9 vs 246.7; p = 0.023). Moreover, fibrotic transformation was significantly more frequent in ET JAK2 (−) vs ET JAK2 (+) (8.9 vs 4.4; p = 0.057).Summary/Conclusion:There was no significant difference between fibrotic transformations in ET on HU vs ET on ANA. The risk of fibrotic transformation of ET seems to be related to analytical data and biological features previous to start the treatment but not with the therapy. The association between HU+ANA therapy and myelofibrosis transformation probably reflects a more advanced disease. Based on our data, contrary to previous observations, ANA does not seem to increase the risk of fibrosis. Concerning higher prevalence of fibrosis in HU+ANA ET therapy, further study is necessary to establish the safety of combined treatment.image

PF684 ANAGRELIDE MODULATES PROPLATELET FORMATION RESULTING IN DECREASED NUMBER AND INCREASED SIZE OF PLATELETS

Background:In patients with essential thrombocythemia (ET) who have high risk factors for thrombotic events, cytoreductive therapy with anagrelide (ANA) or hydroxycarbamide (HC) is one of the treatment options. Despite the clinically evident platelet‐lowering effect of ANA, the detailed mechanism of its effect remains to be elucidated. In addition to the previously reported inhibition of megakaryocytic differentiation, we hypothesized that ANA affects the formation of proplatelets, the final process of platelet release.Aims:To determine platelet size change and proplatelet formation with ANA treatment, we retrospectively analyzed patients’ data and analyzed data obtained from an experimental model using a human megakaryocytic cell line (MEG‐01 cells), an accepted model of thrombopoiesis.Methods:We retrospectively evaluated 48 patients diagnosed with ET in Hokkaido University Hospital (male/female: 14/34, age, 18 to 87 years; median age, 61.5 years; ANA only, 3 patients; ANA after HC due to insufficient decrease in platelets, 11; HC only, 18; observed without ANA or HC, 16). We analyzed changes in platelet counts and mean platelet volume (MPV) after administering each medicine. This study was approved by the Institutional Review Board of Hokkaido University Hospital. Megakaryocytic differentiation of MEG‐01 cells was induced by PMA treatment with or without ANA. Morphological change of proplatelets and size of platelet‐like particles (PLPs) were analyzed.Results:The figure shows the relationship between decrease in platelet count and MPV change in each patient at the time of maximum response to ANA or HC. In patients treated with ANA (n = 14), the degree of platelet count reduction (‐83.4% to −27.1%; median, −56.6%) was strongly correlated with increase of MPV (‐6.6% to +28.7%; median, +16.7%) (left panel; R = 0.777, P = 0.001). On the other hand, in patients treated with HC (n = 29), the degree of platelet count reduction (‐78.7% to −20.8%; median, −55.5%) was not correlated with change of MPV (‐10.3% to +12.9%; median, +1.0%) (right panel; R = 0.245, P = 0.21). PMA‐induced megakaryocytic differentiation of MEG‐01 cells promoted production of PLPs. The number of PLPs was decreased in PMA + ANA‐treated MEG‐01 cells (P &lt; 0.01 vs. PMA alone). PLP size measured by flow cytometry was significantly increased in PMA + ANA‐treated MEG‐01 cells (P &lt; 0.05 vs. PMA alone). These results are consistent with the clinical findings. MGG staining of MEG‐01 cells revealed that ANA modified the formation of cytoplasmic protrusions (proplatelets). We compared morphological parameters including the length, width and number of protrusions. The protrusions were shorter and thicker and the number of protrusions was decreased in PMA + ANA‐treated MEG‐01 cells (P &lt; 0.01 vs. PMA alone).The FAK‐RhoA‐ROCK‐MLC2‐myosin IIA pathway is an important pathway for the formation of proplatelets and production of platelets from megakaryocytes. We evaluated the effect of ANA on phosphorylation of FAK and MLC2. Western blot analysis showed that ANA treatment caused reduced phosphorylation of FAK and increased phosphorylation of MLC2 (PMA vs. PMA+ANA 5 μM, P &lt; 0.01), which presumably resulted in suppression of actin remodeling.Summary/Conclusion:ANA reduces platelet counts and increases platelet size in ET patients. Analysis of ANA‐treated MEG‐01 cells suggested that suppression of proplatelet formation through modulation of the FAK‐RhoA‐ROCK‐MLC2‐myosin IIA pathway is a key mechanism. Morphological observation suggested that ANA induces premature release of platelets, resulting in increased platelet size.image