Essential Thrombocythemia Research Articles

Correlation of TCR Repertoire Diversity with Myeloid Gene Mutations in Chronic and Advanced Phase Myeloproliferative Neoplasms

Introduction: Myeloproliferative neoplasms (MPN) are characterized by a chronic phase (CP), with indolent clinical behavior and, in 10-30% of cases, progression to advanced phases (AP), with aggressive behavior and compromised prognosis. Numerous factors have been associated with progression to AP, including the acquisition of mutations in recurrently mutated myeloid genes (1,2). On the other hand, decreased diversity in the T-cell receptor (TCR) repertoire has been associated with worse prognosis in solid tumors. The purpose of this work is to study the correlation between the acquisition of mutations in myeloid genes and the diversity of the TCR repertoire in chronic and advanced myeloproliferative neoplasms. Methods: Patients with chronic CP and AP myeloproliferative neoplasms were selected. The selected patients underwent an NGS panel of 48 myeloid genes. Only pathogenic (P) or likely pathogenic (LP) variants were considered. In parallel, the study of the TCR repertoire by NGS with the panel “AmpliSeq for Illumina Immune Repertoire Plus, TCR beta Panel” (Illumina™) was performed on the available samples. Finally, the obtained data were correlated. The study was approved by the local ethics committee. Results: A total of 22 patients were selected, 10 in CP and 12 in AP. Regarding diagnosis, 8 were polycythemia vera (PV), 4 essential thrombocythemia (ET), 6 secondary myelofibrosis (MFS) (1 to PV and 5 to ET), 2 systemic mastocytosis (MS) and 1 primary myelofibrosis (MFP). Eighty percent were male and the mean age was 68 years. The AP patients were significantly older (74 vs. 62 years of age, p<0,001). Regarding the myeloid NGS panel result, 41 mutations were found in 19 patients. Fourteen percent of the patients had no mutation, 36% had an isolated driver mutation and 50% had additional mutations. Of these, 9% were in CP and 81% were in AP. The most recurrently mutated genes were JAK2 (45.4%), CALR (18.2%), ASXL1, IDH2, SRSF2, IDH2 (all 13.6%), MPL and KIT (both 9.1%). Regarding the results of the TCR repertoire, it was possible to perform the study in 11 patients, 4 in CP and 7 in AP. Only 6/11 had results of sufficient quality to allow analysis. Of these 6 patients, 3 were in CP and 3 in AP. As can be seen in Figure 1, the diversity of the TCR between CP patients and PC patients was different. Thus, while the pattern of diversity in the CP phase shows the expected distribution, the diversity in AP shows a characteristic pattern. Conclusions: APs of MPNs show an abnormal TCR diversity pattern. This pattern may be related to the acquisition of myeloid gene mutations in AP. To our knowledge, this is the first study demonstrating an alteration in TCR diversity in AP and opens the door to future fields of research.

Signaling Pathway Gene Set Enrichment Analysis in Whole Exome Sequencing Data from Discordant Identical Twins Unveils Relevant Pathways As a Clue to Understand Somatic Mutation Acquisition and Clonal Evolution in Myeloproliferative Neoplasms

Although the role of classic driver mutations (i.e., JAK2, MPL, and CALR) in myeloproliferative neoplasm (MPN) development is well characterized, additional somatic mutations likely modulate the phenotype. Recurrent somatic mutations, such as those in TET2, DNMT3A, ASXL1 genes are commonly identified in MPN using targeted myeloid panels, however identification of somatic mutations using an unbiased approach may unveil novel pathways involved in MPN pathogenesis. We performed whole exome sequencing of blood samples of three sets of discordant identical twins in which one twin has MPN and the other twin is unaffected. The twin sets included all MPN subtypes (1 polycythemia vera, 1 essential thrombocythemia, 1 primary myelofibrosis). We also performed whole exome sequencing on additional family members, including the mother and father of the PV twin and the brother of the ET twin. Sequencing information from the discordant identical twins is a unique opportunity to identify somatic mutations in the affected twin that may contribute to MPN pathogenesis, and also to potentially identify clonal hematopoiesis in the unaffected twin. We mapped all the identified genetic variants to the affected genes, and we filtered all the genes mapped to those variants depending on whether they were present either in the MPN-affected twin or in the unaffected counterpart for further gene set enrichment pathway analysis. The gene set enriched signaling pathways from identified genetic variants for each condition-exclusive (either MPN or unaffected twin) allows us to identify and prioritize key pathways in MPN development in terms of somatic mutations acquisition. Furthermore, besides performing the analysis within each single MPN set, we analyzed the gene sets resulting from combining the different families allowing us not only to identify pathways at each single MPN type level but also among the different MPN diseases (PV, ET, and MF). We divided all the enrichment analyses into coding and silent variants to obtain a higher degree of pathway refinement. In single discordant twin sets we identified several pathways that may play an important role within MPN pathogenesis such as defective mismatch DNA repair, facilitated mitotic progression, extracellular matrix degradation, MAPK signaling, FGFR signaling, O-glycosylation of proteins, collagen metabolism and laminin interactions, neutrophil degranulation, interleukin-33 signaling, Rho GTPases and cGMP regulation, and nuclear pore complex (NPC) assembly. Interestingly, only when we analyze all the MPN-types together all the statistically significant pathways resulting from coding variants correspond only to DNA repair, specifically defective mismatch repair (PMS2, MLH1, MutSα, MutSβ, and TP53). Our findings reveal that DNA repair genes might occupy a central role within MPN acquisition and development, whereas at the same time each single MPN clinical entity might possess its own distinct mechanisms differentiating each one from the others.

Chromosome 9p Duplication Promotes T-Cell Exhaustion and Enhances Stem Cell Clonogenic Potential in JAK2-Mutant Myeloproliferative Neoplasms

Myeloproliferative Neoplasms (MPNs) are a diverse group of clonal hematopoietic disorders originating from a single hematopoietic stem cell, causing an excessive production of mature blood cells. Classic forms of MPNs include polycythemia vera, essential thrombocythemia, and myelofibrosis. The most common mutation is a gain-of-function point mutation in the JAK2 gene, known as JAK2V617F. This mutation leads to constant activation of the JAK-STAT signaling pathway, resulting in the overgrowth of MPN cells. Furthermore, the variant allele frequency (VAF) of JAK2V617F has a significant impact on the severity and phenotype of the disease. Alongside many described genetic mutations, cytogenetic abnormalities are commonly observed in MPNs, particularly involving chromosome 9. As the JAK2 gene is located on the short arm of this chromosome, we hypothesized that chromosome 9 copy number abnormalities might be a disease modifier in JAK2V617F-mutant MPN patients. To characterize the biological effects of chromosome 9 copy number abnormalities on JAK2-mutated MPN cells, we analyzed circulating CD34+ hematopoietic stem and progenitor cells (HSPCs) as well as monocytes and granulocytes from 32 MPN patients. Through Next Generation Sequencing, we categorized the patients into three main groups based on JAK2 mutation and copy number status: patients with two copies of the JAK2 gene with either heterozygous (VAF between 20% and 60%; n=10) or homozygous (VAF > 60% n=10) JAK2V617F mutation, or patients carrying mutant JAK2 and gene amplification (n=12). In-depth analysis of JAK2-amplified patients revealed that the amplification involved the entire chromosome 9p, thus including other gene loci like CD274, which encodes programmed death-ligand 1 (PD-L1). Further investigation of the order-of-events and clonal hierarchies through droplet digital PCR on CD34+ cell-derived colonies showed that most colonies of 9p-duplicated patients had three copies of JAK2, with 2 out of 3 alleles harboring the JAK2 mutation and that point mutations are frequently the initial pathogenic event in clonal evolution, followed by amplification of the JAK2-mutated allele. Functionally, CD34+ cells from +9p patients displayed high clonogenicity and gave rise to a greater number of primitive colonies (Colony Forming Unit-Granulocyte, Erythrocyte, Monocyte, Megakaryocyte, CFU-GEMM). As JAK2 hyperactivation had been previously reported to lead to increased PD-L1 expression, we further explored the functional significance of CD274 amplification in +9p patients. Our analysis showed increased PD-L1 messenger RNA and protein levels in +9p patient CD14+ monocytes compared to those from patients carrying only two copies of chromosome 9. Moreover, immunofluorescence analysis demonstrated significant re-localization of PD-L1 to the cytoplasmic membrane in monocytes from +9p patients, but not in JAK2V617F-homozygous patients (panel A). Increased levels of PD-L1, an immune checkpoint known to curb T cell activation, led us to analyze the T cell compartment, which resulted enriched in CD3+/CD8+/CD57-/PD-1+ exhausted T-cells in +9p patients compared to other MPN patients and healthy donors (panel B). In conclusion, our comprehensive characterization of the molecular interplay between JAK2V617F and chromosome 9 alterations, along with their immunological implications due to PD-L1 hyperactivation, fills a critical knowledge gap and provides valuable insights into the disease progression of 9p-MPNs. Further analysis is ongoing to explore the associations between 9p duplication and hematological parameters in 9p-MPN patients for a better understanding of the clinical implications of this genetic abnormality in MPNs.

Ras Activity Is Significantly Elevated in Patients with Primary Myelofibrosis and Is Agnostic to the Presence of RAS and JAK pathwaymutations

Myelofibrosis (MF) is a myeloproliferative stem cell disorder arising from various clonal mutations in early myeloid-committed hematopoietic progenitor cells. The subsequent acquisition of additional mutations and cytokine signaling then heralds the recruitment of fibroblasts to the marrow space, leading to collagen deposition. MF disease behavior varies widely and hematopoietic stem cell transplantation remains the only curative therapeutic option. In non-transplant candidates, managing cytopenic disease is uniquely challenging as patients develop resistance to commonly-utilized Jak-inhibitors. Expanding the molecular landscape of the disease beyond Jak/Stat signaling with the goal of identifying new therapeutic targets has, accordingly, been an area of growing interest in MF research. Recently-published studies, including results from our own institution, have identified mutations that potentially activate Ras signaling as being associated with features of advanced MF (higher somatic mutation burden) as well as with shorter overall survival and progression to AML. The goals of our study is to expand on this prior data, characterize the activity of Ras signaling and determine its role in patients with MF. Peripheral blood mononuclear cells (PBMCs) from 27 patients with myelofibrosis (MF) at a single institution underwent integrated genomic-transcriptomic sequencing analyses using the University of Michigan MiOncoSeq platform. PBMCs were separated by Ficoll® followed by cell lysis and protein quantification. Ras activity was measured by the binding of effector Ras binding domain (RBD) via ELISA. Patients with MF were compared to healthy donors and to patients with either polycythemia vera (PV) or essential thrombocythemia (ET). Ras activity in MF was examined across different mutations and with respect to various clinical parameters. Multivariate analyses were performed to assess the impact of each individual mutation on Ras activity. Accompanying statistical analyses for all assessments were conducted using two-way unpaired t-tests. Overall, Ras activity in 21 assessable patients with MF was 1.64 fold higher than in PV/ET (12 patients, p=0.0462) and 1.74 fold higher than healthy donors (13 volunteers, p=0.0304). While the majority of patients in the MF cohort had driver mutations, including in JAK2 (73%) and CALR (15.4%), the individual types of driver mutations did not impact Ras activity. Within the Ras/MAP Kinase signaling pathway, mutations were present at a range of variant allele frequencies (VAF), including 2 patients with NRAS (one with VAF 42%, one <5%), 2 KRAS (both <5%), 2 NF1, 1 CBL and 1 RAF1 (VAFs not assessed). The individual impact of various driver mutations, epigenetic regulators and splicing factors as assessed by multivariate analysis identified a trend toward enhanced Ras activity with mutations in RAS, CALR, ASXL1, TET2 and TP53, suggesting that in addition to RAS, other mutations also activate Ras signaling in MF. With regards to clinical parameters, Ras activity was weakly correlated with spleen size and high molecular risk mutation (HMR) presence, although this did not reach statistical significance in this cohort due to a relatively small sample size. Next, we performed a differential enrichment of myeloid and lymphoid cells from PBMCs using EasySep™ and measured Ras activity in lymphoid and myeloid enriched cells. We observed that the ratio of myeloid:lymphoid activity in patients with MF with RAS/MAP Kinase pathway mutations is 3.8 times higher than in patients without such mutations (p=0.0012), suggesting that RAS pathway mutations have a predominant role in myeloid cells. Our results show that the majority of patients with MF have hyperactive Ras signaling compared to PV/ET or healthy donors, regardless of the presence of alterations in the Ras signaling pathway. This underscores the importance of functional assessment of Ras activity in patients with MF and provides evidence that targeting Ras signaling in MF may lead to therapeutic benefit. We are currently expanding our analysis to a larger cohort of MF patients and performing preclinical testing of Ras/MAP Kinase pathway inhibitors as a means of overcoming Jak-inhibitor resistance.

Oral Anticoagulant Therapy in Patients with Philadelphia-Negative Myeloproliferative Neoplasms: A Real-Life Comparison between Vitamin K Antagonists and Direct Oral Anticoagulants

BACKGROUND Chronic myeloproliferative neoplasms (MPN) are characterized by an high risk of thrombosis and bleeding. Historically, vitamin K inhibitors (VKAs) have been the preferred oral anticoagulant therapy (OAT) for these patients (pts). However, nowadays, VKAs are being used less frequently, and direct oral anticoagulants (DOACs) are becoming more popular. Despite this trend, there is still limited evidence in the literature regarding the efficacy and safety of these drugs in the context of MPN. Specifically, there is a lack of data directly comparing VKAs and DOACs in a real-world clinical setting. AIMS The study aimed to conduct a real-world analysis comparing the efficacy and safety of DOACs and VKAs in patients with MPN. Both medications were prescribed for the treatment and secondary prevention of venous thromboembolism (VTE) or for the prevention of thrombosis in pts with atrial fibrillation (AF). The secondary endpoints were to assess the influence of common demographic, clinical, and hematological variables on the incidence of thrombotic events (TE) and bleeding events (BE) METHODS We conducted an observational retrospective monocentric study that enrolled all consecutive pts with MPN, followed at the MPN Clinic of the IRCCS Foundation S. Gerardo dei Tintori - Monza between 1993 and 2022, who received OAT with VKAs or DOACs for VTE or AF. For each pts, we collected demographic, clinical, biological, therapeutic, and hematological data. TE were categorized based on their type (arterial or venous) and location. BE were classified as minor, major, or clinically relevant non-major bleeding, according to the ISTH classification. Thrombosis-free survival (TFS) and bleeding-free survival (BFS) were assessed from the initiation of OAT until the occurrence of the first TE or BE, respectively, and estimated using Kaplan-Meier analysis. The other analyses were conducted using common statistical methods. Multivariable analyses included previously established risk factors for BE and TE. RESULTS A total of 156 pts were enrolled: 89 were women (57%). The median age was 69 years (range 25-92); 61 were affected by myelofibrosis, 35 by polycythemia vera, and 48 by essential thrombocythemia. 64 pts received OAT with VKAs (41%), and 92 with DOACs (59%). For 82 pts OAT was prescribed for AF (52.6%), and for 74 for VTE (47.4%). 17 pts treated with DOACs for VTE received a reduced-dose extended treatment (45.9%). The median exposure time to OAT was 5.8 years in the VKAs group (range 0.2-20 years) and 1.77 years in the DOACs group (range 0.2-10 years). After a median follow-up of 3,2 years, 14 TE were reported: 5 (35.7%) in the DOAC-treated group, of which 4 were arterial (80%), and 9 (64.3%) in the VKA-treated group (7 arterial - 77.8%). The incidence rate of TE was 2.11 thrombosis/pts/year in the VKAs group and 2.14 thrombosis/pts/year in the DOACs group. There were no significant differences in terms of TFS between the two groups, whether considering the entire population or analyzing the therapeutic indications separately (respectively p=0.72, p=0.53, p=0.55). Overall, there were 27 BE: 13 in the DOACs group (51.9%), 14 in the VKAs group (48.1%). Among these, we report 8 major BE, 4 in both groups. The incidence rate of BE was 5.6/pts/year in the DOACs group and 3.3/pts/year in the VKAs group. We did not observe significant differences in terms of BFS analyzing the whole population and the VTE group (respectively p=0.13 and p=0.75), while we found a trend for lower BFS in pts treated with DOACs for AF (p=0.08, Fig. 1). In a multivariate model, adjusted for time of exposition to OAT, age, gender and antiplatelet treatment, the risk of BE was associated solely with DOACs administration (hazard ratio 3.7, p=0.01) We found no significant differences in the risk of TE and BE when considering the various types of DOACs used. Moreover, 45 (28.8%) patients received concomitant antiplatelet therapy, and we found no significant differences in the risk of BE or TE compared to the other group (p=0.39 and p=0.41, respectively). CONCLUSIONS To our knowledge, this is the largest study investigating the safety and efficacy of DOACs compared to VKAs for the treatment and prevention of TE in pts with MPN and VTE or AF. Our findings suggest that DOACs are as effective as VKAs, but they appear to exhibit a trend towards a higher overall risk of BE, especially in the AF group. More data from prospective multicentric studies are necessary to validate our results.

Familial Polycythemia Associated with JAK2 Mutation Previously Known As Variant of Unknown Significance

Introduction Mutations in the Janus Kinase 2 gene (JAK2) are commonly encountered in patients with myeloproliferative neoplasms (MPN) including polycythemia vera (PV) and essential thrombocythemia. JAK2 mutations particularly V617F is associated with PV in 98% cases. Gain of function mutations in the JAK2 exon 12 are reported to be associated with isolated erythropoiesis. Atypical mutations in JAK2 including (c.3323A>G, p.N1108S) were reported in patients with MPN who tested negative for the established driver mutations including exon 14 JAK 2, exon 9 CALR and exon 10 MPL genes. Primary familial congenital polycythemia (PFCP) is a rare disease inherited in an autosomal dominant fashion caused by hypersensitivity of EPO receptor. In such cases, an EPO receptor mutation is only found in 12-15% of affected patients. Due to associated increased risk of thrombosis, bleeding, leukemic involvement and myelofibrosis in PV, bone marrow evaluation is essential in this patient population. In patients with polycythemia, the goals of treatment are to maintain appropriate level of hemoglobin/hematocrit and to reduce severity of symptoms. We present a family affected by polycythemia and leukocytosis associated with an atypical heterozygous variant of JAK2 [c.3323A>G (p.Asn1108Ser)] currently reported as benign or variant of unknown significance (VUS) in literature. Methods Case series, retrospective electronic medical record review with institutional board review exemption Results We report a case series of three Caucasian family members affected with JAK2 VUS mutation (mother and two sons). The proband is a 17-year-old male who presented to an outside facility at the age of 14 with complaints of fatigue, headache, and intense pruritis after hot showers. Initial workup was significant for marked erythrocytosis and reticulocytosis without evidence of abnormal cells on peripheral film. Bone marrow evaluation showed trilineage hematopoiesis with no morphological features of a myeloproliferative neoplasm. Genetic testing was negative for JAK2 V617F and exon 12 mutations. However, whole exome sequencing (WES) revealed a heterozygous mutation of JAK2 gene, c.3323A>G (p.Asn1108Ser). Additional investigation on proband's younger brother, who complained of similar symptoms, also revealed the same JAK2 variant. The proband's mother reported non-specific symptoms including headache, flushing, and occasional blurry vision that she previously attributed to hypertension. Her labs were significant for leukocytosis, monocytosis and thrombocytosis in the presence of normal red blood cell count and WES showed the same mutation. Bone marrow evaluation of the brother and the mother showed normal trilineage hematopoiesis and was negative for malignancy (Table 1). Proband's father is reportedly negative for the aforesaid mutation and is clinically asymptomatic. The proband and his brother are currently managed with phlebotomy every 3 to 4 weeks to maintain hematocrit below 45%, leading to improvement in symptoms. Cytoreductive therapies have been discussed but not initiated. Proband's mother is managed by an adult hematologist. Scheduled phlebotomies with close surveillance is recommended for clinical practice along with interval research sequencing. Conclusion Our case series reports a family with clinical features and laboratories suggestive of a familial form of polycythemia, WES detected the presence of an atypical JAK2 mutation: c.3323A>G, (p.Asn1108Ser). The phenotypical and clinical significance of this mutation has been considered non canonical per reports and classified as likely benign by reputable commercial genetic testing laboratories. However, other reports identify this JAK2 variant as having a significant increased prevalence in patients with MPNs and AML compared to the general population. It may result in a gain of function of the JAK2 protein and subsequent development of MPNs. Enrollment in research sequencing protocols is encouraged in such patients to closely follow the pathologic potential and spectrum of genetic variants rarely reported in literature. Additional research to determine the pathogenicity of this genetic variant is highly warranted.

NK Cells Contribute to Therapeutic Effects of Type I Interferons in Polycythaemia Vera

Myeloproliferative neoplasms (MPN) are clonal hematopoietic stem cell (HSC) disorders resulting in massive overproduction of mature peripheral blood cells, e.g. myeloid cells in chronic myeloid leukemia (CML), erythrocytes in polycythemia vera (PV) or platelets in essential thrombocythemia (ET), eventually occurring with myelofibrosis (MF). The JAK2V617F mutation is highly abundant in PV and has triggered the development of JAK inhibitors relieving PV induced symptoms and splenomegaly. However, neither JAK inhibitors nor phlebotomy or chemotherapy as other treatment options affect the allelic burden of mutated JAK2 as indicator of the clone size of malignant HSCs and thus cannot alter the natural course of the disease. In contrast, interferon alpha (IFNa) is well known to be effective in all MPNs and can even induce long-term remissions but have only been recently approved in the EU for the treatment of PV. Concerning the mode of action, type I interferons (IFNs) can directly inhibit proliferation of the malignant hematopoietic stem cell (HSC) clone. As being an immune stimulatory, type I IFNs drive the activation of immune and stromal cells in general. Thus, it is reasonable to assume that indirect effects of IFNs may also contribute to therapeutic effects. However, the cell specific contribution of type I IFNs on normal and malignant HSCs is unexplored so far. To investigate the immunological mechanisms by which type I IFNs attack malignant HSCs in MPNs, we use a mouse model for PV, in which mice suffer from increased hematocrit and enlarged spleens and in which we can reduce disease symptoms by the injection of IFNa. Performing high-dimensional flow cytometry and single cell sequencing we show here a comprehensive analysis of the contribution of cells and pathways in the therapeutic effect of IFNa in P. vera mice. We identified NK cell frequencies and numbers to be increased in the bone marrow of P. vera mice upon IFNa therapy. In the absence of NK cells after antibody mediated depletion the reduced disease symptoms in IFNa treated P. vera mice were completely reversed, indicating that NK cells play a critical role in the beneficial effects of IFNa treatment of P. vera mice. Thus, NK cells may represent a previously unexplored target in the development of future treatments of MPNs.

Genetic Evolution from Chronic Myeloproliferative Neoplasms to Acute Myeloid Leukemia: An Analysis of Forty-Six Paired Samples

Background: Secondary acute myeloid leukemia (sAML) may arise from chronic myeloproliferative neoplasm (MPN). The genetic evolution pattern may be understood by paired sample analysis. Knowledge of genetic change in MPN/sAML is still limited and more extensive studies should be done. Patients and Methods: Forty-six paired samples (27 males) at diagnosis of MPN (12 polycythemia vera, 6 essential thrombocythemia, 25 primary myelofibrosis and 3 MPN-unclassified) and sAML transformation were available. Mutational analyses of 27 genes ( JAK2V617F, CALR, MPL, ASXL1, IDH1, IDH2, TET2, DNMT3A, EZH2, SRSF2, U2AF1, SF3B1, ZRSR2, SMC1, STAG2, SMC3, RUNX1, SETBP1, IKZF1, WT1, K-RAS, N-RAS, C-CBL, TP53, RAD21, BCOR and BCORL1) were performed. Mutations and their variant allele frequencies (VAF) were measured by next generation sequencing. Results: At baseline (MPN), the mutation number was 2 (maximal 5). Forty-five samples harbored driver gene mutations (31 JAK2V617F, 13 CALR and 1 MPL). Twenty-eight samples harbored at least one epigenetic regulator mutations (11 TET2, 8 DNMT3A,7 ASXL1, 5 EZH2, 2 IDH2, 1 IDH1). Twelve samples harbored spliceosome mutations which were mutually exclusive (6 SRSF2, 3 SF3B1, 2 U2AF1 1 ZRSR2). Other mutations were RUNX1, BCORL1 (n=2 each), CBL and BCOR (n=1 each). In sAML, the median mutation number was 3 (maximal 7). Loss of mutation was found for JAK2V617F (9/31), TET2 (1/11) and SRSF2 (1/6). Acquisition was most common for RUNX1 (n=9), followed by TP53, ASXL1 (n=5 each), K-RAS (n=4) , ZRSR2, STAG2 (n=3 each). The median number of acquired mutation was 1 (maximal 5). The frequencies of gene mutations are summarized in Figure 1A. The baseline VAF of JAK2V617F was high (>35%) in 80.6% (25/31). In progression to sAML, VAF was constant in 29.0% (9/31, VAF changes <10%), increased in 20.0% (9/31, increased VAF> 10%), decreased in 41.9% (13/31, decreased VAF>10%) (Figure 1B). The baseline VAF in CALR mutation was high (>35%) in 91.3% (12/13). VAF remained stable in 76.9% (10/13), and loss of CALR mutation was not observed (Figure 1B). TET2 mutations tended to be stable (7/16), but clonal expansion (5/16 of all mutation events at MPN), or acquisition (n=2) could be found during transformation to sAML. DNMT3A mutations remained stable (5/8), exhibited clonal expansion (2/8), or acquisition (n=1) in sAML progression. ASXL1 mutations remained stable (6/7), with clonal expansion (1/7), or acquisition (n=5) during MPN/sAML progression. All EZH2 mutants had clonal expansion (5/5) and one acquired in sAML (Figure 1B). We found that CALR mutationwas often the founding clone with high VAF at MPN phase, stable clone during sAML, and might acquire mutations in signaling pathways (1 CBL, 1 KRAS), epigenetic regulators (2 ASXL1, 1 IDH2), transcription factors (2 RUNX1, 1 BCOR), spliceosome (2 ZRSR2, 1 SF3B1), and cohesin complex (1 STAG2). JAK2 mutationwas often preceded by other ancestral mutations such as TET2, ASXL1, DNMT3A, EZH2, IDH2, SRSF2 or ZRSR2. During evolution, leukemia arose from non- JAK2 mutated clones with mutations in EZH2, DNMT3A, RUNX1, EZH2, SETBP1, or acquisition of TP53, SF3B1, BCOR and STAG2 mutations) in sAML. In outcome, the median time from MPN to sAML was 82 (range 3.7 to 338.5) months. Female gender, loss of JAK2V617F, presence of U2AF1 and absence of IDH1 were associated with shorter time to sAML transformation. No other genetic mutation in our gene panel or the number of mutations harbored by patients in our cohort had significant impact on time to sAML. Marked increase of VAF was observed for JAK2 (7/22) , DNMT3A (1/7) , IDH1 (2/3), IDH2 (1/3), TET2 (3/10) , EZH2 (4/5), C-CBL (1/1) and SRSF2 (1/4). Marked decrease of VAF was observed for JAK2 (4/22), MPL (1/1), DNMT3A (1/7) , ASXL1 (1/8), SF3B1 (2/3), SRSF2 (1/4) and U2AF1 (1/2). Multiple mutational clones were detected for TET2 (two clones in 4 samples and 3 clones in 1). Clonal expansion and reduction were observed in two pairs of samples upon sAML transformation. Conclusions: At the MPN stage, most patients had driver mutations with frequent co-mutations of epigenetic modifier or spliceosome genes. CALR mutant clones were stable while for JAK2, sAML often arose from clones without JAK2V617F which acquired RUNX1, TP53, ASXL1 and K-RAS. Research funding:Ministryof Science and Technology Council,Taiwan (NSTC 112-2314-B-182-055) and Ministry of Health and Welfare, Taiwan (112-TDU-B-222-124001)

Preclinical Evaluation of JAK2 Specific Investigational Oligonucleotide for the Treatment of MPNs

Background: Myeloproliferative neoplasms (MPN) are clonal hematopoietic stem cell malignancies with cytokine independency or hypersensitivity. Polycythemia vera (PV), an acquired MPN characterized by increased blood cell mass and hematocrit and leukocyte count, is associated with incidental myelofibrosis (MF). Myelofibrosis (MF) is a chronic and progressive disorder characterized by bone marrow fibrosis, extramedullary hematopoiesis, leukoerythroblastosis and splenomegaly. Essential Thrombocythemia (ET) is another type of MPN associated with increased number and size of circulating platelets. All MPNs leave patients at risk for progression to leukemia. Abnormal cytokine signaling due to an aberrant JAK2-STAT pathway has a vital role in pathogenesis of various MPNs. JAK2 mutations can result in hematologic malignancies, where hyperactive signaling of the JAK2-STAT pathway promotes tumor cell proliferation, invasion, and angiogenesis. Increased JAK2 kinase activity is observed in hematologic malignancies; somatic JAK2 V617F gain-of-function mutations are found in large numbers between different subtypes of MPNs patients (40-95%) and are also implicated in MDS-MPN cases. We hypothesize that preventing JAK2 transcriptionby antisense oligonucleotide (ASO) mediated exon masking of the JAK2 intron-exon junction will result in reduced JAK2 mRNA, and thus JAK2 protein, by providing nonsense mediated decay (NMD) in the reading frame. This may alleviate disease manifestations of many patients suffering from different forms of MPNs. Methods: This study utilized a HEL92.1.7 cell line harboring the V617F JAK2 gain-of-function mutation. Antisense Oligonucleotides (ASOs) are small fragments of DNA/RNA that have the ability to reduce production of target protein via interaction with RNA. We designed a series ASOs targeting JAK2 sequence and tested these at a range of concentrations. The ASOs were designed and prioritized based on in silico binding affinity and limited off-target activity. HEL92.1.7 cells underwent treatment with several ASO-prototypes and functional readout at the end of 72h of incubation. Results: We observed significant JAK2 protein decrease (~50%) in ASO-treated samples compared to untreated samples. JAK2 qPCR results confirmed reduction of target transcript in 40-60% range. STAT5 phosphorylation status further confirmed this effect, and we report a ~35% pSTAT5 reduction. Furthermore, this ASO showed limited off-target effects in silico. siRNA and CRISPR knockout protein lysates were used as internal controls. Our preclinical data support our ASO as a highly specific JAK2 agent, affecting direct levels of JAK2 as well as downstream STAT signaling. Conclusions: Around 50-60% of primary MF patients harbor the JAK2 V617F gain-of-function mutation. ASOs offer the capability to directly target JAK2-driven neoplasms with highest precision and effectively reduce JAK2 protein production, without off-target effects linked with small molecules targeting various kinase families. The ability to reduce JAK2 protein may alleviate the disease burden that patients with hematologic malignancies face, resulting in a higher quality of life through prevention and treatment.

Cooperation between SF3B1 and JAK2V617F Mutations Accelerates Fibrotic Progression in Myeloproliferative Neoplasms By Enhancing STAT1 Signaling

Myeloproliferative neoplasms (MPN) are heterogenous clonal hematologic neoplasms where current therapies show limited disease-modification. We previously reported that the splicing factor, SF3B1, is mutated in 5-10% of MPN correlating with myelofibrotic progression in essential thrombocythemia. This adverse phenotype contrasts with SF3B1 mutationsin myelodysplasia where it is associated with milder disease. Moreover, SF3B1 K666 is the dominant hotspot in MPN in contrast to K700E in MDS (abstract #185043). The mechanism by which SF3B1 mutations accelerate myelofibrotic progression in JAK2V617F-mutated MPN is not understood and will be a key step towards the development of disease-modifying therapies. To address this, we applied two complimentary single-cell multiomic methods to study 15 individuals (healthy donor [HD, n=5], JAK2V617F-single mutated [J+, n=5] and JAK2V617F- SF3B1K666double mutated MPN cases [JS+, n=5]). First, we characterised the cellular landscape of JS+ MPN using CITE-seq/10X genomics platform. Then, we analyzed SF3B1-mutant ( SF3B1mut) disease-driving hematopoietic stem and progenitor cells (HSPCs) and their associated aberrant splicing signatures using TARGET-seq, which permitted single-cell genotyping with allelic resolution and intra-patient comparison of mutant versus wild-type (wt) cells. In an analysis of 109,498 cells, we observed abnormally expanded erythroid (ERP), megakaryocyte (MK) and unexpectedly, eosinophil-basophil-mast cell (EBM) progenitors in JS+ as compared with J+ or HD HSPCs (Fig 1A). Two HSC clusters were present, one of which (HSC_mpp2) principally comprised of JS+ cells and had a distinct molecular program (Fig 1A); upregulation in MK (eg. PF4, VWF) and EBM lineage signature genes (eg. TPSB2), interferon signaling genes but downregulation in apoptosis genes and canonical stemness markers (eg. CD133). These signatures suggest the JS+ driven HSC cluster may be MK/EBM-transcriptionally primed and may therefore, promote expansion of these abnormal progenitors which are implicated in driving fibrosis development. Through genotyping of >5000 HSPCs, we resolved the clonal architecture of JS+ MPN, observing that in most cases double mutant cells were present and clonally dominant, with mutant SF3B1 the initiating event. Mutations arose from the HSC level and genotypes were equally distributed in the HSPC compartment. Highlighting the importance of single-cell analysis, and not apparent at the bulk level, JAK2V617F and SF3B1K666mutationsresided in separate clones for one case with a WHO fibrosis score 0 in contrast to the other 4 cases with double mutant cells and fibrosis scores 1-3. This reinforces the cooperative effect of thesemutationsto drive fibrosis. We performed full-length transcriptomics of select HSPCs to study SF3B1mut-specific aberrant splicing events (ASE). In a global splicing analysis, more events were detected in JAK2V617F- SF3B1K666 double mutant than single mutant HSPCs. Retained intron (RI) events were the most prevalent ASE, followed by skipped exon and alternative 3' splice site events. We identified previously described SF3B1mut-specific ASE ( MAP3K7, ERGIC3 and SEPTIN6) confirming the validity of our dataset to study new ASE. Inter- and intra-patient comparison of SF3B1mut versus SF3B1wt cells uncovered a number of novel biologically relevant ASE, including two RI events in STAT1. These RI events were reduced in SF3B1mut HSPCs with the predicted consequence of increased STAT1 expression and activation, of direct relevance to MPN pathobiology since STAT1 is known to play a key role in enhancing megakaryopoiesis in MPN (Fig 1B). We validated these events using long-read Nanopore sequencing of single-cell cDNA libraries and have developed a cell model system with transient expression of SF3B1K666 mutation versus SF3B1wt in HEK293T to corroborate STAT1 ASE occurring as a direct consequence of mutant SF3B1. In further support of upregulated STAT1 activation, we observed upregulation of STAT1 downstream targets in abnormally expanded JS+ HSPCs. In conclusion, this analysis resolved the clonal architecture and hematopoietic cellular composition of JS+ MPN, identifying abnormally expanded and transcriptionally primed HSPC populations with abnormal splicing of STAT1 enhancing JAK-STAT signaling, uncovering the mechanism by which these mutations promote an accelerated fibrotic phenotype.

Assessment of Minimal Clinically Important Difference in Patient-Reported Myelofibrosis-Associated Symptoms Using an Anchor-Based Analysis Based on MANIFEST Arm 3 Data

Background Myelofibrosis (MF) is a life-threatening hematologic neoplasm that can arise as a primary condition or as a progression from polycythemia vera or essential thrombocythemia. The MF Symptom Assessment Form (MFSAF) v4.0 is a seven-item questionnaire that assesses symptom severity from the patients' (pts) perspective. Each item is rated on a scale from 0 (Absent) to 10 (Worst Imaginable). The MFSAF Total Symptom Score (TSS) ranges from 0 to 70, with higher scores indicating worse symptoms. A ≥50% reduction in TSS (TSS50) is a commonly used binary endpoint in clinical trials. However, the clinical meaningfulness of the dichotomization of response at 50% cutoff across the spectrum of responses remains uncertain. The relevance of considering TSS as a continuous endpoint in MF studies is assessed here to capture the full spectrum of symptom changes, enabling a potentially more accurate assessment of treatment effects. Pelabresib (CPI-0610) is an investigational oral small-molecule BET inhibitor, while the Janus kinase inhibitors (JAKis) ruxolitinib or fedratinib are the current standard of care for intermediate- or high-risk pts with MF.InArm 3 of the Phase 2 MANIFEST trial of JAKi treatment-naïve pts with MF treated with pelabresib and ruxolitinib, 56% achieved TSS50 response at Week (Wk) 24. Aims This anchor-based analysis aims to determine the minimal clinically important difference (MCID) of change in TSS as a continuous endpoint, based on data from JAKi treatment-naïve pts with MF treated with pelabresib and ruxolitinib, to better evaluate the incremental clinical benefit of treatment effects. Methods Data from Arm 3 of the MANIFEST Phase 2 study of treatment-naïve pts with MF treated with pelabresib combined with ruxolitinib were used (July 29, 2022 data cut). Anchor-based methods compare the change in a scale-based outcome measure with that of an established patient-reported outcome (PRO). These methods are commonly used to establish the MCID, defined as the smallest difference in score that pts consider beneficial. In this analysis, the Patient Global Impression of Change (PGIC), a seven-point scale reflecting overall improvement compared with baseline (BL), was used as the anchor PRO. On the PGIC, pts rated their change from ‘very much improved’ to ‘very much worse’ (Table 1). The relationship between TSS changes (percentage and absolute) at Wk 24 and PGIC categories was examined using correlation and graphical analyses. Linear and quantile regressions were applied to assess the impact of a one-point difference in PGIC score on TSS change at Wk 24, adjusting for BL TSS score. Only pts with available TSS and PGIC data at Wk 24 were included, with no imputation for missing values. Results In Arm 3 of the MANIFEST trial, 78 of 84 pts treated with pelabresib and ruxolitinib had complete TSS data and PGIC at Wk 24. At BL, mean TSS was 16.4 (SD=8.4), and median TSS was 15.5 (range 2.0-38.3). A -58.8% and -7.7 median percentage and absolute reduction in TSS at Wk 24 was observed, respectively. For PGIC at Wk 24 (Table 1), only six pts (7.7%) scored in the three worsening PGIC categories. Percentage and absolute changes in TSS, and the percentage TSS50 responders for each PGIC category are shown in Table 1. Linear regression demonstrated that a one-category increase in PGIC (indicating worsening) corresponded to an 11.89% increase (95% CI 3.12-20.65) in TSS (indicating worsening) at Wk 24. The quantile regression estimate for this relationship was 11.36% (3.53-19.19). For absolute change at Wk 24, a one-category increase in PGIC resulted in a 1.67 (range 0.74-2.60) increase in mean TSS, with a 1.87 (range 0.62-3.12) increase in median TSS. Conclusion Assuming a one-category change in the seven-category PGIC represents a clinically meaningful difference, the analyses suggest that the MCID in TSS for JAKi treatment-naïve pts with MF could be approximately 11-12% for percentage change from BL and approximately 1.5-2 points for absolute change from BL. These findings provide valuable guidance for assessing treatment outcomes and evaluating symptom improvements' clinical significance in this patient population. Further exploration of these findings requires larger data sets, which will be addressed in the forthcoming Phase 3 MANIFEST-2 study evaluating pelabresib and ruxolitinib versus placebo and ruxolitinib in JAKi treatment-naïve pts with MF.

Dynamic Thrombocytopenia Has a Negative Impact with Distinctive Genetic and Immunologic Features in Patients with Myelofibrosis

Background: Myelofibrosis (MF) is a distinct subtype of the Philadelphia-negative myeloproliferative neoplasms (MPNs) characterized by an overproduction of differentiated hematopoietic cells. MF consisted of primary myelofibrosis (PMF) and secondary myelofibrosis (SMF) progressed from polycythemia vera and essential thrombocythemia. The pathologic and clinical features of MF include myeloproliferation, bone marrow fibrosis, anemia, splenomegaly, and MF-associated symptoms. But, some cases of MF shows cytopenic features, which were known to effect on inferior outcomes. The previous studies evaluating the prognostic impact of cytopenia at diagnosis have a limitation because the progression of cytopenia through disease course cannot be reflected. Therefore, the study by a time-dependent model is required. In the present study, we evaluated prognostic implication of dynamic thrombocytopenia as a time-dependent variable in patients with myelofibrosis. And, we attempt to determine the genetic and immunologic factors associated with dynamic thrombocytopenia. Methods: A total of 226 patients diagnosed with myelofibrosis and treated at Seoul St. Mary's Hematology Hospital from December 2001 to August 2021, who were performed DNA samples for next-generation sequencing (NGS) analysis were included in this study. To evaluated the correlation of various immune subsets and dynamic thrombocytopenia, immunophenotypic analysis by multiparameter flow cytometry was performed. Results: The patients with myelofibrosis were composed of SMF (n=66, 29.2%), PMF (n=160, 70.8%). Among the patients, 100 × 10 9/L or more platelet count group (PLT≥100) was the most common (n = 131, 60%), followed by progression to thrombocytopenia (defined as a PROG) (n = 64, 28.3%), and less than 100 × 10 9/L platelet count group (PLT<100) (n = 31, 13.7%). We observed that the 4-year overall survival of patients was 57.7%, 89.4%, and 93.9% in the PLT<100, PROG, and PLT≥100 groups ( p<0.001), respectively. In the PLT≥100 groups, patients with PMF were fewer than SMF. Next, we performed a time-dependent covariate analysis between the PLT≥100 group and PROG group. The progression of thrombocytopenia showed an inferior overall survival (P=0.004). In the multivariate analysis, the progression of thrombocytopenia ( p = 0.042, HR =7.7 [95% CI 1.04-7.70]), ASXL1 mutation ( p= 0.041, HR = 9.91 [95% CI 1.05-9.91]), and IDH1 mutation ( p =0.002, HR = 1103 [95% CI 5.19-1103]) were associated with poor OS. The frequency of CD45RA +CD4 + T cells was lower in PROG group compared to the PLT>100 group ( p = 0.015). Among the patients in the PROG group, detection of ASXL1 mutation was higher than the other groups ( p=0.021). Genemic alteration of ASXL1 showed a trend for a decreased CD45RA +CD4 + T cells than counterpart ( p=0.01). Conclusion: Our study demonstrated that dynamic thrombocytopenia, as a time-dependent variable has a prognostic value for the patients with MF, and low frequency of CD45RA +CD4 + T cells and genetic alteration of ASXL1 correlated with the progression to thrombocytopenia. Further investigation is warranted to determine the crucial clones and immune microenvironment signature associated with cytopenic features in patients with MF.

SHP2 Inhibition Enhances JAK2 Inhibitor Therapy in Preclinical Models of Myeloproliferative Neoplasms

The classic myeloproliferative neoplasms (MPNs) polycythemia vera, essential thrombocythemia, and primary myelofibrosis are driven by aberrant activation of the JAK2 kinase, primarily by mutations in 3 different genes, JAK2, MPL, and CALR. JAK2 inhibitor therapy is generally effective at reducing MPN patient constitutional symptoms and thus can improve quality of life but has minimal effect on reducing mutant driver allele burden and does not readily alter the course of disease, reflecting the ability of MPN-driving cells to persist during therapy. Thus, the ability of current anti-JAK2 therapies to significantly modify underlying disease biology is limited. Long-term studies of the JAK2 inhibitor ruxolitinib in myelofibrosis patients have, however, identified survival benefits, yet this may be due to anti-inflammatory and spleen reducing impacts. Ultimately, the inability of ruxolitinib and other approved JAK2 inhibitors to effectively antagonize allele burden indicates alternative therapeutic approaches are needed to overcome the persistent survival of MPN-driving cells during JAK2 inhibitor therapy. Efforts to identify mechanisms that can thwart the effects of JAK2 inhibition in MPN cells have highlighted activation of the RAS-ERK pathway, including mechanisms of RAS-ERK signaling that are independent of JAK2. ERK or MEK inhibition have been shown to enhance the activity of ruxolitinib in MPN mouse models, but monotherapeutic inhibition of these kinases displayed limited efficacy. The SHP2 (Src homology region 2 domain-containing phosphatase-2) tyrosine phosphatase mediates RAS activation and subsequent MEK/ERK signaling downstream of cytokine and growth factor receptors. SHP2 plays a role in oncogenic signaling and in resistance to kinase targeted therapies, exemplified in both solid tumor and leukemia models. Given the evidence that activation of RAS-ERK signaling can antagonize the effects of JAK2 inhibition and the fact that SHP2 mediates RAS activation via multiple mechanisms downstream of numerous growth factor and cytokine receptors, we hypothesized that SHP2 may contribute to the survival of MPN cells during JAK2 inhibitor treatment and may provide a therapeutic target to improve current anti-JAK2 therapies. Using preclinical MPN models, we aimed to block RAS activation by inhibiting SHP2 to assess the potential SHP2 inhibition may have as a monotherapy or in combination with JAK2 inhibition. We found that the SHP2 inhibitors RMC-4550 and SHP099 enhanced growth inhibition of MPN model cell lines (e.g., SET2 and UKE1) in combination with ruxolitinib, effectively preventing ruxolitinib persistent growth. Concomitant treatment with both inhibitors led to enhanced inhibition of RAS-GTP levels and ERK activation and increased apoptosis compared to single agents. SHP2 inhibition antagonized the neoplastic growth of primary hematopoietic progenitor cells from MPN patients and synergized with ruxolitinib in all patient samples assessed. In an MPN mouse model driven by MPL-W515L, RMC-4550 antagonized MPN phenotypes, including leukocytosis, hepatosplenomegaly, and allele burden. RMC-4550 treatment diminished levels of a subset of pro-inflammatory cytokines, including IL-1β, CXCL9, and TNFα, among others. The combination of RMC-4550 with ruxolitinib not only suppressed advancing leukocytosis but led to white blood cell levels lower than at treatment initiation (Panel A) and suppressed thrombocytosis compared to ruxolitinib alone. The combination also led to enhanced inhibition of hepatomegaly, extramedullary hyperplasia, megakaryocyte hyperplasia, and allele burden, and a trend toward improved fibrosis. The SHP2 inhibitor RMC-4550 enhanced the survival of mice (following termination of treatment analyses) as both a monotherapy (p < 0.0001 compared to vehicle) and in combination with ruxolitinib (p < 0.01 compared to ruxolitinib monotherapy). Importantly, the combination of SHP2 inhibition using RMC-4550 with JAK2 inhibition using ruxolitinib for 4 weeks in wildtype mice was well tolerated with respect to hematologic parameters and exemplified by no effect on body weight (Panel B). Given SHP2 inhibitors are already undergoing clinical evaluation in patients with solid tumors, our findings suggest that SHP2 is a therapeutic target with potential to be rapidly translated to clinical assessment for MPN patients.

Importance of Diagnosing Polycythemia Vera (PV) Patients (pts) Whose Hematocrit (HCT)/Hemoglobin (HGB) Are Less Than the Who 2022 Stipulated Criteria

Introduction: HCT/HGB values stipulated by WHO in 2022 1* (and 2016) are required for distinguishing pts with PV from those with either essential thrombocythemia (ET), cellular phase primary myelofibrosis (MF), or JAK2-mutated clonal hematopoiesis. We verified WHO values have 100% specificity but only approximately 75% sensitivity 2. Thus, PV pts exist with values less than WHO criteria for HCT/HGB. Distinguishing these PV pts from ET, MF or CHIP is important since prognosis and management differs. No studies to date, however, have defined the frequency and characteristics of these specific PV pts. We therefore reviewed our cohort of PV pts to identify those whose HCT and HGB values were lower than WHO. We report their clinical, hematologic, and molecular findings and their survival to date. Methods : We queried our database of PV pts, extracting data as previously reported 3 for those with HCT/HGB values below WHO threshold at diagnosis (dx). These pts met PV diagnostic criteria by either marrow morphology, red cell mass (RCM), and/or erythropoietin (EPO) values. We recorded clinical, hematologic and JAK2 V617F variant allele frequency (VAF) findings, therapy, clinical course, MF free (MFS) and overall survival (OS). Results: Twelve of 470 (2.6%) pts had criteria of PV with HCT/HGB values below WHO. All were JAK2 V617F positive. 10 had quantitative values, with median VAF 18% (range 6.2-46.1%). Median age was 45 years. Mean erythrocyte values for males (N=4) and females (N=8) respectively were: HCT 47.5 and 42.4%, HGB 16.1 and 14.2 g/dL, RBC 5.18 and 5.13 x10 6 /uL, MCV 91.6 and 83.0 fL (Table 1). Cr-51 RCM was increased in 3. 11 pts had marrow biopsies showing varying degrees of hypercellularity and increased megakaryocytes with the characteristic morphologic changes described in PV. Patient 1 did not have Cornell review of marrow but otherwise had lab and clinical values consistent with PV (Table 2). 5 of 11 pts had an MF score of 0, 5 MF-1, and 1 MF-2. Stainable iron was absent in 5, present in 4, and not assessed in 2. Since low JAK2 VAF is associated with ET, in the 9 pts with VAF under 20%, or qualitative values only, the PV dx was made on marrow morphology and increased RCM, low EPO level, high RBC count or combinations thereof (Table 2). Seven pts were symptomatic at dx, including fatigue, headache, and generalized abdominal discomfort. Spleen was palpable at dx in 3 pts, 1, 8, and 9 cm below the left costal margin, respectively. The last two pts presented with splanchnic vein thrombosis. Another pt had a cerebrovascular accident at dx. Maintenance treatment was phlebotomy in 1, hydroxyurea 2, ruxolitinib 2, and rIFN 7. None of the pts progressed to MF; 1 pt died of colon cancer. Over a median follow up of 7 years, median MFS and OS have not been reached (100% MFS and 92% OS to date). Conclusion: A small number of PV pts present with HCT/HGB below WHO threshold; however, recognition is important because of their symptoms and risk of significant thrombosis. Our 12 pts were diagnosed with PV based on a combination of either marrow morphology, EPO level, and RCM. As isotopic RCM studies are difficult to obtain, RBC and marrow morphology becomes extremely important for correct dx. The presence of marrow iron stores in 4 pts is significant since most PV pts are iron deficient at dx 4. This suggests that the HCT/HGB values observed may represent “early” disease. The HCT/HGB values in the 2 splanchnic vein thrombosis pts with significant splenomegaly represent well-known splenic sequestration and increased plasma volume, masking true polycythemia. In these cases, marrow studies and EPO values are mandatory to distinguish PV from ET. This study emphasizes the appropriate use of RCM and should prompt development of alternative methods to measure blood volume. *2022 WHO PV diagnostic criteria 1: Major Criteria: 1. HGB > 16.5 g/dL (men) HGB > 16.0 g/dL (women) or HCT >49% (men) HCT > 48% (women) or increased red cell mass more than 25% above mean normal predicted value 2. BM biopsy showing hypercellularity for age with trilineage growth (panmyelosis) including prominent erythroid, granulocytic and megakaryotic proliferation with pleomorphic, mature megakaryocytes (differences in size) 3. Presence of JAK2 or JAK2 exon 12 mutation Minor criteria: subnormal serum erythropoietin level

Timing of Thrombosis with Myeloproliferative Neoplasm Diagnosis Among Veterans and Civilians

Rates of Arterial (AT) and Venous Thrombosis (VT) are increased in Veterans with Myeloproliferative Neoplasms (Vet-MPN), including Essential Thrombocythemia (ET), Polycythemia Vera (PV), and Primary Myelofibrosis (PMF), compared to matched controls (J Clin Oncology 2023 41:16_suppl, 7011). The timing of first AT or VT episode in Vet-MPNs remains undefined when compared to Civilians with MPN (Civ-MPN). These findings could elucidate optimal timing for risk mitigation strategies to prevent thrombotic complications and should help to determine duration of thrombotic risk and need for systemic anticoagulation prophylaxis. The Veterans Affairs Informatics and Computing Infrastructure (VINCI) database was queried from January 1, 2006 to January 26, 2023 to identify Vet-MPNs, AT, and VT through ICD-9 and -10 codes. This process yielded 2,752 Vet-MPN and 583,803 Veterans without MPN from Illinois, the state most representative of the US population. These cohorts were compared to data derived from a retrospective chart review of Civ-MPN cared for between January 1, 2019 to December 31, 2022, at MedStar Georgetown University Hospital and MedStar Washington Hospital Center. Service-Connected Exposures (SCE) to potential carcinogens among Veterans were verified through their duration and location of service. We subdivided time to first AT and VT from MPN diagnosis as follows: more than 3 months prior to MPN diagnosis, within 3 months of diagnosis, 3 months to 1 year post diagnosis, 1 to 5 years, and more than 5 years after MPN diagnosis. Mean data from various cohorts were analyzed utilizing Fisher's exact test. Vet-MPN experienced a statistically higher incidence of AT compared to Civ-MPN (p=.0019) and Civ-MPN experienced a statistically higher incidence of AT compared to Veterans without MPN (p=0.02) which served as a control. In contrast, there was a higher incidence of VT among both Vet- and Civ-MPN cohorts compared to Veterans without MPN (p<.0001) (Table 1). The mean ages at MPN diagnosis and onset of first AT were similar (≥63 years) in both Civ- and Vet-MPN. In contrast, the mean age of MPN diagnosis and age of first VT were considerably earlier for Civ-MPN (51 years) versus both Vet-MPN with VT and Veterans without MPN with VT (≥67 years). There was a higher prevalence of hypertension, hyperlipidemia, diabetes, heart failure, and smoking among Veterans with or without MPN compared to Civ-MPN with either AT or VT (Table 1). A trend toward statistical significance was observed for the presence of hypertension and hyperlipidemia (p=0.049 and p=0.02, respectively) in Vet-MPN compared to Civ-MPN, while Veterans without MPN had a statistically higher prevalence of hyperlipidemia (p=0.02) and heart failure (p=0.001) in AT. Notably, a trend toward statistical significance in VT was observed for the presence of diabetes among all Veterans with or without MPN compared to Civ-MPN (p=0.01 and p=0.007, respectively). There was a biphasic distribution in time to onset of first AT and VT in all Vet- and Civ-MPN cohorts (Figure 1). There was a higher cumulative incidence of first AT and VT in Vet-MPN at more than 3 months prior to MPN diagnosis compared to Civ-MPN with AT and VT (Figure 1). On the other hand, the Civ-MPN cohort had a late surge of first VT more than 5 years after MPN diagnosis compared to the other MPN cohorts. A biphasic distribution in time to onset of first AT and VT was also observed among the Vet-MPN cohort when stratified by SCEs from World War II, the Korean War, the Vietnam War, and the Gulf War. The vast majority of both AT and VT in Vet-MPN from all SCE examined experienced onset of thrombotic events more than 3 months prior to MPN diagnosis. Most VT in Gulf War Vet-MPN occurred within 3 months of MPN diagnosis. In conclusion, the predominant risk for developing thrombotic complications in MPN, particularly in Vet-MPN, occurs in a biphasic pattern, beginning more than 3 months prior to MPN diagnosis and becoming apparent again at more than 5 years after MPN diagnosis. These findings suggest that the MPNs convey an enduring hypercoagulability potential throughout the natural history of the disease. There appears to be an accelerated onset of thrombotic complications earlier in the course of disease in Vet-MPN, perhaps reflecting their SCE with toxic carcinogens. More work is necessary to determine epigenetic influences on MPN natural history.

BCL-Xl Represents a Novel Therapeutic Target in Type 2 Mutant Calr-Driven Myeloproliferative Neoplasms

Myeloproliferative neoplasms (MPNs) are clonal disorders that arise in the hematopoietic stem cell (HSC) compartment and that result in the excess production of mature blood cells of the myeloid lineage. All MPNs are unified by their reliance on the constitutive activation of the JAK/STAT pathway. Nevertheless, JAK inhibitors have proven insufficient to significantly improve disease outcome, highlighting the need to further understand the molecular mechanisms driving these diseases and to discover novel therapeutic targets. Calreticulin (CALR), an endoplasmic reticulum (ER) chaperone protein, has been found to be mutated in ~40% of essential thrombocytosis (ET) and myelofibrosis (MF) cases. The most common mutations in CALR are type 1 (52 base pair deletion; CALRdel52) and type 2 (5 base pair insertion; CALRins5) mutations, which both confer distinct clinical and prognostic characteristics. We have previously demonstrated that mutant CALR-driven MPNs rely not only on aberrant JAK/STAT signaling but also on the upregulation of BCL-2 family proteins and activation of the unfolded protein response (UPR) pathway (ASH abstract #147859, 2021). Specifically, we have shown that CALRins5 mutations lead to activation of and dependency on the activating transcription factor 6 (ATF6) branch of the UPR. In parallel, we found that CALRins5 cells exhibit differential up-regulation of the anti-apoptotic protein BCL-xL, but not BCL-2 (ASH abstract #3588, 2021). Based on these findings, we hypothesized that the active form of ATF6, which functions as a transcription factor, may directly transcriptionally activate BCL-xL leading to its up-regulation specifically in CALRins5 but not CALRdel52 MPN cells, and that the ATF6/BCL-xL axis may represent an important pro-survival mechanism that can be targeted for therapeutic gain in CALRins5 MPN patients. To test this hypothesis, we first validated that BCL-xL is significantly up-regulated in CALRins5 versus CALRdel52 and CALR wild type (CALRwt) human cell lines and peripheral blood mononuclear cells (PBMCs) from MPN patients. Because BCL-xL can also be regulated downstream of JAK/STAT signaling, we next tested whether the increased expression of BCL-xL in CALRins5 cells is due to ATF6 activation, JAK/STAT activation, or both. In cells treated with JAK inhibitor ruxolitinib, we found that while BCL-xL expression levels decreased nearly 90% in ruxolitinib treated CALRdel52 cells, this decrease was much more modest in CALRins5 cells, suggesting JAK/STAT activation is only partially responsible for BCL-xL up-regulation in CALRins5 MPN cells. Conversely, in ATF6 knockout cells, we found that deletion of ATF6 did not affect BCL-xL levels in CALRwt or CALRdel52 cells, but did significantly abrogate BCL-xL up-regulation in CALRins5 cells. This suggests that in CALRins5 cells, while JAK/STAT signaling contributes in part to BCL-xL up-regulation, ATF6 is the key mediator of BCL-xL expression. CUT&RUN studies further revealed that ATF6 directly regulates BCL-xL transcription. Lastly, we sought to test whether inhibition of BCL-xL alone is sufficient to induce apoptosis in CALRins5 cells. BH3 profiling studies revealed that CALRins5 cells are more primed for apoptosis via BCL-xL peptide antagonism compared to CALRdel52 cells. We next evaluated the activity of A-1331852, a BCL-xL selective inhibitor, alone or in combination with ruxolitinib, in human cell lines and PMBCs from MPN patients. We observed that CALRins5 cells displayed decreased viability in response to A-1331852 alone and in combination treatments, suggesting an increased sensitivity to BCL-xL inhibition. In conclusion, we demonstrate that CALRins5-driven MPN cells display an enhanced sensitivity to BCL-xL inhibition, which may represent an effective therapeutic approach for CALRins5+ MPN patients.

Newly Identified Roles for PIEZO1 Mechanosensor in Controlling Normal Megakaryocyte Development and in Primary Myelofibrosis

Introduction: Mechanisms through which mature megakaryocytes (Mks) and their progenitors sense the bone marrow extracellular matrix (ECM) to promote lineage differentiation in health and disease are still partially understood. We found PIEZO1, a mechanosensitive cation channel, to be expressed in Mks. Human mutations in PIEZO1 have been described to be associated with blood cell disorders. Primary Myelofibrosis (PMF) is a Chromosome Philadelphia-negative Myeloproliferative Neoplasm (MPN), characterized by altered stiffer ECM and megakaryocytosis, often driven by the gain-of-function mutation in JAK2 V617F. Here, we study a role for PIEZO1 in megakaryopoiesis and proplatelet formation under normal physiological conditions and in the context of PMF. Methods: Bone marrow Mks from C57BL/6J control and myelofibrotic mice carrying the human JAK2 V617F+ mutation, or Mks derived from stem cells of patients carrying the same mutation were analyzed at mRNA and protein levels for Piezo1 expression. Effects of Piezo1 activation or inhibition on Mk maturation and ploidy were assessed by flow cytometry, and proplatelet formation by fluorescent microscopy. Human peripheral blood samples from PMF patients, diagnosed according to established criteria, or healthy individuals were used to compare the two categories for Piezo1 relative expression on mRNA and protein levels and platelet biogenesis. Mk-specific double knockout mice of Piezo1/2 were generated to compare platelet counts under normal conditions or bone marrow ablation with 5-FU challenge. Culture of mouse and human Mks in different 3D microenvironments was used to demonstrate in vitro association of Piezo1 expression levels with different ECM stiffness substrates. Results: Mouse Mks express PIEZO1 and negligible levels of PIEZO2. Pharmacological activation of mouse PIEZO1 increases the number of immature CD41 + Mks, while the number of mature CD41 +CD42 + Mks and Mk ploidy level are reduced. Piezo1/2 knockout mice show an increase in Mk size and platelet count, both at basal state and upon marrow regeneration. Similarly, in human samples, PIEZO1 is expressed during megakaryopoiesis. Its pharmacological activation reduces Mk size, ploidy, maturation, and proplatelet development. Resulting effects of PIEZO1 activation on Mks resemble the profile in PMF. Intriguingly, Mks derived from PMF mice bearing the Jak2 V617F mutation show significantly elevated PIEZO1 expression, compared to wild-type controls. Pharmacological activation of PIEZO1 in these cells significantly augments the number of immature CD41 + Mks, and sharply reduces the number of mature CD41 +CD42 + cells and ploidy level. Accordingly, Mks isolated from bone marrow aspirates of JAK2 V617FPMF patients show increased PIEZO1 expression compared to Essential Thrombocythemia. Additionally, PIEZO1 was overexpressed in 3D culture of human and mouse Mks in stiffer microenvironments as compared to softer ones and liquid medium. Most importantly, PIEZO1 expression in bone marrow Mks is inversely correlated with patient platelet count. The ploidy, maturation, and proplatelet formation of Mks from JAK2 V617FPMF patients are rescued upon PIEZO1 inhibition with GsMTx4. Discussion: Our data show that PIEZO1 might serve as a break of Mk maturation and platelet formation in physiology, and its upregulation in PMF Mks might contribute to aggravating some hallmarks of the disease. Our finding that inhibition of PIEZO1 activity partially rescues the aberrant Mk phenotype associated with PMF suggests that GsMTx4 holds potential as a therapeutic strategy to mitigate the pathological effects observed in Mk development in PMF patients.

High Rate of Disease Progression in Patients with Low-Risk Myelofibrosis (MF) Enrolled in the Prospective, Real-World MOST Study

Background: Patients (pts) with low-risk MF are typically excluded from interventional clinical trials, thus data regarding the prognosis of this pt population are largely derived from retrospective analyses. The Myelofibrosis and Essential Thrombocythemia Observational Study (MOST; NCT02953704) is a prospective observational study that enrolled pts with low-risk MF defined by Dynamic International Prognostic Scoring System (DIPSS) criteria. In this analysis, we report disease progression, defined by worsening of clinical or laboratory parameters, in MF pts during the MOST study. Methods: Criteria for enrollment in MOST were confirmed locally and centrally, and included a physician-reported diagnosis of MF (PMF, post-PV, or post-ET) and lack of any DIPSS risk factors except for age >65 y. Of the 232 MF pts enrolled, 205 met this criteria upon central review and were included in this analysis as Cohort A; the remaining 27 pts were enrolled despite having ≥1 DIPSS risk factor (other than age >65 y) and were analyzed separately as Cohort B. Data were collected as previously described (Gerds A. CLML. 2022;22:e532). Disease progression on study was defined as the acquisition of 1 of the following criteria: hemoglobin (Hb) <10 g/dL, platelets <100×10 9/L, presence of constitutional symptoms (weight loss, fever, or sweats), new or worsening splenomegaly, blasts >1%, WBC count >25×10 9/L, death due to disease progression, leukemic transformation (LT), or >1 RBC transfusion. Median (range) follow-up was 52.9 months (42-68). Results: In Cohort A, 120/205 pts (58.5%) had evidence of disease progression during the study. Baseline characteristics comparing pts with vs without disease progression were similar; although compared with pts without progression, those who progressed were older at diagnosis (median [range], 65.9 [21-88] vs 62.9 [35-86] y), older at enrollment (median [range], 70.5 [35-88] vs 66.0 [35-88] y), and had longer median (range) time from diagnosis to enrollment (1.9 [0-38] vs 1.4 [0-24] y). Similar percentages of pts with vs without progression were receiving MF-directed therapy at enrollment (59.2% vs 61.2%); most common treatments were ruxolitinib (33.3% vs 14.1%) and hydroxyurea (22.5% vs 36.5%). Of pts in Cohort A tested for JAK2 mutations, 66/93 (71.0%) with disease progression and 42/65 (64.6%) without were positive. Of pts tested for CALR mutations, 16/21 (76.2%) with disease progression and 17/23 (73.9%) without were positive. Among the 120 pts with progression, 64 (53.3%) met 1 criteria, 27 (22.5%) met 2 criteria, and 29 (24.2%) met ≥3 criteria during the study. The most common progression criteria met were Hb <10 g/dL (47.5%), platelets <100×10 9/L (31.7%), constitutional symptoms (30.8%), and new/worsening splenomegaly (28.3%) (Table 1). Median (95% CI) durations to 1st, 2nd, and 3rd progression criteria were 24.9 (19.2-34.7), 28.2 (14.3-NE), and 11.6 (8.9-26.5) months, respectively (Figure 1). Of pts who progressed, 6 (5.0%) developed LT and 12 (10.0%) died due to disease progression. In Cohort B at time of enrollment, 25/27 pts (92.6%) met 1 progression criteria. The most frequent was Hb <10 g/dL (13/27 [48.1%]), followed by WBC count >25×10 9/L (7/27 [25.9%]); constitutional symptoms (4/27 [14.8%]), and blasts >1% (1/27 [3.7%]); 1 pt (3.7%) met 2 progression criteria (Hb <10 g/dL and constitutional symptoms), and 1 pt (3.7%) met 3 progression criteria (blasts >1%, constitutional symptoms, and WBC count >25×10 9/L). Eight of 27 pts (29.6%) had evidence of further disease progression while on study (Table 1). The most common new progression criteria met were Hb <10 g/dL and platelets <100×10 9/L (each 5/8 [62.5%]) and constitutional symptoms and new/worsening splenomegaly (each 3/8 [37.5%]). Conclusions: This real-world observational analysis from MOST is the first prospective analysis of disease progression in low-risk myelofibrosis. The results show that with over 4 y of follow-up, nearly 60% of pts with low-risk MF had evidence of disease progression; 17/205 pts (8.3%) developed LT or died (both occurred in 1 pt). Median time to acquisition of 1st and 2nd disease progression criteria was approximately 2 y, respectively, decreasing to approximately 1 y for the 3rd criteria; this indicates a slow but increasing rate of progression over the course of the study. These data provide real-world insight into disease progression rate in pts with low-risk myelofibrosis.

Platelet-Restricted Clonal Hematopoiesis

Clonal hematopoiesis (CH) is a common age-related condition in which somatic mutations accumulate in hematopoietic stem cells (HSCs), leading to the clonal expansion of mutated cells. CH has been causatively linked with risk of disease, including atherosclerotic cardiovascular disease (CVD), and reduced overall survival. The current model of hematopoiesis suggests substantial heterogeneity and lineage-priming within the HSC compartment, including the existence of multipotent but platelet (PLT)-biased HSCs. However, all CH studies to date are based on whole blood or granulocyte (GRA) DNA, and therefore they do not assess for PLT-restricted clones, which we reasoned could be particularly important given the association of CH with CVD. We aimed to assess for presence of PLT-biased CH in a cohort of normal individuals, hypothesizing that PLT-biased CH might represent a distinct subgroup of persons with CH. We analysed a cohort of 151 individuals, aged 22.0 to 98.6 years ( x̅ = 74.9; 95% CI, 50.0 to 91.8), with normal blood counts, and no known hematological malignancy. We first established a method for isolation of high-purity cDNA from PLTs alongside isolation of gDNA and cDNA from matched GRAs. Myeloid cell contamination was extremely low in the PLT fraction as assessed by flow cytometry and molecular analysis. We first performed targeted analysis for presence of the JAK2V617F mutation in PLT and GRA fractions due to the known association of JAK2V617F with CVD and myeloid neoplasms, including essential thrombocythemia. We developed a high sensitivity allele specific JAK2V617F droplet digital PCR (ddPCR) assay (variant allele frequency [VAF] limit of detection 0.1%) for parallel assessment of gDNA and cDNA from GRAs and PLTs respectively. When including all cases with a VAF > 0.1%, JAK2V617F CH was detected in 21 (13.9%) individuals, consisting of 2 cases of non-lineage-restricted CH (present in both GRA and PLT fractions), 6 cases of GRA-restricted CH, and 13 cases of PLT-restricted CH. Only 6 cases showed VAF ≥ 2% (consistent with CH of indeterminate potential) and all were PLT restricted. Comparison of median JAK2V617F VAF in PLT cDNA with VAF in GRA gDNA revealed significantly higher VAF in the PLT-derived material (1.00% vs. 0.26%; p = 0.04). We next developed a custom, targeted, hybridisation capture next-generation sequencing (NGS) panel for parallel ultra-deep sequencing of both gDNA and cDNA, covering the major genes and hotspot mutations involved in CH ( DNMT3A, TET2, ASXL1, IDH2, JAK2, CALR, MPL, CBL, KRAS, GNB1, SF3B1, SRSF2, U2AF1, PPM1D and TP53). The panel comprised a total of 250 120-nucleotide-long probes (including 27 cDNA-specific probes) with a combined footprint size 25,083 bp. Performance studies showed acceptable off-target effect and good coverage, alignment rate, uniformity, and complexity. Agreement between the NGS-measured VAF for GRA gDNA and GRA cDNA was high (ρ c = 0.952, alpha = 0.05). In total, we screened GRA gDNA and matched PLT cDNA samples from 42 individuals. At the resolution of VAF ≥ 2%, analysis of PLTs substantially enhanced the number of individuals with detected CH from 12 (28.6%) to 26 (61.9%) (Fig. 1). Amongst the detected variants (n = 44), mutations detected in the GRA fraction (n = 20) accounted for less than half of the detected variants, while the PLT fraction included ~75% of the variants (n = 33), including 55% (n = 24) that were PLT-restricted. Notably, lineage-restriction for the above variants held true when taking into consideration clones with VAF < 2% too. Median VAF for all CH variants was 3.9% (95% CI, 2.2% to 32.3%), with no difference between GRA and PLT CH. The detected variants were distributed across DNMT3A, TET2, ASXL1, JAK2, SF3B1, and PPM1D genes, with evidence of lineage bias towards PLTs for TET2- and JAK2-related CH. PLT-restricted clones included high-VAF (> 5%) variants in DNMT3A (n = 4; x̅VAF = 13.2%), TET2 (n = 2; x̅VAF = 7.7%), JAK2 (n = 2; x̅VAF = 21.6%), and SF3B1 (n = 1), including an individual with DNMT3AQ842R and the SF3B1K666N at VAFs 16.7% and 12.5% respectively. Selected NGS-detected variants were orthogonally validated. We describe PLT-restricted CH as a frequent phenomenon in persons with normal blood parameters. Given the known association between CH and risk of CVD and hematological malignancy, further studies on larger patient cohorts are warranted to determine the importance of PLT vs. GRA CH in relation to clinical endpoints.

The Reticulin, Collagen, Osteosclerosis (RCO) Score Is Clinically and Prognostically Relevant in Patients with Secondary Myelofibrosis (SMF)

Introduction: Polycythemia vera (PV) and essential thrombocytemia retain an inherent risk for progression to secondary myelofibrosis (SMF; PPV- and PET-MF, respectively). Histopathological examination of bone marrow (BM) biopsy constitutes an essential step in SMF diagnosis and can show a variety of stromal changes, including reticulin fibrosis, collagen deposition, osteosclerosis, and microvascular proliferation. In 2017, Gianelli et al. ( Histopathol 2017;71:89) developed a comprehensive, prognostically informative score, named reticulin (MF), collagen (Co), osteosclerosis (Ost) score (RCOs), aimed at the histological evaluation of primary MF (PMF). Aims&Methods: This retrospective, single-center study aimed to validate the RCOs and explore its clinical, genetic and prognostic correlates in a cohort of patients (pts) with clinically and molecularly annotated SMF diagnosed according to the 2022 ICC criteria. The RCOs was assessed by two independent experienced histopathologists (U.G, R.S.), as previously described ( Histopathol 2017;71:89). Results: The study included 72 pts with a diagnosis of SMF: 28 (39%) PPV-MF and 44 (61%) PET-MF. Median age at diagnosis was 66 years (range 35-85), 44 (61%) were male. Histological analysis of BM stromal changes according to the RCO algorithm provided the following results (Fig. 1A): MF-0 n=0, MF-1 n=0, MF-2 n=35 (49%), and MF-3 n=37 (51%); Co-0 n=8 (11%), Co1 n=25 (35%), Co-2 n=22 (31%), and Co-3 n=17 (24%); Ost-0 n=25 (35%), Ost-1 n=25 (49%), Ost-2 n=7 (10%), and Ost-3 n=5 (7%). The corresponding figures for PPV- and PET-MF separately are reported in Fig. 1A. Overall, morphological features were homogeneously distributed with no significant differences between the two disease subtypes. The computation of the RCOs returned the following results: RCOs 0 n=0, RCOs 1 n=0, RCOs 2 n=6 (8%), RCOs 3 n=15 (21%), RCOs 4 n=10 (14%), RCOs 5 n=9 (13%), RCOs 6 n=14 (19%), RCOs 7 n=9 (13%), RCOs 8 n=5 (7%), and RCOs 9 n=4 (6%). Median RCOs was 5 (range, 2-9) for both PPV- and PET-MF. First, we investigated the correlations of the RCOs with distinct clinical and molecular characteristics in the whole SMF cohort. A higher RCOs was associated with lower hemoglobin value (Pearson's r, -0.3, P=.0173), lower platelet count (Pearson's r, -0.3, P=.0106), and higher LDH value (Pearson's r, 0.3, P=.0308). We found no significant correlations with regards to driver and other co-occurring mutations. Median overall survival (OS) was 71 months (95% CI 45-99), with no significant difference between the PPV- and PET-MF cohorts. In univariate Cox regression analysis, the RCOs directly correlated with the risk of death (HR 3.9, 95% CI 1.2-13.8, P=.0282). In multivariate analysis, the predictive relevance of the RCOs was independent of the MYSEC-PM stratification. By analyzing the single components of the RCOs, both the MF and Ost variables correlated with survival (respectively: HR 3.9, 95% CI 1-12.9, P=.0315; HR 2.2, 95% CI 1.1-4.7, P=.0337), unlike the Co variable. ROC analysis with death as endpoint identified 6 as the optimal cut-off value. Pts with RCOs >6 (n=32) had a significantly worst OS compared to pts with RCOs <6 (n=40), with median values of 42 vs 90 months (HR 2.5, 95% CI 1.3-5.2, P=.0097). ROC analysis separately in the PPV- and PET-MF cohorts returned 5 and 7, respectively. Due to the small study population, we opted to keep the two cohorts together and ascertained whether the integration of the dichotomous RCOs >/<6 (RCOs 6) improved the performance of the MYSEC-PM. To this end, we computed the C-index, Brier score, and time-dependent AUC to assess the prognostic performance of standard and RCOs 6-integrated MYSE-PM (Fig. 1 B). Overall, the combination of MYSEC-PM and RCOs 6 was remarkably superior in predicting death for all parameters and at all time points considered (24, 48, 72, and 96 months). Conclusions: The results of the current study indicate that the RCOs, although formerly developed for pts with PMF, is clinically and prognostically relevant in SMF. Most importantly, we showed that integrating the discrete RCOs 6 into the SMF-specific MYSEC-PM can significantly improve the predictive performance of the prognostic model.