Marrow Fibrosis Research Articles

MRI Reliably Captures Bone Marrow Metrics in Myelofibrosis

Purpose Biopsy remains the clinical standard for evaluating bone marrow of patients with myelofibrosis (MF). However, randomly sampling a small volume of bone marrow, typically from the ilium, fails to capture heterogeneity of disease across anatomic sites. The invasive, painful nature of the procedure limits patient acceptance of repeated biopsies to monitor disease status and response to therapy. Here, we present a non-invasive, MRI technique for evaluating bone marrow in MF. Participants and Methods We analyzed bone marrow of 66 study participants (45 with MF; 15 with essential thrombocytosis or polycythemia vera; and 6 healthy controls). We assessed participant bone marrow across three anatomic sites (vertebral bodies, ilium, and femoral heads), using three quantitative MRI sequences: proton density fat fraction (PDFF) for fat content; apparent diffusion coefficient (ADC) for cellular effect on water mobility; and magnetization transfer (MT) for macromolecular structure and fiber deposition. We correlated these MRI metrics with patient prognosis (dynamic international prognostic scoring system; DIPSS) and iliac biopsy data for cellularity and fibrosis (MF grade). Results Participants with MF had elevated MT ratio (MTR) and ADC and lower PDFF compared to healthy participants. Non-MF MPN participants demonstrated the same trend, though to a lesser extent. Individual MRI metrics correlated strongly across anatomic sites (r pearson from 0.57 to 0.89, p-value < 0.01), with notable heterogeneity both within and among different regions. DIPSS risk groups had limited correlation with MRI metrics. To understand to what extent quantitative MRI biomarkers identify fibrotic marrow, we developed a multivariate logistic regression model to stratify the 45 participants with MF by early fibrosis (MF grades 0-1) and overt fibrosis (MF grades 2-3). Of the seven total anatomy-MRI metric combinations, three had the greatest contributions toward grading marrow fibrosis: PDFF in the vertebral bodies and ilium along with ADC in the ilium. We developed the logistic regression model to predict early and overt fibrosis with these three parameters using a 70%/30% train/test split with 5-fold cross validation. Based on this model, quantitative MRI metrics are associated with the severity of fibrosis in the bone marrow (test set performance: accuracy = 84.6%, sensitivity to predict overt fibrosis = 88.9%, specificity = 75%, area under the receiver operator curve = 0.94). Conclusion Quantitative bone marrow MRI reliably captures relevant metrics of MF bone marrow, including cellularity and fat replacement by fibrosis, and may be useful in clinical drug trials and patient management for MF.

Red Cells Distribution Width and Prognosis in Myelofibrosis Patients Treated with Ruxolitinib

Introduction Myelofibrosis (MF) is a BCR::ABL1-negative myeloproliferative neoplasm characterized primarily by genetic abnormalities and an aberrant production of pro-inflammatory cytokines (including IL-2, IL-6, TNF-α, and TGF-β among others) released from pathological hematopoietic stem cells leading to the deposition of reactive fibrosis in the bone marrow (BM), at the same time the development of extramedullary hematopoiesis, mainly in the spleen and liver, and finally the production of red blood cells (RBC) of often bizarre shape, i.e. dacryocytes. Consequently, increased variability in RBC volume, so-called anisocytosis, is a hallmark of MF. A small number of studies have already demonstrated that quantitative measurement of anisocytosis in terms of red cells distribution width (RDW) may have a negative prognostic role in newly diagnosed MF patients. Aim In this study we further evaluate the prognostic impact of RDW in a series of patients with primary (PMF) and secondary myelofibrosis (SMF) at diagnosis and during treatment with ruxolitinib (RUX). Methods Between October 2014 and May 2023, the RDW of 49 consecutive patients with PMF or SMF (32 males and 17 females; median age at RUX initiation, 70.5 years) were retrospectively assessed at MF diagnosis, at RUX start and after 3, 6, 12, 24 and 36 months from therapy initiation. All patients were treated with RUX according to current indications, requiring an IPSS risk of at least intermediate-1. Red cells distribution width was routinely reported by automated cell counters as part of RBC count, with an upper limit of normal of 14.5% used as a cut-off in our laboratory. At the time of RDW evaluation, complete blood cells count and spleen size [expressed as the distance from the left costal margin (BCM) in cm] were available for all patients. Results Among 49 patients, 24 had PMF (10 prefibrotic and 14 overt fibrotic) and 25 SMF [15 had post-polycythemia vera (PPV)- and 10 post-essential thrombocythemia (PET)-MF]. The JAK2V617F mutation was detected in 38 (77.6%) cases, CALR mutations in 8 (16.3%), and MPL in 1 (2%), while 2 patients (4.1%) were “triple-negative”. Elevated RDW values were present in all but 3 patients at MF diagnosis with a median RDW of 18.7% (range, 13.3% - 25.7%). RDW was higher in patients with splenomegaly ( p=0.04) and with JAK2V617F mutation ( p=0.09). Furthermore, looking specifically at JAK2V617F-mutated cases, the greater the RDW values, the higher the JAK2V617F allele burden. Interestingly, a higher RDW value at diagnosis also correlated with a shorter time from MF diagnosis to RUX start (-3.5 months per one RDW unit, 95%CI: -7.5 to +0.4; p=0.08). We observed a modest (<1%) increase in RDW during the first 6 months of RUX treatment. In a multivariable random-intercept model that considers all time points and contains the covariates time, RUX dose, and hemoglobin (Hb), we also observed a clear decrease in RDW with increasing Hb (slope: -0.35% per g/dL of Hb, 95%CI: -0.56 to -0.14; p=0.001). Median overall survival (OS) from MF diagnosis was 6.5 years with a median duration of RUX treatment of 33.3 months. The median RDW at diagnosis of 18.7% was used as a cut-off to identify two subgroups of patients [Group 1 (N=22): RDW 18.7% to 25.7%; Group 2 (N=23): RDW 13.3% to 18.6%] showing a difference in OS [Group 1 vs. 2: Hazard Ratio (HR) 2.74, 95%CI: 1.13 to 6.65; p=0.03] ( Figure 1); the median OS was 8.5 (14 deaths) and 11.8 (9 deaths) years for the Group 1 and 2, respectively. Using continuous RDW at diagnosis, the crude HR was 1.21 per one RDW unit (95%CI: 1.06 to 1.37; p=0.004). In a Cox model adjusted for gender, age, and Hb, the HR was 1.13 (95%CI: 0.99 to 1.29; p=0.06). Notably, RDW at diagnosis was associated with a 35% increase in infectious disease mortality in a crude analysis (HR 1.35, 95%CI: 1.07 to 1.71, 8 deaths; p=0.01) and a 27% increase (HR 1.27, 95%CI: 0.99 to 1.63; p=0.07) in the adjusted analysis. Conclusions Red cells distribution width may represent a good integrative measure of several disease processes and also has prognostic significance at the time of MF diagnosis and during RUX treatment. Even though our preliminary observations are limited by the small number of patients and need to be further confirmed by larger studies, we can speculate that RDW may help in the rapid detection of MF patients with a poor prognosis, which can only partially be improved from currently available targeted therapies with JAK inhibitors.

Phase 1/2 Study of TP-3654, a Selective PIM1 Kinase Inhibitor: Preliminary Data Showed Clinical Activity and Cytokine Reductions in Relapsed/Refractory Myelofibrosis Patients

Background: PIM1 expression is significantly upregulated in hematologic malignancies including myelofibrosis (MF) and has been shown to play a role in modulating the activity of cytokine induced PI3K/AKT and JAK/STAT pathways. The distinct clinical features of MF are partly attributed to the elevated circulating cytokines; a subset of which are shown to correlate with poor prognosis. TP-3654, an oral investigational highly selective PIM1 kinase inhibitor, is currently being evaluated in a phase 1/2 study in patients (pts) with relapsed/refractory MF. In preclinical studies, TP-3654 alone and in combination with ruxolitinib showed spleen size and bone marrow (BM) fibrosis reduction in murine MF models. Importantly, TP-3654 also showed reduction of cytokine response genes and serum TGF-β in progenitor cells and JAK2V617F murine MF model, respectively. Methods: This phase 1/2 study evaluates the safety and efficacy of TP-3654 monotherapy in pts with MF (NCT04176198). Key eligibility criteria include primary or secondary MF; previously treated with or ineligible for JAK inhibitor treatment; DIPSS intermediate 1, 2 or high-risk MF; platelet ≥25x10 9/L; splenomegaly (≥450 cm 3 by imaging); and ≥2 measurable symptoms per MFSAF v4. The study aims to identify the MTD and/or RP2D of TP-3654 monotherapy and to assess safety, clinical activity (spleen volume reduction [SVR], total symptom score [TSS] improvement), PK, and PD markers (cytokine profile, BM fibrosis etc.). We investigated the patterns of similarity of 43 cytokines changes after 12 weeks of TP-3654 treatment using principal component analysis (PCA). Results: As of 20 June 2023, a total of 23 pts enrolled across 5 dose levels in the phase 1 dose escalation part. At baseline, median age 73 years (range 61, 80), spleen volume 2008.2 cm 3 (range 609, 6006), TSS 20.8 (range 4, 62), platelet 115 x10 9/L (range 64, 520), hemoglobin 10.1 g/dL (range 5.9, 13.7; 5 pts requiring transfusion). All pts, except one, had received ≥1 prior JAK inhibitor treatment (median 12.9 months [range 2.3, 81.0]). Median duration of TP-3654 treatment was 14.9 weeks (range 2, 96). No DLT occurred. Treatment-related adverse events (TRAEs) occurring in ≥20% of pts included grade 1/2 nausea, vomiting, and diarrhea. Grade 3 TRAEs were diarrhea (n=1) and platelet count decreased (n=2). Mean hemoglobin and platelet counts remained stable throughout the 24-week treatment period. SVR observed in 10 of 13 evaluable pts treated for ≥12 weeks (median best change -11%, range +97% to -63%); 3 pts showed ≥35% SVR. TSS improvement were observed in 12 of 13 evaluable pts (median best change -70%, range +9 to -100%); 7 pts showed ≥50% TSS response (Fig 1A); 5 pts had durable response for ≥12 weeks. Broad reductions in cytokines observed as early as within the first 24 hours after TP-3654 treatment. At week 12, pts with higher cytokine reductions correlated with greater TSS improvement. Two components of the PCA, PC1 and PC7, include MF associated cytokines such as IL-8, IL-18, CD40, ENRAGE amongst others, were reduced with TP-3654 treatment and these reductions correlated with symptom improvement (Fig 1B). BM fibrosis was reduced in one pt who also achieved spleen and symptom responses, showed MF associated cytokine reductions, and is on active treatment for 2 years. Conclusions: This preliminary data of TP-3654 in relapsed/refractory MF pts showed early signs of clinical activity including spleen volume reduction, TSS improvement, and correlating cytokine reductions. TP-3654 is well tolerated with limited myelosuppressive adverse events. Enrollment is ongoing as monotherapy and current data support the development of TP-3654 in combination with JAK inhibitors given the preliminary clinical activity and minimal cytopenia.

First-Hit SETBP1 Mutations Cause a Myeloproliferative Disorder with Bone Marrow Fibrosis

Somatic SETBP1 mutations are found in various myeloid disorders covering both myeloproliferative neoplasms (MPN) and myelodysplastic syndromes (MDS). To characterize the early steps of SETBP1-mediated leukemogenesis, we generated a conditional mouse model expressing SETBP1 G870S mutant in the entire hematopoietic tissue through Cre-mediated recombination driven by the Vav1 promoter. In all mice signs of a hematological disease appeared between 30 and 90 days: longitudinal analysis revealed accumulation of white blood cells (WBC) in virtually all heterozygous SETBP1 G870S mice, with a marked imbalance between the lymphoid and myeloid lineages in favor of the latter, and an increase in mature myeloid cells in absence of circulating blasts or non-segmented myeloid precursors. Kaplan-Meier analysis revealed a dramatic decrease in event-free survival in SETBP1 G870S mice. BM histology showed overt myeloid hyperplasia with fibrosis and no evidence of dysplasia except for the megakaryocytic lineage. Exploration of visceral organs highlighted the presence of severe hepatosplenomegaly with massive infiltration by myeloid elements, disruption of normal tissue architecture, and signs of extramedullary hematopoiesis. Single-cell RNA-sequencing (scRNA) on BM Lin - cells identified Mecom, Setbp1 and Hoxa9 among the top upregulated genes in SETBP1 G870S precursors. Pseudotime analysis of scRNA data revealed the presence of 261 spatially autocorrelated genes. Of them 158 were also differentially expressed in SETBP1 G870Svs control mice. Spi1, encoding for the master regulator of hematopoietic differentiation PU.1, was significantly upregulated in SETBP1 G870S precursors. PU.1 promotes the maturation of bone marrow early precursors towards the granulocytic/monocytic lineages by directly impairing the transcription of Gata2 and Gata1. In line with these data, Gata2 and Gata1 expression was profoundly suppressed in the early myeloid precursors of SETBP1 G870S mice, which associated with the down-modulation of markers of the erythroid lineage, such as the Carbonic Anhydrase 1, in the MEP differentiation branch. Ourmouse model recapitulates many clinical features of primary myelofibrosis (PMF). As up to 10% of PMF are triple-negative for the classical JAK2, CALR, and MPL mutations (TN-PMF), we set out to assess SETBP1 mutations in this context. We analyzed 36 TN-PMF patients by exome sequencing. In 29 we did not find any evidence of somatic mutations; in the remaining 7 (7/36; 19.4%) high VAF SETBP1 degron mutations were identified. In two patients SETBP1 was found as a single somatic variant, while in the others it coexisted with ASXL1, NRAS TET2 SRSF2, RIT1 CBL or CSF3R mutations. Notably, SETBP1 mutations were the only shared alterations among all seven patients. A markedly reduced overall survival was observed for SETBP1 positive patients, with a median survival time of 24 months (median survival not reached at 60 months for SETBP1 negative patients). To dissect the clonal architecture of SETBP1 positive TN-PMF at single-cell resolution, we applied single-cell targeted DNA sequencing on 3 SETBP1-mutated TN-PMF samples using the Tapestri technology, showing that, as opposite to MDS/MPN, in all TN-PMF cases SETBP1 is a very early clonal event. Therefore, our study suggests a clear partition of TN-PMF into two groups, the first one characterized by oncogenic, high VAF SETBP1 mutations accompanied by other oncogenic variants and poor prognosis and the other one characterized by no evidence of an active clonal process or of driver oncogenic events and much lower aggressiveness. SETBP1 positive TN-PMF disorders lie in a gray zone comprised between the MPN and MPN/MDS boundary. In MDS/MPN, SETBP1 mutations are often found as mid or late events. In contrast, we show here that in TN-PMF SETBP1 appears to be one of the earliest hits, therefore highlighting a potentially relevant biological difference occurring in the two subsets. In this context the presence of early SETBP1 mutations seems to promote the occurrence of a myeloid disorder characterized by the triad: leukocytosis without differentiation block or dysplasia, BM fibrosis and progressive splenomegaly, hence recapitulating many clinical features of overt or prefibrotic/early PMF and resembling the SETBP1 mouse model.

Thrombocytopenia Associated with Myelofibrosis Is Characterized By a Reduced Proportion of Megakaryocyte (MK) Biased Hematopoietic Stem Cells, Defects in MK Maturation and Increased Levels of Pro-Inflammatory Cytokines

The clinical course of patients with advanced forms of myelofibrosis (MF) can frequently be punctuated by thrombocytopenia. Marrow biopsies from these MF patients uniformly reveal megakaryocytic (MK) hyperplasia and dysplasia irrespective of the patient's platelet count. We have performed studies to better understand the origins of MF associated thrombocytopenia. We first examined the expression of G6b-B (MK-lineage specific immunoreceptor tyrosine-based inhibition motif) which is essential in promoting MK maturation, normal platelet release, platelet size and function (Becker. Blood Adv 2022). Recent reports have indicated that genetic deletion in G6b-B and loss-of-function mutations in both murine models and man lead to severe macro-thrombocytopenia and increased bone marrow fibrosis (Becker. Blood Adv 2022; Hofmann. Blood 2018; Melhem. Eur J Hematol 2017), indicating a possible role for this receptor in the development of thrombocytopenia in MF patients. We generated populations of MKs from mononuclear cells or CD34 + from thrombocytopenic or non-thrombocytopenic MFs and healthy donors (HDs) using a two-step culture system as previously described (Mosoyan. Leukemia 2017). A progressive increase of MK maturation (CD41 +CD42 +) was observed in HDs (28.5%±7) and non-thrombocytopenic MFs (32%±13.6) with a concomitant increase of total G6B expression 27%±9 and 38%±14 respectively. By contrast, MKs generated from thrombocytopenic MFs patient cells had limited CD41 +CD42 + (7%±5) and G6B (8.7%±4.7) expression (p<0.05). In addition, morphological analysis revealed that MKs from thrombocytopenic MF patients were small with hypolobulated nuclei while the MKs generated from HDs and non-thrombocytopenic MFs were larger and contained greater numbers of lobes ( Figure 1A). Psaila and coworkers have previously reported that MF is characterized by MK biased hematopoietic stem cells (HSCs) identified by increased expression of G6B (Psaila. Mol Cell 2020). We, however, determined that a reduced proportion of CD34 +CD38 - HSCs from thrombocytopenic patients expressed G6B as compared to non-thrombocytopenic MF or HD CD34 +CD38 - cells (p<0.05) ( Figure 1B). These studies indicate a reduced proportion of MK biased HSCs in thrombocytopenic MFs which likely leads to impaired MKs maturation. We next explored if the MF associated pro-inflammatory milieu also contributes to the MF MK maturation block. Thrombocytopenic MF plasma contained higher levels of YKL-40 (chitinase-3-like-1 protein), TNFα and TPO than HD plasma. For instance, MF plasma was characterized by higher levels of YKL-40 (median141,321 pg/mL) in comparison to HDs (median 19,380 pg/mL). Furthermore, YKL-40 levels were significantly higher in thrombocytopenic (median 187,696 pg/mL; p<0.05) than non-thrombocytopenic (median 78,082 pg/mL) MFs. In addition, TNFα and TPO plasma levels were also significantly higher in plasma from thrombocytopenic than non-thrombocytopenic MFs (p<0.05). We also observed that addition of either rYKL40 or rTNFα to HD-MK cultures resulted in delayed MK maturation as assessed by a reduced proportion of cells expressing CD41 +CD42 + (from 30% to 16.5% and 6.5% respectively) and G6B (from 35.5% to 22.5% and 9.5% respectively). We have previously reported that the treatment of MFs with a protein trap for TGFβ1 led to increases in platelet numbers (Mascarenhas. Clin Cancer Res 2023). The plasma from these patients prior to treatment not only had elevated levels of TGFβ1 (median 6433,917 pg/mL) but also YKL40 (median 275,640 pg/mL). Treatment of the patients with the TGFβ protein trap reduced both TGFβ1 (median 608,087 pg/mL) and YKL-40 (median 160,733 pg/mL) plasma levels, suggesting a regulatory loop between YKL-40 and TGFβ. We further explored this loop by treating the JAK2V617F+ HEL cell line with rYKL-40 in combination with rTGFβ1, which resulted in greater expression of YKL-40 (1.6 FI) than treated cells with rYKL-40 alone. In addition, treatment of HEL cells with a pan TGFβ inhibitor reduced YKL-40 protein expression (1.55 to 0.38). These data suggest the existence of an interplay between YKL40 and TGFβ on MF-MK maturation. Overall, these experiments indicate that thrombocytopenia in MF patients is due to intrinsic abnormalities in G6B originating at the level of the HSC compartment resulting in impaired MK maturation which can be further enhanced by the actions of a number of pro-inflammatory cytokines.

Retrospective Analysis of the Relationship between Transfusion Independence and Bone Marrow Fibrosis Reduction in Patients with Myelofibrosis Treated with Pacritinib Versus Ruxolitinib

Background Pacritinib is a JAK1-sparing JAK2/IRAK1/ACVR1 inhibitor for treatment of myelofibrosis (MF). In addition to improving spleen volume and symptoms, pacritinib is associated with anemia benefit in MF patients. Recent in vivo studies have shown that dual JAK2/IRAK1 inhibition is associated with improvement in both cytopenias and bone marrow reticulin fibrosis (BMF) in an inflammation-driven murine MF model ( Cuenca Zamora E, et al. EHA 2023; P987 and P990). Here, we retrospectively analyzed the relationship between achieving transfusion independence and reduction in BMF in MF patients treated with pacritinib 200 mg twice daily (BID) vs ruxolitinib (RUX) on the phase 3 PERSIST-2 study. Methods: PERSIST-2 enrolled patients with platelet counts ≤100 x 10 9/L. This analysis focused on pacritinib 200 mg BID and on patients who received RUX as best available therapy (BAT) who enrolled ≥12 weeks prior to study termination and who required red blood cell (RBC) transfusions at baseline. The proportion of patients who achieved transfusion independence response (TI-R, any 12-week interval with no RBC transfusions) was ascertained for pacritinib vs RUX. The proportion of patients with BMF reduction (≥1 grade decrease in reticulin fibrosis from baseline at week 24) was reported among patients on pacritinib achieving TI-R vs. non-response (NR). Results: The analysis included 41 patients on pacritinib (median dose intensity 100% through week 24) and 18 on RUX (median daily dose 10mg at week 24). Baseline characteristics were similar between the groups, including median platelet count (41 vs 38 x 10 9/L) and median hemoglobin (8.7 vs 8.6 g/dL). All patients required RBC transfusion at baseline. A significantly greater proportion of patients treated with pacritinib vs RUX achieved TI through week 24: 37% (n=15/41) vs 6% (n=1/18), P=0.023. Nominally, this trend held for those with baseline platelets <50 x 10 9/L: 28% vs 8%, P=0.222. In addition, a greater percentage achieved a 50% reduction in RBC transfusions over any 12 weeks (49% vs 6%, P=0.001). Paired bone marrow assessments at baseline and week 24 were available for 18/41 of patients on pacritinib, of whom 44% (8/18) achieved TI-R on study. The proportion of patients who experienced BMF reduction (>=1 grade at any point) was significantly greater among TI-R (62.5%, n=5/8) compared to TI-NR (10%, n=1/10) on pacritinib ( P=0.043). Of the 5 patients who achieved TI-R and BMF reduction, all had grade 2-3 fibrosis at baseline, and 2 experienced a reduction from grade 3 to grade 1 ( Figure 1). By contrast, paired bone marrow biopsies were available for 5 patients on RUX, and there was no association between fibrosis reduction in TI-R (0%, n=0/1) and TI-NR (25%, n=1/4). Conclusions: In cytopenic MF patients from PERSIST-2, TI-R on pacritinib was associated with BMF improvement. Though these results are based on a small sample size, they contrast with recent data suggesting no correlation between BMF reduction and TI-R on the JAK1/2 inhibitors momelotinib and ruxolitinib ( Oh ST, et al. Blood 2022;140 (Supp 1):821-23). While differences in study design could have impacted these results, these findings suggest that distinct inhibitory profiles may further distinguish the clinical impact of these treatments. Further studies are warranted to confirm the relationship between BMF reduction and anemia benefit in patients treated with pacritinib. Study funded by CTI BioPharma Corp., a Sobi company

Meter: A Multi-Country, Real-World Chart Review Study to Explore Treatment Patterns, Effectiveness and Healthcare Resource Utilization for Patients with Myelofibrosis

Background : Myelofibrosis (MF) is a rare myeloproliferative disorder associated with significant morbidity and mortality. The hallmarks of MF pathobiology include aberrant myeloproliferation, cytokine-induced inflammation, bone marrow reticulin fibrosis, extramedullary hematopoiesis, cytopenias, and constitutional symptoms. Hydroxyurea was the most commonly used drug in patients with MF prior to the approval of ruxolitinib (RUX), a first-in-class Janus kinase 1/2 inhibitor (JAKi) that is widely approved for symptomatic patients with MF. In addition to RUX, the JAKis fedratinib and, more recently, pacritinib have also been approved by the US Food and Drug Administration. Real-world treatment patterns and impact of currently available JAKis on patients with MF are not well understood. Several novel agents are in advanced development and will further reshape the treatment landscape of MF. A better understanding of current treatment patterns will assist in positioning novel treatments within the MF therapeutic landscape. Methods : The METER study (NCT05444972) is an ongoing multicountry noninterventional retrospective chart review assessing treatment patterns, effectiveness, and healthcare resource utilization (HCRU) in patients diagnosed with MF. Data from patients aged ≥18 years with primary or secondary MF treated on or after the local first date of RUX approval until December 31 , 2021 were assessed. Patients who received treatment for MF in a clinical trial were excluded. The primary objective is to describe real-world MF treatment patterns, including patient characteristics, time from MF diagnosis to first-line (1L) therapy, choice, duration and reason for change/discontinuation of initial and subsequent treatments for MF, and treatment procedures. Secondary objectives include assessments of days hospitalized as part of HCRU. Results : As of June 1, 2023, 928 patient charts were included, met eligibility criteria, and had initial treatment information available. Most patients were male (54%) and White (65%), and the median (range) age for patients ≤89 years of age was 66 (58-73) years; the most common geographic locations were Europe (53%), Latin America (23%), and North America (13%). Of patients, 66% (614/928) had primary MF, and 10% (97/928) were transfusion-dependent. Of patients tested for high molecular risk mutations, 50% (81/162) were positive. Of patients with available bone marrow fibrosis data, 82% (554/680) had grade ≥2 bone marrow fibrosis at diagnosis. The average interval from MF diagnosis to start of initial treatment (index date) was approximately 8.5 months. RUX was the most commonly used 1L therapy (47%; 437/928) followed by hydroxyurea (41%; 380/928). RUX was also the most common therapy used in second-line (2L), third-line, fourth-line, and fifth-line treatment for 62% (217/353), 63% (107/171), 66% (56/85), and 59% (24/41) of patients, respectively. Mean (SD) time from index date to procedural intervention was 556 (519) days; the most common procedure was stem cell transplant (n = 71) followed by splenectomy (n = 20). Of the 927 patients who received 1L therapy, 98% remained on 1L therapy through Week 24, and 66% did not initiate 2L therapy until Week 156. Median (95% CI) duration of 1L therapy was 48 (45-52) months (Figure). Median (95% CI) survival from the start of 1L therapy was 79 (71-NR) months. The estimated survival rate (95% CI) was 76% (73-79) at Week 156. Median (95% CI) overall survival was 120 (93-147) months. Median (Q1-Q3) total number of days hospitalized after the index date was 2 (0-19), and median (Q1, Q3) total number of days in the intensive care unit after the index date was 0 (0, 0). Analyses will be updated for presentation. Conclusions : In patients with MF, RUX was the most commonly used agent in all lines of therapy. The greatest reduction in duration of MF treatment occurred from 1L to 2L. Most patients remained on 1L therapy through Week 24 and did not initiate 2L therapy until Week 156. Median survival time from start of 1L therapy to death was 79 months (approximately 6 years). There was a high degree of advanced bone marrow fibrosis as well as transfusion dependence among this real-world patient population. Together, these results highlight the importance of optimizing 1L therapy.

Estimating Transfusion-Related Medical Costs and Associated Time Burden in Patients with Myelofibrosis: A Comparison of Momelotinib Vs Ruxolitinib Based on Simplify-1

Introduction: Myelofibrosis (MF) is a myeloproliferative neoplasm characterized by splenomegaly, debilitating symptoms, and bone marrow fibrosis. Transfusion-dependent (TD) anemia is present in one-quarter of patients (pts) with MF at diagnosis and becomes more common over time. Transfusion dependence imposes a high cost burden on healthcare systems, with annual total medical costs up to 9 times higher for TD pts than for non-TD pts (Gerds A, et al. ASH 2022. Abstract 1729). Momelotinib (MMB) is a Janus kinase (JAK) 1/JAK2/activin A receptor type 1 inhibitor that has demonstrated symptom, spleen, and anemia benefits in pts with MF across 3 phase 3 trials. Evidence in JAK inhibitor-naive pts comes from SIMPLIFY-1 (NCT01969838), a randomized, double-blind, phase 3 trial of MMB vs ruxolitinib (RUX). Eligible pts had International Prognostic Scoring System high-risk or intermediate-2-risk MF, or intermediate-1-risk MF associated with symptomatic splenomegaly, hepatomegaly, or anemia (hemoglobin <10 g/dL) and/or unresponsiveness to available non-JAK inhibitor therapy. MMB was associated with higher red blood cell (RBC) transfusion independence rates (66.5% vs 49.3%), lower transfusion dependence rates (30.2% vs 40.1%), and fewer RBC units transfused (median of zero units/month vs 0.4 units/month) compared with RUX by week 24. This study estimated the projected differences between MMB and RUX in total medical costs as well as cost and time burden associated with outpatient transfusion visits, based on results from SIMPLIFY-1, in all pts with MF and separately in those aged ≥65 years. Methods: Analyses were based on transfusion status (TD, transfusion independent [TI], or transfusion requiring [TR]) and mean rates of outpatient transfusion visits over 24 weeks in SIMPLIFY-1. TD was defined as ≥4 RBC units transfused or hemoglobin <8 g/dL in the prior 8 weeks, TI was defined as no RBC transfusions and all hemoglobin ≥8 g/dL in the prior 12 weeks, and TR was defined as being neither TD nor TI. Projected cost offsets for MMB relative to RUX were calculated based on observed transfusion status and rates over 24 weeks from SIMPLIFY-1 combined with cost estimates for TD vs TI/TR from the IBM MarketScan Database used to inform commercial health plans. Projected time savings were calculated similarly using transfusion rates from SIMPLIFY-1 and previously reported time burden estimates for transfusion visits in pts with TD β-thalassemia (Knoth RL, et al. EHA2022. Poster P1741). For pts aged ≥65 years, individual pt data from SIMPLIFY-1 and cost estimates from the Medicare fee-for-service database were used instead. Results: Based on the IBM MarketScan Commercial Database, projected average annual total medical cost savings based on week 24 transfusion status with MMB vs RUX were $20,215 per pt with baseline TD status and $23,991 per pt with baseline TI/TR status. Fewer outpatient transfusion visits with MMB vs RUX resulted in projected annual savings of $25,704 per pt with baseline TD status and $12,083 per pt with baseline TI/TR status. Among pts with baseline TD status, projected annual time savings in transfusion visits for MMB vs RUX (147 vs 253 hours) totaled 106 hours per pt (42 hours, preparation and waiting; 50 hours, transfusion and recovery; 15 hours, travel), a 42% reduction for MMB vs RUX. In the subgroup analysis that focused on pts aged ≥65 years, based on the Medicare fee-for-service database, projected average annual total medical cost savings with MMB vs RUX were $11,102 per pt with baseline TD status and $17,373 per pt with baseline TI/TR status. Fewer outpatient transfusion visits with MMB vs RUX resulted in projected annual savings of $28,826 per pt with baseline TD status and $18,255 per pt with baseline TI/TR status. Among pts aged ≥65 years with baseline TD status, projected annual time savings in transfusion visits accounted for MMB vs RUX (160 vs 279 hours) totaled 119 hours per pt (47 hours, preparation and waiting; 56 hours, transfusion and recovery; 16 hours, travel), a 43% reduction for MMB vs RUX. Conclusions: Reductions in transfusion dependence and transfusion visits for MMB vs RUX were associated with projected healthcare system savings in transfusion costs and transfusion-related time burden in JAK inhibitor-naive pts with MF, including in subgroup analyses of pts aged ≥65 years.

Platelet Response in Pacritinib-Treated Patients with Cytopenic Myelofibrosis: A Retrospective Analysis of PERSIST-2 and PAC203 Studies

Background: Myelofibrosis (MF) is a myeloid malignancy characterized by clonal hematopoisis, bone marrow fibrosis (BMF) and ineffective extramedullary hematopoiesis, resulting in progressive cytopenias. Thrombocytopenia is both prognostic of poor outcomes and predictive of treatment intolerance with the JAK1/2 inhibitor ruxolitinib, which exacerbates cytopenias. Pacritinib is a JAK1-sparing inhibitor of JAK2/IRAK1/ACVR1 that can be administered at full dose to patients regardless of baseline platelet count (PLT). While prior studies have shown that pacritinib is associated with PLT stability in most patients, PLT improvement has not been described outside of a recently published case report ( Yacoub A, et al. JCO Precis Oncol. 2023;7:e2200523). Here, we report rates of hematologic improvement in PLTs (HI-P) with pacritinib across two clinical trials. Methods: Patients with baseline PLT ≤100x10 9/L on the pacritinib 200 mg BID arms of the phase 3 PERSIST-2 study (randomized ≥12 weeks prior to study termination) or the phase 2 dose-finding PAC203 study were included. HI-P was defined per International Working Group (IWG) criteria (baseline PLT <20x10 9/L: increase to >20x10 9/L and by at least 100%; baseline PLT 20-100 x 10 9/L: absolute increase of ≥30x10 9/L) in the absence of PLT transfusions, sustained over any 8 weeks while on treatment. Efficacy outcomes (reduction in spleen volume, symptom score, and BMF) were compared between HI-P responders vs non-responders (those who did not meet IWG criteria). Rates of PLT improvement were also analyzed on the best available therapy (BAT) arm of the PERSIST-2 study over the treatment period (end of study treatment). Results: Of 117 patients randomized to pacritinib (75 from PERSIST-2, 42 from PAC203), 16% (n=19) experienced HI-P on study (as defined in methods). Additionally, 14 of the 19 HI-P patients had sustained platelet improvement over ≥12 weeks). By contrast, only 5% (4/77) of patients on BAT achieved HI-P response prior to end of study treatment. HI-P responders on pacritinib compared to non-responders, had numerically higher median baseline PLT count (63 vs 45x10 9/L) and hemoglobin (Hb; 10.3 vs 8.8 g/dL). A similar percentage had prior (<30 days) JAK2 inhibitor exposure (68.4% vs 66.3%) and JAK2V617F mutation (78.9 vs 74.5%); median JAK2 allele burden was low in both responders and non-responders (26.7% vs 36.9%). Among HI-P responders with prior ruxolitinib exposure, most (85%, 11/13) had recent exposure within the prior 30 days, with median dose of 10 mg BID. PLT improvement was noted within the first 12 weeks in most HI-P responders, whereas PLT count remained stable, on average, among non-responders ( Figure 1a). To evaluate if recent ruxolitinib exposure and washout of drug was responsible for HI-P, we analyzed the subset of patients with ruxolitinib exposure in previous 30 days (11/19). The mean platelet count increased from 56 to 141x10 9/L at week 12. In the remaining 8/19 patients without recent ruxolitinib exposure (n=6 were treatment naïve), platelets increased from 59 to 102 x 10 9/L at week 12. While there was no difference in the magnitude of spleen volume or symptom score reduction in HI-P responders vs non-responders, a correlation with change in BMF was noted. Among the 36 patients with available bone marrow data from baseline and week 24, HI-P responders were numerically more likely to have BMF reduction (67%, n=4/6) compared to non-responders (23%, n=7/30, P=0.057, Figure 1b). There was no difference in the rate of hemorrhagic events (by standardized MedDRA queries) in PLT responders vs non-responders (47% vs 46%), though grade ≥3 bleeding was observed at lower frequency in responders compared to non-responders (10.5% vs 16%). Other commonly reported adverse events with pacritinib occurred at similar frequency between responders and non-responders. Discussion: PLT improvement meeting IWG criteria occurs in a subset of MF patients treated with pacritinib and does not seem to be explained by changes in spleen volume. Further studies are warranted to assess the correlation between hematologic improvement and BMF reduction with pacritinib. It is possible that pacritinib's unique mechanism of action as an IRAK1 inhibitor could result in modulation of the bone marrow microenvironment and thrombopoiesis. Study funded by CTI BioPharma Corp., a Sobi company.

Selinexor Plus Ruxolitinib in JAK Inhibitor (JAKi)-Naïve Patients with Myelofibrosis: Long Term Follow up from XPORT-MF-034 Suggestive of Disease Modification

Background: Myelofibrosis (MF) is a myeloproliferative neoplasm characterized by splenomegaly, constitutional symptoms, and inflammation due to unregulated, clonal proliferation of hematopoietic stem cells in the bone marrow. Common genetic drivers include JAK2, CALR, and MPL. Although improvements in splenomegaly and symptoms occur with the current approved therapies, additional assessments are needed to understand the impact of therapies on disease modification. Increased burden of variant allele frequency (VAF) of driver mutations is associated with poor disease outcomes, and increased expression of pro-inflammatory cytokines is implicated in disease pathophysiology and symptoms burden. Selinexor (SEL) is an investigational oral XPO1 inhibitor that may inhibit multiple MF-relevant pathways including STAT, ERK, and AKT. Preclinical studies have shown potential synergy between SEL and ruxolitinib (RUX) treatment in vivo. Safety and efficacy analysis of SEL plus RUX in JAKi treatment-naïve MF in a Phase 1 study supported a 60 mg SEL dose as the recommended dose in a Phase 3 study. Here we include exploratory analyses of a range of biomarkers in the Phase 1 study of SEL and RUX in JAKi-naïve myelofibrosis. Methods: Phase 1 of XPORT-MF-034 (NCT04562389) is an open-label study evaluating the safety and efficacy of SEL at 40mg and 60mg once weekly plus RUX with JAKi-naïve MF. Assessments included safety and rates of spleen volume reduction of ≥35% (SVR35) and total symptom score reduction of ≥50% (TSS50). Platelet and hemoglobin levels were also assessed. Longitudinal clinical biomarker assessments included %VAF change at week 24 for driver genes and plasma cytokine levels at week 4 (Cycle 2 Day 1). Bone marrow (BM) biopsies were obtained at screening and week 24, then evaluated for bone marrow fibrosis (BMF) and other BM cellular markers (CD61, CD71). Results: As of April 10, 2023, a total of 24 patients (pts) received at least one dose of SEL (40 mg: n=10; 60 mg n=14). The most common adverse events (AEs) overall of any grade were nausea (75% majority grade 1-2), fatigue (58%), anemia (54%), and thrombocytopenia (54%). Those receiving a prophylactic anti-emetic reported lower incidence and severity of nausea AEs compared to those that did not; pts experienced a median weight increase of 3.3 kg at week 24. Two pts discontinued treatment due to treatment-related AEs (thrombocytopenia and neuropathy). Generally stable hemoglobin levels were observed in 48% of transfusion-independent pts. Median hemoglobin levels were 10.1 g/dL at baseline, 9.3 g/dL at week 12, and 10.2 g/dL at week 24. Median platelet counts remained generally stable with median values of 258 k/uL at baseline, 164 k/uL at week 12, and 193 k/uL at week 24. SVR35 at week 24 in the intent-to-treat (ITT) population was achieved by 40% of the 40 mg cohort and 79% of the 60 mg cohort and TSS50 was achieved by 10% and 58%, respectively. Mutation analysis showed no pts were triple negative, with 18 harboring JAK2 mutations, 5 with CALR, and 1 with MPL. Of 13 pts with available data at week 24, 5 pts (38%) had a reduction of VAF that was ≥20%, and 3/5 of these pts had high VAF (>50%) driver mutations at baseline and were also characterized as HMR. A panel of 74 cytokines was evaluated in 21 MF pts with baseline and week 2 samples (including 10 with end-of-treatment [EOT] samples), and in 10 healthy donors. Comparing baseline MF to healthy samples, 50% of the analyzed cytokines were elevated. A rapid and durable decrease of pro-inflammatory MF-relevant cytokines including TGF-β1, IFNɣ, TNF-α, IL-7, IL-1Ra, and IL-16 was observed in the majority of pts at week 2 and continued into EOT. Notably, the reduction in IL-18 correlated to SVR and TSS response at week 24, with R 2 values of 0.29 and 0.28, respectively. BM analysis at week 24 showed an increased number of erythroid CD71+ elements in 3/14 pts. Conclusions: SEL plus RUX was generally well tolerated and manageable.Evidence of potential disease modification in pts with JAKi-naïve MF patients was seen via rapid stabilization of platelets and maintaining hemoglobin levels, VAF reduction, and pro-inflammatory cytokine reduction. Promising biomarker and efficacy data suggests that SEL in combination with RUX has the potential to become a novel, first-line treatment for pts with MF. Updated clinical data with longer follow-ups and additional subgroup analyses will be available at the time of presentation.

Impact of Graft Source [Cord Blood Supported By CD34+ Selected Cells (Haplo-Cord) Vs. Matched Donor] on Transplant Outcomes in Myelofibrosis

Introduction: Allogeneic hematopoietic cell transplantation (allo-HCT) is curative for patients with myelofibrosis (MF). Here, we evaluated the impact of donor type [cord blood transplant supported by CD34+ cells (haplo-cord) vs adult matched donor] on outcomes in MF. Methods: All adult patients with primary or secondary (post-ET or post-PV) MF, including those progressing to acute leukemia (AML) who underwent their first allo-HCT at NewYork-Presbyterian Hospital/Weill Cornell Medicine between 01/2015 and 06/2022 were included. Results: 40 patients were transplanted. Median age was 63 years (range 21-77). 23% had a Karnofsky performance scale (KPS) of <80. Two had undergone splenectomy prior to HCT. The median spleen size was 18cm (10-28). Median number of pre-HCT molecular mutations was 2 (range 0-7). 16 (40%) had MF-3 (Table 1). 24 (60%) underwent a matched sibling donor (MSD) (n=9) or unrelated donor (MUD) (n=15) HCT, and 16 (40%) underwent a cord-based (CB) HCT. 39 received Fludarabine/Melphalan +/- low dose total body irradiation (TBI), one underwent myeloablative conditioning with TBI/etoposide based on extensive extramedullary disease. The median time to neutrophil engraftment in the MSD/MUD group was 14 days (IQR 14-18) vs 30 days in the CB group (IQR 15-36) and (p=0.1). The median time to platelet engraftment was 22 days (IQR 17-38) in MSD/MUD vs 49 days (IQR 26-60) in the CB group (P=0.07). The CI of absolute neutrophil count (ANC) and platelet engraftment by day 60 for MSD/MUD vs CB was 92% (95% CI 65-98%) vs 80% (95% CI 45-94%) (p=0.09) and 75% (95% CI 51%- 88%) vs 59% (95% CI 27-81%) (p=0.36), respectively. One patient in the MSD/MUD died without neutrophil engraftment compared to 3 in CB group. 5 patients in each group died without platelet engraftment. Median follow-up for all alive patients was 53 months. Overall survival (OS) for the cohort was 62% at 1 year (95% CI 49-79) and 34% at 3 years (95% CI 21-55). OS at 3 years was similar between MSD/MUD group and CB (32% vs 37%, P=0.9) (figure 1). Graft versus host disease relapse free survival (GRFS) at 3 years was not significantly different in MSD/MUD group, compared to CB group (15% vs 33%, P=0.64). Cumulative incidence of relapse at 3 years in MSD/MUD group was 28% (95% CI 10-49), and 12% (95% CI 1.8-34) in CB group (p=0.356). Non-relapse mortality (NRM) at 1 year was 33% (95% CI 15-52) in MSD/MUD group and 38% (95% CI 15-61) in CB group. NRM at 3 years was 54% (95% CI 29-73) in MSD/MUD group and 48% (95% CI 19-72) on CB group (P=0.95). Discussion: In our small cohort, we showed that OS after haplo-cord transplant was comparable to adult matched donors' grafts (MSD or MUD). While the haplo-cord platforms have been shown to decrease the time to engraftment (11 days for neutrophils and 22 days for platelets) compared with double cord transplants or haploidentical transplants (van Besien K et al . Haematologica 2016, van Besien K et al. Blood Adv 2019), in patients with MF, we saw a trend towards delayed engraftment compared to patients transplanted with adult matched donors' grafts (MSD or MUD). This suggests that the CD34 selected cells may not be able to provide an adequate myeloid bridge and overcome the low cellular content in the CB for adult patients with extensive marrow fibrosis. Larger studies are needed to compare best alternative graft (CB-based versus haploidentical or mismatched unrelated donors) for patients with MF that lack a matched donor.

TP53 mutations and Their Impact on Survival in Patients with Myeloproliferative Neoplasms

Introduction: TP53 mutations in patients with myeloproliferative neoplasms (MPN) are associated with poor prognosis, including progression to blast phase MPN. However, low variant allele fraction (VAF) TP53 mutations have been reported to remain stable over years in chronic phase MPN. A major unmet clinical need in MPN is the ability to discriminate patients with TP53-mutant MPN who are at high-risk of secondary AML (sAML) and warrant immediate intervention from those who are at lower risk of sAML in whom active surveillance can be employed. Therefore, we sought to identify parameters associated with leukemic transformation and overall survival in the context of MPN with genetic aberrations in TP53. Materials and Methods: We retrospectively analyzed a cohort of 947 MPN patients from the Dana-Farber Cancer Institute Hematologic Malignancies Data Repository (HMDR) with at least one clinical next-generation sequencing (NGS) panel performed. Patient characteristics such as age at MPN diagnosis, gender, MPN subtype and driver mutations were recorded. Furthermore, information about the course of disease was extracted including occurrence of sAML and overall survival (Figure 1). We also analyzed type and number of additional mutations as well as cytogenetics. With respect to TP53-specific parameters, we evaluated the number of TP53 mutations, TP53 VAF, loss of heterozygosity (LOH) at the TP53 locus, phenotypic annotations of TP53 (i.e. PHANTM score) and 17p deletion. We defined “multi-hit” TP53 as the presence of two or more TP53 mutations, TP53 VAF higher than 50%, TP53 mutation plus 17p deletionor TP53 mutation and documented LOH. Results: A total of 947 patients were analyzed, of which 40 harbored at least one detectable TP53 mutation. A total of 13 patients were found to have a multi-hit TP53 mutations defined by > 50% VAF in 6 patients, two or more TP53 mutations in 5 patients and TP53 mutation + 17p deletion in 5 patients. The MPN diagnosis at time of TP53 mutation detection was post ET/PV myelofibrosis (secondary MF) (n=23, 58%), primary myelofibrosis (MF) (n=7, 18%), pre-fibrotic MF (n=2, 5%), essential thrombocythemia (ET) (n=6, 15%) and polycythemia vera (PV) (n=2, 5%). Two patients with ET and one patient with PV did not have a concurrent in-house bone marrow biopsy performed at the time the TP53 mutation was detected. Two patients with ET developed sAML within 12 months of TP53 mutation detection, without prior mention of fibrosis. Age at first MPN diagnosis was not significantly different between patients with or without TP53 mutation. The average time from initial MPN diagnosis to detection of the first TP53 mutation was 9 years (range: 0-33 years). The most common MPN driver mutation among patients with TP53 mutations was JAK2 (75%), followed by CALR (13%)and MPL (5%). Out of all TP53-mutated patients, 8% showed a triple negative status. The most frequent additional mutations among patients with TP53 mutations were TET2 (25%), U2AF1 (15%), ASXL1 (13%), and DNMT3A (10%). There was no significant difference between single-hit and multi-hit TP53 status regarding MPN subtype, driver mutations and co-mutations (Table 1). Seven patients (single-hit: 15%, multi-hit: 23%) with a TP53 mutation developed sAML during the course of their disease, compared with only 3% of all patients without a TP53 mutation and 50% (single-hit: 41%, multi-hit: 69%) were deceased at the time of the last follow-up compared to 18% of all patients without a TP53 mutation. We focused on overall survival from the initial MPN diagnosis and considered whether patients developed bone marrow fibrosis during their disease course (Figure 1). Survival did not differ significantly between single-hit TP53 and patients with multi-hit TP53 (p=0.2), but survival did differ significantly between multi-hit TP53 patients and TP53 wildtype patients with MF/prefibrotic MF/Secondary MF (p=0.02) as well as compared to all MPN patients without a TP53 mutation (p<0.001). Survival was not significantly different between single-hit TP53 and TP53 wildtype MF/prefibrotic MF/Secondary MF patients (p=0.4). Conclusions: In a large cohort of 947 molecularly characterized MPN patients, 4% of the cohort developed a TP53 mutation during their course of disease. 18% of all TP53-mutant patients developed sAML with an adverse effect on overall survival for patients with multi-hit but not single-hit TP53 mutations.

Quantitative Analysis of Bone Marrow Features Highlights Heterogeneity in Myelofibrosis Patients Treated with Zinpentraxin Alfa in a Phase II Clinical Study

Quantitative image analysis has potential to transform the interpretation of bone marrow trephine (BMT) samples in myeloproliferative neoplasms (MPN) and improve the evaluation of anti-fibrotic therapies. To investigate the utility of recently developed algorithms evaluating reticulin fibrosis and megakaryocyte features in myelofibrosis (MF), we analysed samples from a multi-center, phase II study of zinpentraxin alfa (ZPN, PRM-151). ZPN is a recombinant form of human pentraxin-2 (PTX2) that has shown clinical activity as monotherapy and in combination with ruxolitinib (RUX) in a phase II trial in patients with Int-1/-2 or high risk MF (NCT01981850). In stage 2 of this study, patients ineligible for, intolerant of, or with an inadequate response to RUX were randomised to receive 0.3, 3.0, or 10.0 mg/kg ZPN on Days 1, 3, and 5 of Cycle 1, and every 4 weeks thereafter for up to 9 cycles. We demonstrate the potential of quantitative image analysis to augment and refine conventional expert histological assessment of MF. Reticulin and H+E-stained BMTs from three timepoints (screening, C4D1 and C9D29) were analysed from 50/97 patients enrolled. Manual assessment (MA) of marrow fibrosis and megakaryocyte features was performed by blinded, independent central review. Assessment of fibrosis using the Continuous Indexing of Fibrosis (CIF) score and analysis of megakaryocytes was performed by automated analyses ( Ryou H, Leukaemia, 2022). There was a moderate correlation between the automated average CIF score and manual fibrosis grade (Spearman's rho = 0.39). However, there was significant overlap in the distribution of CIF scores between samples manually assigned to MF-2 and MF-3. Approximately 38% of MF-2 samples fell within the interquartile range of CIF distribution observed in MF-3, and around 48% of MF-3 samples fell within the interquartile range observed in MF-2. Next, we visualised the screening BMTs in fibrosis PCA (principal component analysis) space to capture both average CIF score and Shannon entropy (‘unevenness‘) of CIF score for each sample. This revealed significant heterogeneity in reticulin fibrosis at screening when compared to an independent reference cohort of 130 MPN samples ( Ryou H, Leukaemia, 2022), likely reflecting the effects of pre-treatment [ Figure 1]. To track fibrosis across all timepoints we plotted the average CIF score for each sample. This revealed striking variability in fibrotic response within the trial cohort, with 15 of 42 patients (36%) demonstrating improvement in average CIF score [ Figure 2]. Notably, improvements in average CIF score by cycle 9 appeared to be most marked in patients with higher CIF scores at screening. No significant ZPN dose-dependent effect on fibrosis was observed. We next measured megakaryocyte density (megakaryocytes per unit area of intertrabecular space) and compared it to MA. We found a strong correlation (Spearman's rho = 0.81) between automated and MA of megakaryocyte density, with no significant change across any of the treatment arms. For megakaryocyte clustering (defined as ≥ 3 megakaryocytes in direct contact) we observed a moderate to strong correlation (Spearman's rho = 0.62) between MA and automated assessment, with no significant change across any of the treatment arms. Finally, we sought to compare the sample megakaryocyte cell features (cytomorphology and topology) at screening with those of an independent reference cohort of 88 MPN samples ( Sirinukunwattana, Blood Adv., 2020). Strikingly, not only was there marked heterogeneity in megakaryocyte features between patient samples at screening, but the observed features were significantly different to those of newly diagnosed MF. No significant changes in megakaryocyte cytomorphology or topology were seen across any of the treatment arms. In summary, our findings reveal considerable BMT morphological heterogeneity in patients participating in a Phase II trial of ZPN. This likely reflects poorly understood and under-recognised variation in morphological features encountered in patients with longstanding and/or pre-treated MF when compared to newly diagnosed patients. This highlights the potential of such variability to confound the evaluation of novel therapeutics in MPN, and emphasises the utility of robust quantitative methods to analyse and visualise morphological features in clinical study samples that can complement conventional manual assessment.

The Efficacy and Safety of Selinexor in Combination with Ruxolitinib in Ruxolitinib-Treated Myelofibrosis Patients： the Interim Analysis of a Prospective, Open-Label, Multicenter, Parallel-Cohort, Phase 2 Study

Background: Myelofibrosis is a chronic hematologic malignancy characterized by constitutional symptoms, bone marrow fibrosis, extramedullary hematopoiesis resulting in splenomegaly and a propensity toward leukemic progression. Although JAK inhibitors have improved patients' splenomegaly and symptom burden, the clinical management of MF is still challenging for its limited treatment options, especially when patients become less responsive or intolerable to Ruxolitinib (RUX) treatment. Selinexor (SEL) is a novel, first-in-class, oral selective inhibitor of nuclear export agent that binds the karyopherin protein exportin 1 (XPO1). Preclinical studies showed SEL decreased viable cells and colony formation both in newly diagnosed and RUX-exposed MF cells. ESSENTIAL trial showed SEL's sustained spleen responses as a single agent in JAKi refractory MF. Besides, a phase I study reported, with SEL plus RUX in treatment-Naïve MF, 92% patients(pts) achieved SVR35, 69% had symptom response and 65% had stable or improved Hgb levels. We have presented promising efficacy and safety data in real-world setting of SEL plus RUX regimen in MF patients in 2023 EHA. Here we are to report the efficacy and safety of the regimen in RUX-treated patients in a prospective clinical trial. Methods: This is a prospective, open-label, multi-center, parallel-cohort phase 2 study, examining SEL plus RUX regimen in RUX-treated MF pts. The study includes MF pts with suboptimal RUX responses or intolerable to RUX treatment. Pts are given SEL 40mg or 60mg QW plus RUX (dosage per investigator judgement, RUX intolerable pts will receive reduced dose of RUX). A total of 40 pts will be included into the study. The primary endpoint is spleen response assessed by palpation or CT/MRI scan per IWG-MRT and ELN response criteria. Key secondary endpoints include anemia response, symptom response defined as symptom resolution or alleviation according to TSS or physician's judgement, and safety. Results: Characteristics As of July 2023, 28 pts had received at least one dose of oral weekly SEL, including 12 suboptimal RUX responses pts and 16 RUX intolerable pts. The median age was 65 years old. Mean duration of prior RUX treatment was 24.5 months (range: 4-77). 18 pts had primary MF, 4 had post-PV MF, and 6 had post-ET MF. Median time from MF diagnosis to study enrollment was 5 years (0.25-29). JAK2, CALR and MPL mutations were present in 18 (64.28%), 5 (17.85%) and 1 (3.57%) pts respectively. All pts presented constitutional symptoms, 25 (89.29%) had splenomegaly, and 7 (25.00%) had packed red blood cell (PRBC) transfusion-dependent anemia. DIPSS risk category was intermediate 23 (82.14%), high risk 3 (10.71%) and unknown 2 (7.14%). Median treatment duration was 134 days (14-525) (Table 1). 15 pts (53.57%) discontinued SEL. Reasons for treatment discontinuation were death in 4 pts (3 from covid-19, 1 sepsis), participation in clinical trial in 2 pts, unsatisfactory response in 2, transplantation in 1, toxicity in 2, economic burden in 1 patient and lost to follow-up in 3. Efficacy Analysis The analysis of spleen response included 21 pts with spleen length assessment data by either palpation or CT/MRI. Spleen response is defined as length reduction more than 50% per IWG-MRT and ELN response criteria. 15 (71.42%) pts had a reduction in spleen volume and 8 (38.09%) achieved spleen response ( Figure 1). For symptom response, 27 pts are with available data. 23 (85.18%) pts had symptom alleviation, 9 (33.33%) achieved ≥50% reduction in TSS or symptom response assessed by physician, of which 1 achieved symptom resolution. There are 12 pts with PRBC transfusion-independent anemia (Hgb≤100g/L) at baseline and had received study treatment for at least 28 days. 10 (83.33%) pts had maintained stable or improved Hgb levels at the last follow up. 5/7 (71.42%) PRBC transfusion-dependent pts became transfusion independent (1 pt) or had reduced transfusion frequency (4 pts), and 2 pts remained stable. Safety The most common TEAEs were nausea 14 (50.00%), vomiting 9 (32.14%), decreased appetite 8 (28.57%), and anemia 7 (25.00%). The most frequent TEAEs ≥ Grade 3 were anemia 3(10.71%) and thrombocytopenia 2(7.14%). Conclusion: In this preliminary analysis, the combination of SEL and RUX showed encouraging efficacy and manageable safety in RUX-treated pts with myelofibrosis.

Pelabresib in Combination with Ruxolitinib for Janus Kinase Inhibitor Treatment-Naïve Patients with Myelofibrosis: Results of the MANIFEST-2 Randomized, Double-Blind, Phase 3 Study

Background Myelofibrosis (MF) is characterized by bone marrow fibrosis, anemia, splenomegaly and MF-associated symptoms. These hallmarks result from dysregulation of the JAK/STAT pathway and BET-mediated MF target gene modulation. Pelabresib (CPI-0610; pela) is an investigational oral small-molecule drug designed to inhibit BET-mediated gene transcription involved in MF pathogenesis. Preclinical data support the potential of combining pela with therapiesthat target overlapping pathways, such as JAK/STAT, to improve response by inhibiting the molecular drivers of MF. The Phase 3 MANIFEST-2 trial was initiated based on compelling data from Arm 3 of the ongoing Phase 2 MANIFEST study (NCT02158858), which is evaluating the combination of pela and ruxolitinib (rux) in JAK inhibitor (JAKi) treatment-naïve patients (pts) with MF. In Arm 3 of MANIFEST, primary endpoint analyses at 24 Weeks (Wks) for the 84 pts enrolled reported SVR35 (≥35% reduction in spleen volume from baseline) in 68% of pts and TSS50 (≥50% reduction in Total Symptom Score [TSS] from baseline) in 56% of pts (Mascarenhas, et al. 2023). Aims MANIFEST-2 (NCT04603495) is a global, Phase 3, randomized, double-blind, active-control study of pela + rux versus placebo + rux in JAKi treatment-naïve pts with primary MF, post-polycythemia vera MF or post-essential thrombocythemia MF. The aim of this study is to evaluate the efficacy and safety of pela + rux. Here we present the study design and baseline patient stratification for MANIFEST-2. Results from the primary analysis of MANIFEST-2 will be presented at the ASH Annual Meeting 2023. Methods The MANIFEST-2 study schema is shown in Figure 1. Key eligibility criteria include a DIPSS score of Intermediate-1 (Int-1) or higher, platelet count ≥100 × 10 9/L, spleen volume ≥450 cm 3 by CT or MRI, ≥2 symptoms with an average score ≥3 or a TSS of ≥10 using the MFSAF v4.0, peripheral blast count <5% and an ECOG performance status ≤2. Patient randomization was stratified by DIPSS risk category (Int-1 vs Int-2 vs High), platelet count (>200 × 10 9/L vs 100-200 × 10 9/L) and spleen volume (≥1800 cm 3 vs <1800 cm 3). Double-blind treatment of pela (125-175 mg) or placebo was administered once daily for 14 consecutive days, followed by a 7-day break, which is considered one cycle of treatment. Rux (5-25 mg) was administered twice daily based on platelet counts and spleen response for all 21 days of the cycle. The primary endpoint is SVR35 response at Wk 24. Secondary endpoints include TSS50, percentage change in TSS, safety, pharmacokinetics, changes in bone marrow fibrosis, progression-free survival, overall survival, conversion from transfusion dependence to independence and rate of red blood cell transfusion for the first 24 wks. Results A total of 591 pts were screened at 138 sites. Following the screening process, 431 pts from North America, Europe, Asia and Australia were randomized. The number of pts assigned to each randomization stratum are presented in Table 1; the majority of pts presented with DIPSS Int-1 or -2, had a platelet count above 200 × 10 9/L and spleen volume below 1800 cm 3. The study opened for enrolment in November 2020; the first patient received their initial treatment on April 22, 2021, and the last patient received their first treatment on March 2, 2023. The last patient is expected to reach the 24-wk primary endpoint on August 31, 2023, following which a primary analysis will occur. As of June 27, 2023 there were 383 pts ongoing in the study. Conclusion The Phase 3 MANIFEST-2 study was initiated based on compelling data from the ongoing Phase 2 MANIFEST study, in which the combination of pela and rux in Arm 3 demonstrated significant clinical activity and disease-modifying potential without clinically relevant treatment-limiting toxicity. MANIFEST-2 has completed recruitment and will provide definitive efficacy results of combination therapy in JAKi treatment-naïve pts with MF. As DIPSS Int-1 pts have been excluded or underrepresented in prior randomized trials in MF, MANIFEST-2 will also provide important insights in assessing the benefits of starting treatment at an earlier stage of the disease. Primary results from the pivotal Phase 3 MANIFEST-2 trial of pela and rux versus placebo and rux in JAKi treatment-naïve pts may have the potential to influence the MF treatment paradigm and will be presented at the ASH Annual Meeting 2023.

Targeting BRD4 to Counteract Cytokine-Induced Expression of PD-L1 and PD-L2 on Vascular, Osteoblastic and Stromal Niche Cells

The stem cell niche plays a crucial role in promoting the expansion of leukemic stem cells (LSC) and in mediating resistance of LSC against various anti-neoplastic drugs in different types of leukemias. Recent data suggest that targeting niche cells may be an effective approach to overcome niche-mediated drug resistance of LSC. However, niche cells themselves are considered to be often resistant against therapeutic intervention. This may especially be relevant in those myeloid neoplasms where niche cell expansion is a pathognomonic feature and the resulting neo-angiogenesis, bone marrow fibrosis and/or sclerosis are important prognostic variables. We and others have recently shown that several of the anti-neoplastic drugs used to treat myeloid leukemias can also counteract niche cell expansion. So far, however, little is known about mechanisms underlying resistance of niche cells against anti-neoplastic drugs. Recent studies have shown that certain immune checkpoint antigens are overexpressed in cancer cells and/or their microenvironment and that these checkpoint antigens contribute to drug resistance. Other studies have shown that checkpoint molecules, including PD-L1 and CD47, are displayed by LSC in acute myeloid leukemia, chronic myeloid leukemia and other myeloid neoplasms. We asked whether niche-related cells also display these checkpoint molecules. Furthermore, we screened for cytokines that promote expression of checkpoint antigens on niche cells and asked for drugs that counteract growth and cytokine-induced checkpoint expression in these cells. The following niche cells were examined: primary human umbilical vein endothelial cells (HUVEC), the human endothelial cell line HMEC-1, the human osteoblast-like cell line CAL-72, primary osteoblasts, and the human stromal cell line HS5. Expression of the checkpoint antigens PD-L1, PD-L2, PD1, CD28, CD47, CD80, CD83, CD86 and TIM-3 was determined by flow cytometry. All niche cells were found to constitutively express PD-L1, PD-L2, and CD47. The other checkpoint antigens were expressed at low or undetectable levels in these cells. Expression of PD-L1, PD-L2 and CD47 in niche cells was confirmed by quantitative PCR and by immunocytochemistry. Of all cytokines tested (n=12), interferon-alpha (IFN-A; effective range 50-500 ng/ml) and interferon-gamma (IFN-G; 10-500 ng/ml) were found to promote surface expression of PD-L1 and PD-L2 in HMEC-1, CAL72, and HS5 cells in our immunocytochemical and flow cytometry experiments. Moreover, we found that tumor necrosis factor-alpha (TNF-A; 100 ng/ml) enhances IFN-G-induced expression of PD-L1 and PD-L2 in HMEC-1, CAL-72 and HS5 cells. The BRD4-targeting drug JQ1 and the BRD4 degraders dBET1 and dBET6 suppressed cytokine-induced upregulation of surface expression of PD-L1 and PD-L2 in HMEC-1, HUVEC, CAL-72 and HS5 cells, suggesting a role for BRD4 and MYC in checkpoint regulation. In all cell lines examined, dBET6 was found to be a more effective drug compared to JQ1 and dBET1 (IFN-G-induced expression of PD-L1 in: HMEC-1: IC 50 for JQ1 0.5 µM vs dBET1 1 µM vs dBET6 0.05 µM; CAL-72: IC 50 for JQ1 >0.5 µM vs dBET1 1 µM vs dBET6 <0.05 µM). Together, we show that endothelial cells, osteoblasts and stromal cells display PD-L1, PD-L2 and CD47 in a constitutive manner. Our data also show that the pro-inflammatory cytokines IFN-G and IFN-A augment expression of PD-L1 and PD-L2 in these cells, and that TNF-A can further enhance IFN-A/IFN-G-induced expression of PD-L1 and PD-L2 in niche cells. In addition, we show that the BRD4-targeting drugs JQ1, dBET1 and dBET6 suppress cytokine-induced PD-L1 and PD-L2 expression on niche cells. Whether checkpoint expression in niche cells is associated with drug resistance in myeloid neoplasms and whether drug-induced suppression of checkpoint expression in niche cells can overcome resistance remains unknown.

Atrial Fibrillation in Patients with Myelofibrosis

Background: Atrial fibrillation (AF) is the most common arrythmia (1-2% of the general population) and is associated with high risk of arterial thrombotic events. AF is not well characterized in the context of myeloproliferative neoplasms (MPN). Recent studies imply that thrombotic risk stratification among MPN AF patients may be suboptimal using the standard CHA2DS2VASC score, and true predictor of thrombotic risk may be presence of myelofibrosis instead. Clinical context in which AF occurs in myelofibrosis patients had not been investigated so far, but such analysis is needed to better understand why is the presence of myelofibrosis in particular the main predictor of thrombotic events among AF MPN patients. We aimed to evaluate prevalence and clinical associations of AF among patients with myelofibrosis. Methods: We retrospectively investigated a cohort of 154 patients with overt myelofibrosis treated in 6 Croatian hematology centers in period 2004-2022. Diagnoses were reassessed according to the 2016 WHO criteria for primary myelofibrosis (PMF) and the 2008 IWGMRT criteria for secondary myelofibrosis (SMF). Presence of AF documented in medical records and clinical correlations of AF status with main disease related characteristics were analyzed. Results: Among a total of 154 patients, 86 had PMF, 35 had post polycythemia vera (PV) and 33 had post essential thrombocythemia (ET) SMF. Median age was 69 years. There were 91 (59%) males, 111 (75%) patients were JAK2, 9 (7.3%) CALR and 4 (3.3%) MPL mutated. AF was present in 15 (9.7%) patients with myelofibrosis which was significantly more frequent than anticipated in the general population of similar age (P<0.001). There were no significant associations of AF with neither etiology of myelofibrosis, age, sex, mutational status, degree of bone marrow fibrosis, spleen size, history of thrombosis, estimated glomerular filtration rate, hemoglobin, WBC nor platelet count (P>0.05 for all analyses). DIPSS score did not differ between AF and non-AF PMF patients, but MYSEC-PM score was significantly higher among AF SMF patients (P=0.029). AF in comparison to non-AF patients more frequently had constitutional symptoms (87% vs 54%, P=0.015), positive bleeding history (27% vs 6%, P=0.004), heart failure (47% vs 12%, P<0.001), obesity (21% vs 4%, P=0.010), lower MCHC (313 vs 318 g/L, P=0.031), higher urea (median 8.5 vs 6.8 g/L, P=0.009) and higher CHA2DS2VASC score (4 vs 3 points, P=0.003). AF more frequently received anticoagulants and diuretics than non-AF patients (P<0.05), whereas similar rates of cytoreductive therapy and aspirin were utilized. Despite all myelofibrosis patients with AF patients having CHA2DS2VASC score ≥2 points, only 60% of them received anticoagulant therapy, 27% received aspirin and 73% received cytoreductive therapy. No significant difference in survival and thrombosis was observed between AF and non-AF myelofibrosis patients (P>0.05 for all analyses) although study may be underpowered for these analyses. Summary/Conclusion: AF is frequent in patients with myelofibrosis and might be associated with higher frequency of constitutional symptoms, particular cardiovascular comorbidities and consistently high predetermined CHAD2DS2VASC risk. Nevertheless, unexpectedly low proportion of patients receives anticoagulant therapy, possibly due to anticipated risk of bleeding and cytopenias common in myelofibrosis patients. It remains a question whether suboptimal treatment or presence myelofibrosis itself might be a risk factor for unfavorable outcomes among AF MPN patients.

Real-World Use, Effectiveness and Safety of Romiplostim, Eltrombopag and Avatrombopag in Immune Thrombocytopenia: Data from the Norwegian ITP Registry

Introduction: Thrombopoietin receptor agonists (TPO-RAs) are among the most widely used second-line treatments for immune thrombocytopenia (ITP). TPO-RAs counterbalance platelet destruction by stimulating proliferation and maturation of the megakaryocytes. Response rates varies between 60-90% and in the majority of cases continuous treatment with TPO-RAs is required to maintain response. However, it has recently been shown that 10-30% of patients can maintain response after treatment discontinuation (sustained response off-therapy - SROT). Romiplostim, Eltrombopag, and Avatrombopag are 3 TPO-RAs licensed for use in ITP in many countries. However, there is a lack of comparative studies on the efficacy and safety of these agent. Aims: To describe real-world use, effectiveness (rates of initial response, durable response, discontinuation and SROT) and safety of TPO-RAs in Norway for each of the 3 agents. Methods: This is a multicenter, retrospective/prospective study based on data from Norwegian ITP Registry (NOR-ITP). We identified all patients with primary ITP who had received at least one TPO-RA during the course of their disease (n=87). These patients were diagnosed with ITP between 1966 and 2023 and were included in NOR-ITP between 2016 and May 2023. We defined response to treatment as platelet count > 30*10 9/L and double than baseline and absence of bleeding, and complete response as platelet count > 100*10 9/L; time to response as the time to obtain response; durable response as 2 consecutive platelet counts > 30*10 9/L at 6 months without other therapies for ITP; SROT as response for at least 6 months in absence of any therapy for ITP after the discontinuation of TPO-RA. We reported adverse events as registered in NOR-ITP. We stratified the first TPO-RA agents by year ranges (2009 to 2019, 2019 to 2022, and 2022-2023) to evaluate tendency in choice of first agent and treatment line in which first TPO-RA was used. Results: Median (Q1; Q3) age of 87 ITP patients at the time of first TPO-RA was 60 years (38; 68); M:F ratio = 1:1. Phase of ITP in which the first TPO-RA was initiated was newly diagnosed in 14 (16%), persistent in 15 (17%), and chronic in 58 (67%) patients. As first TPO-RA, 23 (26%) received Romiplostim, 50 (57%) Eltrombopag, and 14 (17%) Avatrombopag. As shown in Figure1a, TPO-RAs are increasingly used in in the first and second line in ITP. Eltrombopag was the most widely used agent during 2009-2019, but the use declined from 2019-2022, while the use of Avatrombopag increased to become the mostly used agent in 2022-2023 (Figure1b). Response to first TPO-RA was achieved in 70% (16) for Romiplostim, 68% (35) for Eltrombopag, and 100% (14) for Avatrombopag. A complete response was achieved by 61% (14) for Romiplostim, 56% (28) for Eltrombopag, and 100% (14) for Avatrombopag. Median (Q1; Q3) time to response in days from start of TPO-RA was 7 (5;12) for Romiplostim, 10 (7;14) for Eltrombopag, and 7 (4;14) for Avatrombopag. Durable response rate (at 6 months) was 52% (n=12) for Romiplostim, 74% (n=37) for Eltrombopag, and 64% (n=9) for Avatrombopag. Reasons for discontinuation of Romiplostim were remission in 7 (30%) cases, absence or loss of response in 9 (40%), and was ongoing or discontinued for other reasons in 7 (30%); Eltrombopag was discontinued because of remission in 18 (36%) cases, absence or loss of response in 9 (18%), and was ongoing or discontinued for other reasons in 23 (46%). Avatrombopag was discontinued for remission in 4 (29%) cases, absence or loss of response in 3 (21%), and was ongoing or discontinued for other reasons in 7 (50%). SROT was obtained in 2 (9%) cases for Romiplostim, and 3 (6%) for Eltrombopag. Median duration of SROT was 13 (13; 44) months. Side effects were reported in 22 (25%) patients, 4 fatigue (5%), 2 bone marrow fibrosis (2%), and 2 thrombosis (2%). Conclusion: Our results show a comparable effectiveness between the three agents, however, a pattern of use of first TPO-RA is changing, and it appears to be slowly turning towards Avatrombopag. This trend is supported by current recommendations from Norwegian medicine funding authority towards Avatrombopag as first choice. Moreover, trust of clinicians towards TPO-RAs is increasing in recent years, as these agents begin to be chosen in earlier lines. Safety profiles were excellent, thrombosis and bone marrow fibrosis were rarely found.

Incorporating mutations and bone marrow fibrosis into the revised international prognostic scoring system in myelodysplastic syndromes

The independent prognostic significance of bone marrow fibrosis (BMF) in myelodysplastic syndromes (MDS) is challenged under currently molecular prognostic models. In this study, the clinical and genetic data from 438 MDS patients were analyzed retrospectively. The patients were randomly divided into training (n = 306) and validation (n = 132) cohorts. The independent significant prognostic factors included age, IPSS-R, BMF, TP53 and U2AF1. Using their weighted coefficients, we developed a simplified prognostic system. Four risk groups were produced: low, intermediate, high and very high. The new model yielded more clearly separated survival curves than the IPSS-R. In addition, our model achieved higher C-indexes (0.61 in the training cohort and 0.63 in the validation cohort) than the IPSS-RM model (0.59 and 0.58) and IPSS-R (0.57 and 0.56). In conclusion, BMF was an independent significant prognostic factor for MDS, and adding BMF into the IPSS-R improved its predictive capability.

PRİMER İMMÜN TROMBOSİTOPENİDE ARTMIŞ RETİKÜLER LİF DERECESİ

OBJECTIVE: Bone marrow fibrosis in primary immune thrombocytopenia (ITP) has become a centre of attention since cases with fibrosis were reported during trombopoeitin receptor agonist therapies but, there have been few studies evaluating the fibrosis status of the patients at diagnosis. The aim of the study was to evaluate the impact of marrow fibrosis on especially response to treatment and prognosis in patients with ITP. MATERIAL AND METHODS: Bone marrow reticulin fiber grade, haemoglobin, platelets, age, sex, co-morbidities of the patients, hepatitis and autoimmune markers on admission, response, remission status and duration of remission and treatments were recorded from medical files of the patients and each parameter was evaluated for an association with reticular fiber grade in 53 patients with ITP. RESULTS: 79.3% of patients had marrow reticulin content grade 1 or more. No significant correlations were found between bone marrow reticular fiber grade and total blood count at diagnosis, response times to the first, second- and third-line treatment, platelet counts after treatment and time between two treatment lines, age, gender, presence of comorbidity and antinuclear antibody positivity and response rate and time. There was a significant and positive correlation between platelet count at diagnosis and age (p<0.05) and, there was a statistically significant and negative correlation between white blood and neutrophil count at diagnosis and age (p<0.05) CONCLUSIONS: For the first time, higher grade of fibrosis was found in patients with ITP. Prospective studies with follow-up bone marrow biopsies are needed to validate the link between ITP and autoimmune fibrosis.