Prefibrotic Primary Myelofibrosis Research Articles

A comparative retrospective study of pre-fibrotic primary myelofibrosis versus overtly fibrotic stage in Qatar: clinicopathological, genetic landscape, risk stratification and survival data (2008–2021) – a single center experience

ABSTRACT Background In MENA region, there is a lack of evidence on Primary Myelofibrosis (PMF), leading to its underrepresentation in medical literature. This study marks the first comprehensive report on PMF data in Qatar, presenting findings from a single-center study spanning 13 years (2008-2021). Methods Clinicopathological data, genetic features, and disease progression parameters of pre-PMF and overt PMF subgroups were collected. Overall survival (OS), progression-free survival (PFS), DIPSS plus four categories and merged low and high-risk DIPSS scoring groups were assessed. Results Pre-PMF patients showed higher hemoglobin (P < 0.001), and platelet counts (P < 0.05) but lower blast counts, LDH levels, constitutional symptoms (P < 0.0001), and splenomegaly (P < 0.010) than overt PMF patients. JAK2 V617F mutation was more common in pre-PMF (P = 0.059), while unfavorable karyotypes were exclusive to overt PMF (P = 0.028). Median overall survival was significantly longer at 276.9 months (IQR: 315.9, 276.9 months) to what was previously reported. Overt PMF patients predominantly fell into the higher DIPSS risk category (P < 0.001) and showed greater disease progression than pre-PMF (P < 0.0001). Complications including refractory anaemia (P < 0.001) and leukemic transformation (P = 0.043), increased notably in the high-risk group. Furthermore, 86.2% of high-risk patients required treatment versus 59.4% of the lower-risk group (P = 0.020). Conclusions To the best of our knowledge our research represents the first and largest published dataset on PMF in MENA region to be published. Merged DIPSS plus scoring came to be a pragmatic tool for defining high-risk patients who significantly differ in mortality, progression, need for treatment and leukemic transformation.

A randomized, double-blind, placebo-controlled phase 3 study to assess efficacy and safety of ropeginterferon alfa-2b in patients with early/lower-risk primary myelofibrosis

Primary myelofibrosis (PMF) is the most aggressive of the myeloproliferative neoplasms and patients require greater attention and likely require earlier therapeutic intervention. Currently approved treatment options are limited in their selective suppression of clonal proliferation resulting from driver- and coexisting gene mutations. Janus kinase inhibitors are approved for symptomatic patients with higher-risk PMF. Additionally, most ongoing clinical studies focus on patients with higher-risk disease and/or high rates of transfusion dependency. Optimal treatment of early/lower-risk PMF remains to be identified and needs randomized clinical trial evaluations. Pegylated interferon alfa is recommended for symptomatic lower-risk PMF patients based on phase 2 non-randomized studies and expert opinion. Ropeginterferon alfa-2b (ropeg) is a new-generation pegylated interferon-based therapy with favorable pharmacokinetics and safety profiles, requiring less frequent injections than prior formulations. This randomized, double-blind, placebo-controlled phase 3 trial will assess its efficacy and safety in patients with “early/lower-risk PMF”, defined as pre-fibrotic PMF or PMF at low or intermediate-1 risk according to Dynamic International Prognostic Scoring System-plus. Co-primary endpoints include clinically relevant complete hematologic response and symptom endpoint. Secondary endpoints include progression- or event-free survival, molecular response in driver or relevant coexisting gene mutations, bone marrow response, and safety. Disease progression and events are defined based on the International Working Group criteria and well-published reports. 150 eligible patients will be randomized in a 2:1 ratio to receive either ropeg or placebo. Blinded sample size re-estimation is designed. Ropeg will be administered subcutaneously with a tolerable, higher starting-dose regimen. The study will provide important data for the treatment of early/lower-risk PMF for which an anti-clonal, disease-modifying agent is highly needed.

Multi-omics differences in the bone marrow between essential thrombocythemia and prefibrotic primary myelofibrosis

Essential thrombocythemia (ET) and prefibrotic primary myelofibrosis (pre-PMF) are Philadelphia chromosome-negative myeloproliferative neoplasms. These conditions share overlapping clinical presentations; however, their prognoses differ significantly. Current morphological diagnostic methods lack reliability in subtype differentiation, underlining the need for improved diagnostics. The aim of this study was to investigate the multi-omics alterations in bone marrow biopsies of patients with ET and pre-PMF to improve our understanding of the nuanced diagnostic characteristics of both diseases. We performed proteomic analysis with 4D direct data-independent acquisition and microbiome analysis with 2bRAD-M sequencing technology to identify differential protein and microbe levels between untreated patients with ET and pre-PMF. Laboratory and multi-omics differences were observed between ET and pre-PMF, encompassing diverse pathways, such as lipid metabolism and immune response. The pre-PMF group showed an increased neutrophil-to-lymphocyte ratio and decreased high-density lipoprotein and cholesterol levels. Protein analysis revealed significantly higher CXCR2, CXCR4, and MX1 levels in pre-PMF, while APOC3, APOA4, FABP4, C5, and CFB levels were elevated in ET, with diagnostic accuracy indicated by AUC values ranging from 0.786 to 0.881. Microbiome assessment identified increased levels of Mycobacterium, Xanthobacter, and L1I39 in pre-PMF, whereas Sphingomonas, Brevibacillus, and Pseudomonas_E were significantly decreased, with AUCs for these genera ranging from 0.833 to 0.929. Our study provides preliminary insights into the proteomic and microbiome variations in the bone marrow of patients with ET and pre-PMF, identifying specific proteins and bacterial genera that warrant further investigation as potential diagnostic indicators. These observations contribute to our evolving understanding of the multi-omics variations and possible mechanisms underlying ET and pre-PMF.

CCN2/CTGF expression does not correlate with fibrosis in myeloproliferative neoplasms, consistent with noncanonical TGF-β signaling driving myelofibrosis

The classical BCR::ABL1-negative myeloproliferative neoplasms (MPN) form a group of bone marrow (BM) diseases with the potential to progress to acute myeloid leukemia or develop marrow fibrosis and subsequent BM failure. The mechanism by which BM fibrosis develops and the factors that drive stromal activation and fibrosis are not well understood. Cellular Communication Network 2 (CCN2), also known as CTGF (Connective Tissue Growth Factor), is a profibrotic matricellular protein functioning as an important driver and biomarker of fibrosis in a wide range of diseases outside the marrow. CCN2 can promote fibrosis directly or by acting as a factor downstream of TGF-β, the latter already known to contribute to myelofibrosis in MPN.To study the possible involvement of CCN2 in BM fibrosis in MPN, we assessed CCN2 protein expression by immunohistochemistry in 75 BM biopsies (55 × MPN and 20 × normal controls). We found variable expression of CCN2 in megakaryocytes with significant overexpression in a subgroup of 7 (13%) MPN cases; 4 of them (3 × essential thrombocytemia and 1 × prefibrotic primary myelofibrosis) showed no fibrosis (MF-0), 2 (1 × post-polycythemic myelofibrosis and 1 × primary myelofibrosis) showed moderate fibrosis (MF-2), and 1 (primary myelofibrosis) severe fibrosis (MF-3). Remarkably, CCN2 expression did not correlate with fibrosis or other disease parameters such as platelet count or thrombovascular events, neither in this subgroup nor in the whole study group. This suggests that in BM of MPN patients other, CCN2-independent pathways (such as noncanonical TGF-β signaling) may be more important for the development of fibrosis.

Conventional Cytogenetic Analysis and Array CGH + SNP Identify Essential Thrombocythemia and Prefibrotic Primary Myelofibrosis Patients Who Are at Risk for Disease Progression.

The Philadelphia chromosome-negative myeloproliferative neoplasms (Ph-MPNs) are a heterogeneous group of clonal hematopoietic malignancies that include polycythemia vera (PV), essential thrombocythemia (ET), and the prefibrotic form of primary myelofibrosis (prePMF). In this study, we retrospectively reviewed the karyotypes from conventional cytogenetics (CC) and array Comparative Genomic Hybridization + Single Nucleotide Polymorphism (aCGH + SNP) in patients with ET or prePMF to determine whether the combined analysis of both methodologies can identify patients who may be at a higher risk of disease progression. We performed a comprehensive genomic review on 169 patients with a clinical diagnosis of ET (154 patients) or prePMF (15 patients). Genomic alterations detected by CC or array-CGH + SNP were detected in 36% of patients. In patients who progressed, 68% had an abnormal genomic finding by either technology. There was a shorter progression-free survival (PFS) among patients who were cytogenetically abnormal or who were cytogenetically normal but had an abnormal aCGH + SNP result. Leveraging the ability to detect submicroscopic copy number alterations and regions of copy neutral-loss of heterozygosity, we identified a higher number of patients harboring genomic abnormalities than previously reported. These results underscore the importance of genomic analysis in prognostication and provide valuable information for clinical management and treatment decisions.

Broad Next-Generation Integrated Sequencing of Myelofibrosis Identifies Disease-Specific and Age-Related Genomic Alterations.

Myeloproliferative neoplasms (MPN) are characterized by the overproduction of differentiated myeloid cells. Mutations in JAK2, CALR, and MPL are considered drivers of Bcr-Abl-ve MPN, including essential thrombocythemia (ET), polycythemia vera (PV), prefibrotic primary myelofibrosis (prePMF), and overt myelofibrosis (MF). However, how these driver mutations lead to phenotypically distinct and/or overlapping diseases is unclear. To compare the genetic landscape of MF to ET/PV/PrePMF, we sequenced 1,711 genes for mutations along with whole transcriptome RNA sequencing of 137 patients with MPN. In addition to driver mutations, 234 and 74 genes were found to be mutated in overt MF (N = 106) and ET/PV/PrePMF (N = 31), respectively. Overt MF had more mutations compared with ET/PV/prePMF (5 vs. 4 per subject, P = 0.006). Genes frequently mutated in MF included high-risk genes (ASXL1, SRSF2, EZH2, IDH1/2, and U2AF1) and Ras pathway genes. Mutations in NRAS, KRAS, SRSF2, EZH2, IDH2, and NF1 were exclusive to MF. Advancing age, higher DIPSS, and poor overall survival (OS) correlated with increased variants in MF. Ras mutations were associated with higher leukocytes and platelets and poor OS. The comparison of gene expression showed upregulation of proliferation and inflammatory pathways in MF. Notably, ADGRL4, DNASE1L3, PLEKHGB4, HSPG2, MAMDC2, and DPYSL3 were differentially expressed in hematopoietic stem and differentiated cells. Our results illustrate that evolution of MF from ET/PV/PrePMF likely advances with age, accumulation of mutations, and activation of proliferative pathways. The genes and pathways identified by integrated genomics approach provide insight into disease transformation and progression and potential targets for therapeutic intervention.

Comparative Analysis of Bone Marrow Microbiome and Proteome Differences between Essential Thrombocythaemia and Prefibrotic Primary Myelofibrosis

Background Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) are hematologic malignancies with a hallmark feature of chronic inflammation. MPNs mainly include polycythaemia vera (PV), essential thrombocythaemia (ET), primary myelofibrosis (PMF) and prefibrotic primary myelofibrosis (pre-PMF). In contrast to ET, an accurate morphological diagnosis of pre-PMF is a key issue due to its worse prognosis. However, it is difficult to accurately diagnose pre-PMF, mainly relying on pathological diagnosis. The heavy reliance on morphology for accurate diagnosis poses a challenge to the professional abilities of physicians. The uneven expertise of physicians can lead to improper diagnosis and delay patient treatment. Therefore, accurate diagnosis of these two disease subtypes is of great importance. It is increasingly recognized that the bone marrow microenvironment, including inflammation and metabolic imbalances, influences disease progression. So far, there have been few studies focusing on the combined analysis of the bone marrow microbiome and proteome in MPN patients. We aim to describe the changes of bone marrow microbiome and proteome and their association with pathogenesis in ET and pre-PMF patients Method We analyzed the formalin-fixed, paraffin embedded (FFPE) tissue slide of bone marrow from six patients with untreated pre-PMF (median age: 64 years, m/f=4/2) and ET (n=7, median age: 53 years, m/f=2/5). Proteomic analysis of FFPE tissue slides of bone marrow from 13 patients was performed using 4D-direct DIA. The bone marrow microbiota was analyzed by using 2bRAD-M sequencing technology. Result The clinical and laboratory characteristics of ET and pre-PMF patients are summarized in Table 1. The JAK2 V617F mutations were detected in all patients, and the mutations were more frequent in patients with pre-PMF than in those with ET (17.9% vs. 55.8%, p=0.001). All patients in the ET group are at MF grade 0, while all in the pre-PMF group are at MF grade 1. The lactate dehydrogenase in pre-PMF group patients is higher than in ET group patients, and the difference is statistically significant ( p=0.007). In addition, the neutrophil-to-lymphocyte ratio (NLR), an inflammation-related indicator, is significantly increased in the pre-PMF group. The neutrophil-to-lymphocyte ratio in the pre-PMF group is significantly higher than in the ET group, which is statistically significant ( p=0.015). High density lipoprotein cholesterol (HDL) in the pre-PMF group is significantly reduced, and the difference is statistically significant ( p&amp;lt;0.001). This suggests a difference in lipid metabolism between pre-PMF and ET. Figure 1A shows a significant correlation between NLR and HDL, with a statistical difference ( p= 0.013). Proteomics analysis identified a total of 177 differential proteins in the bone marrow of ET and pre-PMF, of which 108 were up-regulated and 69 were down-regulated in the pre-PMF group (Figure 1B and 1C). Through functional enrichment analysis, we found that the differentially expressed proteins upregulated in the pre-PMF group (such as: CXCR4, CXCR2, GBP5, etc.) are mainly enriched in immune and inflammation-related GO (Gene Ontology) terms, such as immune response (GO:000695). The differentially expressed proteins downregulated in the pre-PMF group (such as APOA4, FABP4, etc.) are mainly enriched in lipid metabolism-related GO terms, such as lipoprotein metabolic process (GO: 0042157), and positive regulation of fatty acid biosynthetic process (GO: 0045723). Association analysis revealed a significant relationship between differential proteins and clinical markers (Figure 1D). We identified 223 differential microbes in the bone marrow, which were classified into 88 genera. The α diversity of the microbiota in bone marrow calculated by Chao1 showed that there was significant difference in species diversity between the two groups (Figure 1E). Microbiome composition showed statistically significant differences between pre-PMF and ET ( p&amp;lt;0.001, R 2=0.302, PERMANOVA). Microbial diversity and richness was higher in pre-PMF, and reaching statistical significance in this small cohort (Figure 1F). Conclusion We have shown that there are multi-level differences, including the proteome and microbiome, between ET and pre-PMF patients.This could potentially provide new insights for distinguishing and treating these two disease subtypes.

Conventional Cytogenetic Analysis and Array CGH+SNP Identify Essential Thrombocythemia (ET)/Prefibrotic Primary Myelofibrosis (pre-PMF) Patients Who Are at Risk for Disease Progression

ET and pre-PMF are myeloproliferative neoplasms (MPNs) that are each characterized by thrombocytosis but are difficult to distinguish by clinical and histopathological criteria. Moreover, each has the propensity to progress at variable rates to more advanced forms of the disease. The cause of progression to more overt forms of myelofibrosis (MF) or accelerated /blast phase (AP/BP) has yet to be fully understood, making prognostication difficult. To identify patients (pts) who are at risk for disease progression, we retrospectively reviewed the karyotypes from conventional cytogenetics (CC) and array Comparative Genomic Hybridization + Single Nucleotide Polymorphism (aCGH+SNP) of 158 pts with a diagnosis of ET or pre-PMF from 2017 to 2022 at our institution. Among these pts, 58% were female (92 pts) and 42% were male (66 pts), and the median age was 61 years (range 13-92). Mutational analysis was performed in 155 pts, and JAK2 mutations were present in 63% of cases, CALR in 19%, and MPL in 7% of cases. The remaining 11% of pts were triple negative. An abnormal karyotype was observed in 15% (24/158), and the remaining 85% (134/158) were either normal or had non-diagnostic results due to failed or limited peripheral blood chromosome analysis. Of the 111 pts that had aCGH+SNP testing, 6% (7/111) had prior abnormalities as detected by conventional cytogenetic analyses, while abnormalities were found in 30% (33/111) with aCGH+SNP exclusively. In this cohort, 29% (46/158) of pts underwent disease progression. Among these cases, 65% (30/46) had progression to either MF or chronic myeloid leukemia or myelodysplasia, and 22% (10/46) progressed to AP/BP. In addition, 13% (6/46) evolved to polycythemia vera. Disease progression, irrespective of its form, was associated with genomic alterations detected by aCGH+SNP, cytogenetic analysis, or both at the time of first encounter. Of the 46 pts undergoing progression during their clinical course, 65% (30/46) had an abnormal genomic finding by either technology compared to 35% (16/46) who were normal (p&amp;lt;0.001). Strikingly, among 24 pts who were abnormal by classical cytogenetics, 79% (19/24) progressed to either AP/BP or MF compared to only 20% (26/125) who were cytogenetically normal (p&amp;lt;0.001). As shown in Figure 1, there was a shorter progression-free survival among pts who were cytogenetically abnormal as compared to normal pts (p&amp;lt;0.001). Among 134 pts with ET/pre-PMF who were normal or had non-diagnostic results by classical cytogenetic studies, aCGH+SNP testing was performed in 104 cases and additional genomic aberrations were detected in 32% (33/104). Within this group, 30% (10/33) progressed to MF with a trend towards a shorter progression-free survival. In those patients that progressed to MF, the most frequent abnormality was 9p CNLOH occurring in 6 pts. In the remaining 71 pts that were cytogenetically and aCGH+SNP normal, only 11% (8/71) had disease progression. Overall, the combination of both conventional cytogenetics and aCGH+SNP increased the sensitivity of detecting genomic abnormalities from 15% (cytogenetics alone) to 37% when incorporating aCGH+SNP. These data indicate that genomic alterations detected by conventional cytogenetic analyses or CGH+SNP array identify a subset of ET/pre-PMF pts that are at a higher risk of disease progression.

Clinical and Prognostic Validation of the Reticulin, Collagen, Osteosclerosis (RCO) Score for Primary Myelofibrosis (PMF)

Introduction: Primary myelofibrosis (PMF) is hallmarked by stromal bone marrow (BM) changes that include reticulin fibrosis, collagen deposition, osteosclerosis, and microvascular proliferation. Accurate evaluation of BM fibrotic changes is key for MF diagnosis and deserves prognostic implications. Gianelli et al. ( Histopathol 2017;71:89) developed a comprehensive score, named reticulin (MF), collagen (Co), osteosclerosis (Ost) score (RCOs), which was prognostically informative and more accurate than the 2005 European Consensus on Grading of Bone Marrow Fibrosis (ECGMF) in identifying high-risk patients (pts). Aims&amp;Methods: This retrospective, single-center study aimed to validate the RCOs and explore its clinical, genetic and prognostic correlates in a large cohort of clinically and molecularly annotated PMF pts diagnosed according to 2022 ICC criteria. The RCOs was assessed by two independent experienced histopathologists (U.G, R.S.). Results: The study included 222 pts with a diagnosis of PMF: 121 (55%) prefibrotic PMF (pre-PMF), 101 (45%) overt PMF. Median age was 66 years (range, 18-91), 127 (57%) were male. BM histological analysis according to the RCO algorithm provided the following results: MF-0 n=20 (9%), MF-1 n=102 (46%), MF-2 n=47 (21%), and MF-3 n=53 (24%); Co-0 n=130 (59%), Co-1 n=33 (15%), Co-2 n=31 (14%), and Co-3 n=28 (13%); Ost-0 n=146 (66%), Ost-1 n=45 (20%), Ost-2 n=18 (8%), and Ost-3 n=13 (6%). Next, we looked at the distribution of the Co and Ost variables according to MF grade and PMF diagnosis. Overall, morphological features evolved harmonically, with pre-PMF showing the less severe stromal changes and higher reticulin fibrosis being associated with the highest values of Co and Ost. Computation of the RCOs returned the following results: RCOs 0 n=20 (9%), RCOs 1 n=94 (42%), RCOs 2 n=15 (7%), RCOs 3 n=19 (9%), RCOs 4 n=9 (4%), RCOs 5 n=16 (7%), RCOs 6 n=13 (6%), RCOs 7 n=17 (8%), RCOs 8 n=11 (5%), and RCOs 9 n=8 (4%). Median RCOs for pts with pre- and overt PMF was 1 (range, 0-2) and 5 (range, 2-9), respectively. We investigated the correlations of RCOs with clinical and molecular variables. Higher RCOs was associated with overt PMF (median, 5 vs 1, P&amp;lt;.0001), male gender (median, 3 vs 1, P=.0005), lower hemoglobin (Pearson's r, -0.5, P&amp;lt;.0001), lower platelets (Pearson's r, -0.4, P&amp;lt;.0001), higher blasts (Pearson's r, 0.4, P&amp;lt;.0001), higher LDH (Pearson's r, 0.5, P&amp;lt;.0001), and higher prevalence of splenomegaly (median, 3 vs 1, P&amp;lt;.0001) and constitutional symptoms (median, 5 vs 1, P=.0004). As to molecular variables, a higher RCOs was associated with abnormal karyotype (median, 3 vs 1, P=.0003), absent JAK2 mut (median, 2 vs 1, P=.0224) but higher JAK2 allele burden in JAK2-mut pts (Pearson's r, 0.3, P&amp;lt;.0006), triple negativity (median, 5 vs 1, P=.0223), EZH2 mut (median, 6 vs 1, P=.0033), ASXL1 mut (median, 6 vs 1, P&amp;lt;.0001), KRAS mut (median, 8 vs 1, P=.0059), and HMR status (i.e., ≥1 mut in ASXL1, EZH2, IDH1/2, SRSF2) (median, 5 vs 1, P&amp;lt;.0001). Median overall survival (OS) was 96 months (95% CI 81-134). In univariate Cox regression analysis, RCOs directly correlated with risk of death (HR 3.1, 95% CI 1.5-6.3, P=.0023). MF and Co variables correlated with OS (respectively: HR 4.0, 95% CI 1.8-8.6, P=.0003; HR 2.7, 95% CI 1.5-5.0, P=.0008), unlike Ost. ROC analysis with death as endpoint confirmed 5 as the optimal cut-off value ( Histopathol 2017;71:89). Pts with RCOs &amp;gt;5 (n=65 overt PMF) had significantly worst OS compared to pts with RCOs &amp;lt;5 (n=121/36 pre-/overt PMF), with median values of 64 vs 134 months (HR 2.3, 95% CI 1.4-3.6, P=.0004) (Fig. 1A). Considering only overt PMF pts, ROC analysis confirmed 5 as the more accurate cut-off value (univariate survival analysis: HR 2.1, 95% CI 1.1-4.2, P=.0257). We finally computed the MIPSS70 by replacing the original BM fibrosis grade ≥2 variable with the dichotomous RCOs 5 ( &amp;gt;/&amp;lt;5) in 177 (80%) pts with full molecular information, and calculated the C-index, Brier score, and time-dependent area under the curve (AUC) (Fig. 1B). The highest values for performance and accuracy in predicting death were achieved by the RCOs 5-integrated MIPSS70 at 12, 36, and 48 months. Conclusions: Our findings confirm the clinical and prognostic consistency of RCOs, supporting the importance of a comprehensive and accurate evaluation of BM stromal changes in PMF. Integration of RCOs within the MIPSS70 might improve the predictive performance of the model.

Classification of Philadelphia-Negative MPN As Low Risk and High Risk MPN Based on Peripheral Blood Values and Molecular Genetics Only

Background: Philadelphia-negative myeloproliferative neoplasms (Ph -MPN) are characterized by overproduction of differentiated hematopoietic cells mediated in the majority of cases by mutations in JAK2, MPL or CALR (MPN driver genes). Ph - MPN include polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PMF) and MPN, unclassifiable (MPN-U) which differ in clinical presentation, prognosis, fibrosis and leukemic transformation rate as well as therapeutic options. However, WHO based classification requires experienced pathologists, in particular to diagnose prefibrotic PMF as high risk MPN, and thus remains observer dependent. Aim: Observer independent classification of Ph - MPN solely based on molecular genetics and peripheral blood (PB) parameters. Patients and Methods: We analyzed 267 Ph - MPN cases diagnosed as ET (n=76), PV (n=74), PMF (n=68) or MPN-U (n=49) following the WHO4R classification including only patients with an MPN driver mutation (median age: 68 years [22-91]; female: 41%; median follow-up: 5 years). All samples were analyzed by cytomorphology and whole genome sequencing (median coverage 100x). Mutation status of 20 genes associated with myeloid malignancies were analyzed in detail. Results: Genomic landscape of the MPN cohort showed mutations in JAK2 in 222 (83%) (n=132: single mutation; n=90: either mutation and copy neutral loss of heterozygosity or mutation and 9p gain), in CALR in 36 (14%), and in MPL in 9 (3%) patients. The median number of mutations per patient was two (range: 1-6). Within the cohort, 141 patients (53%) harbored MPN driver mutations only, while in 126 (47%) one to 5 additional mutations were found. The most frequent additional mutations were ASXL1 (n=50), TET2 (n=49; thereof 15 biallelic), SRSF2 (n=26), DNMT3A (n=18), U2AF1 (n=11) and TP53 (n=9; thereof 3 biallelic). Regarding PB parameters, high WBC (≥11×10 9/ L) were detected in 127 patients (48%), high PLT (≥450×10 9/ L) in 116 (43%) and anemia (HB &amp;lt;♂12 or ♀11 g/dL) in 50 patients (19%). We then performed Cox regression analyses for overall survival (OS) including only genetic abnormalities and PB values. In univariate analyses, biallelic TET2 inactivation ( TET2bi: hazard ratio/HR: 3.789), RUNX1 (n=4; HR: 13.479), ASXL1 (HR: 1.886), SRSF2 (HR: 3.218) mutations as well as high PLT (HR: 0.440), high WBC (HR: 1.998) and PB blasts &amp;gt;1% (n=57; HR: 1.904) showed significant associations with OS (for all p&amp;lt;0.02), while no effect was observed for MPN driver mutations, DNMT3A and monoallelic TET2 mutations. In addition, anemia showed a trend towards poor prognosis (HR: 1.595; p=0.079). Following this, we determined high and low risk MPN. In the absence of thrombocytosis, high risk MPN was defined if any of the four following risk factors were present: i) mutation in ASXL1 or SRSF2 or RUNX1 or TET2bi, ii) PB blasts &amp;gt;1%, iii) high WBC or iv) anemia. In the presence of thrombocytosis, at least two of these risk factors were required (Figure 1). Survival analysis revealed a significantly worse median OS of high risk (n=137; 51%) compared to low risk MPN (n=130; 49%) (8 years vs. not reached; p&amp;lt;0.001). This proposed novel stratification reflected prognosis better than WHO entities (median OS: MPN-U: 7 years; MF: 8 years; PV/ ET not reached; overall p=0.001) (Figure 1A). All 76 ET cases (100%) and 44/74 PV cases (59%) were classified as low risk MPN, while 63/68 PMF cases (93%) and 44/49 MPN-U cases (90%) were classified as high risk MPN (Figure 1B). Within low risk and high risk MPN, no significant OS differences were observed between the WHO entities ( p=0.114 and p=0.355, respectively). Conclusions: JAK2-pathway mutated Ph - MPN can be categorized into low and high risk MPN based on molecular genetics and PB values only. The presence of these objectively assessable risk factors in a patient with ET or PV warrants for a profound reevaluation of bone marrow histology (eventually by a reference pathologist for MPN) and clinical course before a high risk MPN (including prefibrotic PMF) can be ruled out. Whether the predictive value of low risk MPN based on molecular genetics and PB values only could be sufficient to omit BM biopsy in selected patients, needs to be evaluated in further prospective studies.

Morphologic and Genetic Features of Unclassifiable Chronic Myeloid Neoplasms: Five-Year Experience of a Canadian Provincial Referral Center

Introduction: The challenge of diagnosing and definitively classifying myeloproliferative neoplasms (MPNs) and myelodysplastic/myeloproliferative neoplasms (MDS/MPNs) remains a conundrum in the field of hematology. While the World Health Organization has outlined diagnostic criteria for various categories, there remain cases that elude specific classification, falling under the umbrella of MPN, unclassifiable (MPN-U) and MDS/MPN, unclassifiable (MDS/MPN-U). In this study, we aimed to compare the morphological and genetic profiles of cases classified as MPN-U or MDS/MPN-U against more definitively classified cases (including chronic myelomonocytic leukemia (CMML), essential thrombocythemia (ET), MDS/MPN with ring sideroblasts and thrombocytosis (MDS/MPN-RS-T), and primary myelofibrosis (PMF), including prefibrotic PMF (pre-PMF)). Methods: Morphologically unclassifiable myeloid neoplasm cases referred for pathologist review to our laboratory in the largest tertiary care center in British Columbia, Canada were identified through pathology database query over a five-year period. Bone marrow materials were reviewed and reanalyzed with standardized collection of qualitative and semiquantitative data by two hematopathologists. Morphological parameters studied included the myeloid to erythroid ratio, bone marrow cellularity, lineage dysplasia, megakaryocyte histotopography, bone marrow blast frequency, fibrotic changes, and bone marrow vascularity, among others. Clinical data, cytogenetic findings, and results of a targeted sequencing panel assessing a set of approximately 50 genes recurrently mutated in myeloid malignancies were collected by electronic medical record review. A cohort of contemporarily collected myeloid neoplasms with definitive pathologic diagnoses was reviewed for comparison. Statistical analyses were performed using python3. Results: The cohort comprised 29 cases classified as MPN-U, 12 cases of MDS/MPN-U, and 5 cases each of ET, PMF, pre-PMF, CMML, and MDS/MPN-RS-T. MPN-U cases were further subcategorized based on the apparent reason for difficulty in classification, including: overlapping features with another MPN; late stage/overt fibrosis; early stage without fully-developed morphologic features; and coexisting atypical morphologic findings. MPN-U with overlapping features had highly variable reticulin fibrosis, while, as expected, late stage entities had a high degree of reticulin fibrosis and early stage entities showed less overt fibrosis (Figure, Left, demonstrating select morphologic features of evaluated chronic myeloid neoplasms). MPN-U as a whole appeared to show less prominent fibrosis-associated findings than MDS/MPN-U and to, in general, show a greater degree of neovascularization, similar to changes observed in cases with PMF. Splicing factor and epigenetic modifier mutations appeared to be overrepresented in MDS/MPN-U relative to MPN-U, notably excluding MPN-U with atypical morphologic features (Figure, Right, demonstrating the distribution of pathogenic variants identified in evaluated myeloid neoplasms; results are indicated as presence (1) or absence (0) of a variant for each gene). Notably, no CALR mutations were identified in the MDS/MPN-U category or early stage / atypical morphology MPN-U, though they were enriched in overtly fibrotic MPN-U entities. Discussion/Conclusion: In this cohort we observed, as expected, significant overlap in cases classified as MPN-U and MDS/MPN-U with better delineated entities, though patterns of morphologic change and mutational spectra suggest potential directions for further investigation of additional criteria that may guide accurate classification of these neoplasms. These findings reflect the heterogeneity of MPNs described in the literature and the complexity of their genetic underpinnings, particularly in the context of concurrent dyshematopoiesis; suggest enhanced bone marrow vascularization may be an informative differential diagnostic criterion in evaluation of these neoplasms, though further study is required; and emphasize the need for broader genetic testing and perhaps further subtyping of MPN-U cases for a more nuanced analytical approaches for these disorders.

Clinical and Genetic Features By Next-Generation Sequencing and RNA Sequencing in Pre-Fibrotic Primary Myelofibrosis Patients

The 2016 WHO classification recognized pre-fibrotic primary myelofibrosis (pre-PMF) as a distinct entity. Such knowledge is essential to depict the landscape of pre-PMF and develop mechanism-based approaches to slow the progression of pre-PMF. Here, we conducted a retrospective study of 234 pre-PMF patients defined by 2016 WHO diagnostic criteria in 13 hematooncology centers between 01 January 2015 and 31 July 2023 (ChiCTR2200061208). We evaluated clinical characteristics, risk factors and genetic features of progression to overt-PMF in pre-PMF patients. Among 234 pre-PMF patients, 135 (57.7%) harbored JAK2V617F, 36 (15.4%) CALR and 3 (1.3%) MPL mutations; 60 (25.6%) patients were negative for all three mutations. JAK2V617F-mutated patients were older ( P=0.036), had more constitutional symptoms ( P=0.027) and cardiovascular risk factors ( P=0.045), higher white blood cell count (WBC) ( P&amp;lt;0.001), hemoglobin level (HB) ( P&amp;lt;0.001), neutrophile granulocyte (NEU) ( P&amp;lt;0.001), eosinophilic granulocyte (EOS) ( P&amp;lt;0.001), basophilic granulocyte (BAS) ( P&amp;lt;0.001), hematocrit (HCT) ( P=0.002) and red blood cell distribution width (RDW) ( P&amp;lt;0.001) (Figure A). CALR-mutated cases had higher platelet hematocrit (PCT) ( P=0.043) and lactic dehydrogenase (LDH) ( P=0.008). Besides, CALR group had a higher proportion of 25.0% to progress to overt-PMF than JAK2V617F group (9.6%) and TN group (6.7%) ( P=0.025) (Figure A). As for risk factors of progression to overt-PMF, univariable analysis found it was associated with mean platelet volume (MPV) ( P=0.019), HB ( P=0.027), WBC ( P=0.008), LDH ( P=0.004) and CALR mutations ( P=0.007). Multivariable analysis revealed that lower HB value ( P=0.041; HR 0.981, 95% CI 0.962-0.999) and CALR mutation ( P=0.001; HR 3.716, 95% CI 1.331-10.069) had negative impact on the progression to overt-PMF (Figure B). When divided by HB with cut-off point of 128 g/L, patients with HB value ≤128 g/L were more likely to progress to overt-PMF compared to &amp;gt;128 g/L groups ( P=0.007) (Figure C). And patients carried CALR mutation were also more likely to progress to overt-PMF ( P&amp;lt;0.001) (Figure D). We performed next-generation sequencing (NGS) in 18 pre-PMF patients, including 9 JAK2V617F, 3 CALR and 6 TN, 3 of which progressed to overt-PMF and 15 stayed in pre-PMF until the end of the follow-up. JAK2V617F was the most frequently mutated gene (9/38, 23.7%), followed by TET2 (7/38, 18.4%), RELN (4/38, 10.5%) and ASXL1 (3/38, 7.9%). JAK2V617F-mutated patients possessed more mutations related to activation signaling pathway ( P=0.025) and DNA methylation ( P=0.033) (Figure F). TN patients possessed more mutations related to transcription factor ( P=0.048) (Figure F). Patients who progressed to overt-PMF possessed more mutations associated with myeloid related transcription factor ( P=0.025) (Figure H). We further performed RNA-Seq analysis in 6 pre-PMF samples and 14 overt-PMF samples. Compared to pre-PMF, overt-PMF showed 49 up-regulated and 13 down-regulated differentially expressed genes (DEGs), of which CSCF3 and CXCL13 were significantly up-regulated (Figure I,J). Gene ontology (GO) functional enrichment analysis showed that DEGs were enriched in cell-cell signaling, cytokine activity, monocyte chemotaxis and chemokine activity (Figure K). KEGG biofunctional analysis showed that DEGs were significantly enriched in cytokine-cytokine receptor interaction, chemokine signaling pathway, IL-17 signaling pathway and NF-kappa B signaling pathway (Figure L). In conclusion, CALR-mutated pre-PMF patients with HB value ≤128 g/L were more likely to progress to overt-PMF. Genetic analysis found patients with high proportion of progress to overt-PMF tended to possess mutations associated with myeloid related transcription factor. And the upregulation of CSCF3 and CXCL13 may potentially drive the progression of overt-PMF in pre-PMF patients through inflammatory and cytokine related pathways. Acknowledgement: This research was funded by the Key R&amp;D Program of Zhejiang, No. 2022C03137; Public Technology Application Research Program of Zhejiang, China, No. LGF21H080003; Zhejiang Medical Association Clinical Medical Research special fund project, No. 2022ZYC-D09. *Correspondence to: Jian Huang, M.D., Ph.D., Department of Hematology, The First Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou 310003, Zhejiang, China. E-mail: househuang@zju.edu.cn

Ropeginterferon Alfa-2b for Pre-Fibrotic Primary Myelofibrosis and DIPSS Low/Intermediate-1 Risk Myelofibrosis: Durable Responses and Evidence of Disease Modification

Background Most patients with primary myelofibrosis (PMF) in pre-/early fibrotic stage (pre-PMF) and at low or intermediate-1 risk by the dynamic international prognostic scoring system (DIPSS) will progress to higher risks or overt MF. There is currently no consensus on the optimal treatment for these patients. Ropeginterferon alfa 2b (ropeg) is a next-generation monopegylated interferon alfa-2b developed specifically to treat myeloproliferative neoplasms (MPN). Aims P1101MF is an on-going multicenter phase 2 study of ropeg in patients with pre-PMF and DIPSS low/intermediate-risk PMF. Methods Key eligibility included morphologically confirmed pre-PMF, overt PMF, post-polycythemia vera MF (PPV-MF) or post-essential thrombocythaemia MF (PET-MF), DIPSS low/intermediate-1 risk, and the need for cytoreduction. The primary outcome were responses in haemoglobin (Hb, from 10 g/dL to upper reference range), white blood cell (WBC, &amp;lt; 10 x 10 9/L) and platelet (≤ 400 x 10 9/L) at 24 and 48 weeks. Secondary outcomes included adverse events (AEs), changes in mutant allele frequencies (MAF) of driver and non-driver genes as assessed by droplet digital polymerase chain reaction (ddPCR), quality-of-life (QOL), cytokine profiles and bone marrow morphology. Patients received ropeg at a dose of 250 mcg at week 1, followed by 350 mcg at week 2 and 500 mcg every 2 weeks from week 4 onwards. Results At the data cut-off of 30 June 2023, 37 men and 29 women with a median age of 59 (range: 30-86) years were enrolled. The diagnoses were pre-PMF (=46, 69.6%); overt PMF (N=6, 9.1%); PPV-MF (N=5, 7.5%); and PET-MF (N=9, 13.6%). Mutational profile of driver genes was JAK2V617F (N=46, 69.6%); CALR mutations (type 1/type-1 like, N=13, 19.7%; type 2/type 2-like, N=4, 6.1%); MPL mutation (N=1, 1.5%); and triple-negative (N=3, 4.5%). The median time from diagnosis to treatment was 5.8 (1-271) months. The median baseline parameters were white blood cell count (WBC): 6.49 (3.25-33.95) x 10 9/L; haemoglobin (Hb): 12.5 (7.1-15.9) g/dL; platelet count: 495 (113-1114) x 10 9/L; and lactate dehydrogenase: 277 (136-1146) IU/L. The median follow-up was 69 (4-69) weeks, with 61 patients (92%) and 51 patients (77%) having completed 24 and 48 weeks of treatment respectively. Responses in Hb, WBC and platelet were 75%, 82% and 74% respectively at 24 weeks, and 82%, 80% and 71% respectively at 48 weeks. Reduction of MAF at 24 and 48 weeks was achieved for JAK2V617F in 50% (19/38) of patients (≥50%, N=10, 26%; complete, N=3, 8%) and 79% (22/28) of patients (≥50%, N=6, 21%; complete, N=1, 4%) respectively; and for CALR in 44% (7/16) of patients (≥50%, N=1, 6%) and 50% (5/10) of patients (≥50%, N=1, 10%) respectively. Forty-six patients (69.6%) had bone marrow biopsies serially performed, with 42 patients (91%) having stable/improved marrow fibrosis. Eight patients (17.4%) had resolution of marrow fibrosis by 48 weeks. There were 5 discontinuations (personal reasons, N=1; intractable pruritus, N=1; symptom/spleen size progression, N=3). Disease progression (pre-PMF to overt PMF; development of accelerated/blast phase) was not observed. Non-haematological AEs included transaminitis (grade 1-2, N=34); malaise (grade 1-2, N=27; grade 3-4, N=1) and hair loss (grade 1-2, N=21). The most common haematological AE was anaemia (grade 1-2, N=15; grade 3-4, N=6). Conclusion: Ropeg was well-tolerated, effective in cytoreduction and induced molecular and morphologic responses in patients with pre-PMF and low/intermediate-1-risk MF.

Interpretable Artificial Intelligence (AI) Differentiates Prefibrotic Primary Myelofibrosis (prePMF) from Essential Thrombocythemia (ET): A Multi-Center Study of a New Clinical Decision Support Tool

Introduction Overlapping clinical, molecular, and histopathological characteristics pose challenges in differentiating prePMF from ET. The median overall survival, however, significantly differs between prePMF and ET (11.9 vs 22.2 years, Jeryczynski, 2017). The difference in survival highlights the need to distinguish between these two myeloproliferative neoplasms (MPNs) to select disease-specific therapeutic options. This area of unmet need often requires expert assessment at high-volume academic institutions to render a definitive diagnosis. Our aim in this study is to develop and validate a biologically-motivated AI algorithm to rapidly, accurately, and inexpensively diagnose prePMF and ET directly from diagnostic bone marrow (BM) biopsy digital whole-slide images (WSI). Methods Patients with a clinical/histopathological diagnosis of prePMF or ET as determined by the International Consensus Classification of Myeloid Neoplasms were identified at the University of Florence, Italy (Florence) between 06/2007 and 05/2023 and Moffitt Cancer Center, Tampa, FL (Moffitt) between 01/2013 and 01/2022. Diagnostic H&amp;E-stained BM biopsy slides were digitized using Aperio AT2 slide scanners (Leica Biosystems, Deer Park, IL) at each institution . The training cohort comprised of 200 (100 prePMF / 100 ET) patients from Florence, and the external test cohort entailed 26 (6 prePMF / 20 ET) patients from Moffitt. In total, the resultant model was trained on 32,226 patient-derived WSI. Our chosen pretrained neural network, RetCCL, was previously trained on 32,000 diagnostic WSIs to potentially represent a histologically-informed model (Wang, 2023). BM WSI were tessellated into representative image tiles extracted at 10x magnification (302 microns per image dimensions) for model training. Finally, a prediction upon each patient's WSI was calculated by attention-based multiple instance learning, which is a method that automatically assigns a numeric weight to an image portion representing its relative importance to the classification task. Model performance was assessed utilizing the area under the receiver operator curve (AUC). The cutoff threshold for diagnosis classification was determined by maximizing Youden's Index. For qualitative assessment, attention scores were plotted as a heatmap across the BM WSI and reviewed for morphological features by an expert hematopathologist. Custom scripts were written using our open-source AI framework, Slideflow (Dolezal, 2021). Model development was performed on the Minerva High Performance Computer at Mount Sinai Hospital. Evaluation time upon a single WSI was estimated using a consumer-grade computer with an NVIDIA RTX 3080 graphics processing unit. Results Within the training cohort, 5-fold cross validation resulted in a mean AUC of 0.90 and standard deviation of 0.04. A final locked model re-trained on the entire training cohort resulted in an AUC of 0.90 upon evaluation of the test cohort ( Figure 1). We optimized the classification threshold to balance sensitivity and specificity; the final diagnostic classification accuracy on the test cohort was 92.3% with a sensitivity and specificity for prePMF diagnosis of 66.6% and 100%, respectively. Upon review of the slides with highest prediction value per class, attention heatmaps highlighted the model's reliance on areas of cellular marrow without reliance on image artifacts or background ( Figure 2). Using affordable consumer-grade hardware, evaluation upon a previously unseen WSI was completed in approximately 6.1 seconds (4.9 for preprocessing and 1.2 for evaluation). Conclusion We developed a novel AI model with high accuracy for distinguishing between prePMF and ET in distinct clinical cohorts. To our knowledge, this study represents the largest image-based AI study within MPNs with external validation. Our proposed model may assist clinicians in appropriately identifying patient cohorts who would benefit from disease-specific therapies or enrollment onto clinical trials. We imagine that a potential high-speed, low-cost algorithm may reliably distinguish prePMF from ET patients with high specificity which can be democratized to the MPN clinical community in routine practice and drive clinical trial accrual for biologically rational novel therapeutics.

A case of prefibrotic primary myelofibrosis in a child with type-Ⅰ CALR gene mutation

A case of prefibrotic primary myelofibrosis in a child with type-Ⅰ CALR gene mutation

Easily Applicable Predictive Score for Differential Diagnosis of Prefibrotic Primary Myelofibrosis from Essential Thrombocythemia.

Essential thrombocythemia (ET) and prefibrotic primary myelofibrosis (prePMF) initially have a similar phenotypic presentation with thrombocytosis. The aim of our study was to determine significant clinical-laboratory parameters at presentation to differentiate prePMF from ET as well as to develop and validate a predictive diagnostic prePMF model. This retrospective study included 464 patients divided into ET (289 pts) and prePMF (175 pts) groups. The model was built using data from a development cohort (229 pts; 143 ET, 86 prePMF), which was then tested in an internal validation cohort (235 pts; 146 ET, 89 prePMF). The most important prePMF predictors in the multivariate logistic model were age ≥ 60 years (RR = 2.2), splenomegaly (RR = 13.2), and increased lactat-dehidrogenase (RR = 2.8). Risk scores were assigned according to derived relative risk (RR) for age ≥ 60 years (1 point), splenomegaly (2 points), and increased lactat-dehidrogenase (1 point). Positive predictive value (PPV) for pre-PMF diagnosis with a score of ≥points was 69.8%, while for a score of ≥3 it was 88.2%. Diagnostic performance had similar values in the validation cohort. In MPN patients with thrombocytosis at presentation, the application of the new model enables differentiation of pre-PMF from ET, which is clinically relevant considering that these diseases have different prognoses and treatments.

Distinctive Attributes of Indian Patients With Classical BCR::ABL1 Negative Myeloproliferative Neoplasms: Unified Clinical and Laboratory Data.

We report one of the largest single center data from a mixed referral setting in India describing baseline characteristics and outcomes of patients with classical BCR::ABL1 negative myeloproliferative neoplasms (MPNs). Patients diagnosed from June 2019 to 2022 were included. Workup and treatment was as per current guidelines. Diagnosis comprised polycythemia vera (PV) in 51(49%), ET in 33(31.7%) and prefibrotic primary myelofibrosis (MF) pre fibrotic myelofibrosis (prePMF) and myelofibrosis in 10(9.6%) patients each. Median age at diagnosis was 52 years for PV and ET, 65.5 for MF and 79 years for prePMF. Diagnosis was incidental in 63(56.7%) and after thrombosis in 8(7.2%) patients. Baseline next generation sequencing (NGS) was available for 63(60.5%) patients. Driver mutations in PV: JAK2 in 80.3%; in ET: JAK2 in 41%, CALR in 26%, MPL in 2.9%; in prePMF JAK2 in 70%, CALR in 20%, MPL in 10%, and in MF: JAK2 in 10%, MPL in 30% and CALR in 40%. Seven novel mutations were detected of which 5 were potentially pathogenic on computational analysis. After median follow up of 30 months, 2 patients had disease transformation and none had new episodes of thrombosis. Ten patients died, most commonly with cardiovascular events(n=5,50%). Median overall survival was not reached. Mean OS time was 10.19 years(95%CI, 8.6 to 11.74) and mean time to transformation was 12.2 years(95% CI,11.8 to 12.6). Our data indicates comparatively indolent presentation of MPNs in India with younger age and lower risk of thrombosis. Further follow up will enable correlation with molecular data and guide modification of age based risk stratification models.

Molecular prognostication in Ph-negative MPNs in 2022.

The application of genomic techniques, including cytogenetics and DNA sequencing, to decipher the molecular landscape of patients with myeloproliferative neoplasms (MPNs) has radically modified diagnostic approach and management through improved risk stratification. Three driver mutated genes (JAK2, MPL, CALR) are variably harbored by >80% of patients and associated with clinical characteristics, as well as major disease-related complications and different survival outcomes. Therefore, JAK2 V617F mutation is included in the revised International Prognosis Score of Thrombosis for Essential Thrombocythemia score for prediction of thrombosis in patients with essential thrombocythemia and prefibrotic primary myelofibrosis, while a CALR type 1 mutated genotype constitutes a favorable variable for survival in patients with myelofibrosis (MF). Novel, integrated clinical and cytogenetic/mutation scores (Mutation-Enhanced International Prognostic Score System for Transplantation-Age Patients with Primary Myelofibrosis [MIPSS70/v2], genetically inspired prognostic scoring system [GIPSS], Myelofibrosis Secondary to PV and ET- Prognostic Model [MYSEC-PM]) have been devised that guide selection of stem cell transplantation candidates with MF or help predict the risk associated with the transplant procedure (Myelofibrosis Transplant Scoring System), with greater performance compared with conventional scores based on hematologic and clinical variables only. On the other hand, several clinical needs remain unmet despite the great amount of molecular information available nowadays. These include the prediction of evolution to acute leukemia in a clinically actionable time frame, the identification of patients most likely to derive durable benefits from target agents, in primis JAK inhibitors, and, conversely, the significance of molecular responses that develop in patients receiving interferon or some novel agents. Here, we discuss briefly the significance and the role of genomic analysis for prognostication in patients with MPNs from a clinician's point of view, with the intent to provide how-to-use hints.

Neutrophil to Lymphocyte Ratio (NLR) at Diagnosis Is a Predictor of Venous Thrombosis and Poor Outcome in Patients with Prefibrotic Primary Myelofibrosis

Background Thrombosis is a common complication in patients (pts) with myeloproliferative disease: it represents the product of a dysfunctional interaction between erythrocytes, platelets, leukocytes and endothelium. A pro-inflammatory microenvironment appears to be associated with an increased thrombotic risk. Neutrophil-to-lymphocyte ratio (NLR) reflects the balance between systemic inflammation and immunity and is emerging as a prognostic biomarker in several diseases, including hematological ones. Recently Carobbio et al. [BCJ, 2022] described NLR as a predictor of venous thrombosis in Polycythemia Vera. No score was specifically developed to assess thrombotic risk in myelofibrosis (MF), and even less is known in the setting of prefibrotic primary myelofibrosis (pre-MF). Aims In a multicenter cohort of pre-MF pts, we retrospectively investigated the relationship between NLR values at diagnosis and the risk of: 1) thrombosis 2) disease progression into overt-MF, accelerated (AP) or blast phase (BP); 3) overall survival (OS). Methods We collected clinical, histopathological and hematological data of 176 pts with a diagnosis of pre-MF, revised according to 2016 World Health Organization criteria, followed at 4 Hematological Centers between 2002 and 2022. NLR values were calculated using data obtained from the complete blood count at diagnosis, as the ratio between absolute neutrophilic count (ANC) and absolute lymphocyte (ALP) count. Pts were dichotomized using the best threshold identified by ROC analysis. Thrombotic complications included arterial (ischemic stroke, myocardial infarction, peripheral arterial disease) and venous (deep vein thrombosis, pulmonary embolism, cerebral and splanchnic vein thrombosis) events. Thrombosis-free survival (TFS) and OS were determined from pre-PMF diagnosis to the first thrombotic event and to death or last contact, respectively. TFS/OS were estimated by Kaplan-Meier analysis; the log-rank test was used to compare TFS/OS differences between risk stratified groups. Results Overall, 176 pts were included in this study. Median age was 63.6 (range 25-80) years; 88 (50%) were male. Majority of pts were JAK2V617F mutated (76,1%). Median NLR value at diagnosis was 3 (range 0,64-41,5). By ROC analysis, 6 was identified as the best cut-off value to dichotomize pts based on NLR; 23 pts (13%) presented NLR >6 at diagnosis. A major thrombotic event occurred before or at diagnosis in 53 pts (30,1%): 29 arterial and 25 venous (one patient had both a venous and arterial event). During the follow-up (FU), 30 pts (17%) developed a thrombosis, for a total of 38 thrombotic events (20 venous and 18 arterial events, accounting for an incidence rate of 1,67 and 1,50 per 100 pts-year, respectively). In statistical analysis, factors significantly associated to thrombosis during the FU were previous thrombosis (p=0.05) and JAK2 V617F mutation (p=0.03). Total leucocyte and platelets count, as well as age, sex and IPSET thrombosis score did not correlate with thrombotic events. Pts with ANC > 9 x10^9/L or ALP count < 1,5x10^9/L at diagnosis showed a trend towards a greater incidence of thrombosis in FU (p=0.06 and p=0.07, respectively). Pts with NLR value > 6 at diagnosis had a significantly higher incidence of venous thrombotic events (p=0.03) and a shorter venous TFS (p=0.004, Figure 1) compared to patients with NLR <6. The NLR value was not predictive of arterial thrombotic risk. After a median FU of 6.14 years (range 0,5-27,96) years, 32 pts (18,2%) died; 22 pts (12,5%) had disease progression: of them, 13 (7,4%) experienced overt MF and 9 (5,1%) evolved into an AP or BP. Pts with NLR value > 6 at diagnosis had a significant shorter OS compared to the group with NLR <6 (median OS 4,93 vs 6,22 months respectively, p=0.02). Conclusions This multicenter study investigated for the first time whether the NLR value at diagnosis could be a predictive marker of thrombotic risk and poor outcome in pre-PMF setting. Previous thrombosis and JAK2V617F mutation are confirmed to be risk factors for thrombotic events also in pre-MF setting. Notably, NLR > 6 at diagnosis emerged as a simple marker to identify pts at higher risk of thrombosis and poor outcome in the pre-PMF setting: pts with a NLR value > 6 have a significantly higher venous thrombotic risk and a shorter venous TFS. Furthermore, NLR group > 6 appears to have a shorter OS. These data are promising but require to be confirmed on a larger scale. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clinical Features and Outcomes of Pre-Fibrotic Primary Myelofibrosis Patients with Triple-Negative, JAK2V617F and Calr Mutations

The 2016 WHO classification recognized pre-fibrotic primary myelofibrosis (pre-PMF) as a distinct entity. However, little is known about the clinical characteristics and prognosis of pre-PMF patients with JAK2V617F mutations, CALR exon 9 mutations, and triple-negative (TN) mutations. Thrombotic prognostic model specific for pre-PMF is still lacking and the applicability of International Prognostic Score for Thrombosis in Essential Thrombocythemia (IPSET-thrombosis) needs further validation in pre-PMF patients. We conducted a retrospective study of 168 pre-PMF patients defined by 2016 WHO diagnostic criteria in the First Affiliated Hospital of Zhejiang University School of Medicine and the Fourth Affiliated Hospital of Zhejiang University School of Medicine (ChiCTR2200061208). We retrospectively evaluated the clinical characteristics, prognosis of hemorrhage and thrombosis, and the evolution risk to overt-PMF and acute leukemia in 168 pre-PMF patients. We also assessed the risk factors for thrombosis events and validated the availability of IPSET-thrombosis scoring system in pre-PMF patients. Among 168 pre-PMF patients, 98 (58.3%) harbored JAK2V617F, 22 (13.1%) CALR and 2 (1.2%) MPL mutations; 46 (27.4%) patients were negative for all three mutations. Compared to JAK2V617F-mutated cases, TN pre-PMF patients were younger (P<0.001), had fewer constitutional symptoms (P=0.003), lower white blood cell count (WBC) (P=0.01), hemoglobin level (HB) (P=0.007), mean platelet volume (MPV) (P=0.03), hematocrit value (HCT) (P=0.045), red blood cell distribution width (RDW) (P<0.001) and lactic dehydrogenase (LDH) (P=0.021). They also had fewer thrombotic events (1, 2.2%) compared to JAK2V617F group (25, 26.3%) and CALR group (5, 22.7%) during the follow-up and at diagnosis (P<0.001) (Figure A). A proportion of 7.1% and 18.2% progressed to overt-PMF in JAK2V617F and CALR group, respectively, while no TN pre-PMF patients developed overt-PMF progression (0, 0.0%) (P=0.015) (Figure B). We also found, although not statistically significant, a lower incidence of hemorrhagic events (2% vs. 12%) and acute leukemia (2% vs. 5%) in TN pre-PMF groups compared with JAK2V617F pre-PMF groups (Figure C, Figure D). As for risk factors for thrombosis, univariable analysis found thrombosis was associated with constitutional symptoms (P=0.003), thrombosis history (P<0.001), smoking status (P=0.007) and JAK2V617F mutations (P=0.008). Patients with higher HB (P=0.013), MPV (P=0.042) and HCT (P=0.040) were also more likely to develop thrombotic events. In multivariable analysis, smoking history (P=0.036; HR 0.902, 95% CI 0.05-0.212), higher HB value (P=0.048; HR 1.336, 95% CI 1.001-1.156) and thrombosis history (P=0.001; HR 0.313, 95% CI 0.009-0.053) had negative impact on the development of thrombosis events (Figure E). When divided by HB value with cut-off point of 144.5, subjects with HB value <144.5 g/L were less likely to develop thrombosis events compared to ≥144.5 g/L (P=0.003) (Figure F). And subjects with no history of smoking were less likely to develop thrombosis events compared to subjects with a history of smoking (P=0.004) (Figure G). We also validated IPSET-thrombosis scoring system in pre-PMF patients, results showed significant statistical difference among very low-, low-, intermediate- and high-risk groups (P<0.001) (Figure H). In conclusion, TN pre-PMF patients were younger, had fewer constitutional symptoms, lower WBC, HB, MPV, HCT, RDW and LDH, and less likely to develop thrombotic events and overt-PMF compared to JAK2V617F and CALR groups. TN pre-PMF patients with smoking history, higher HB and thrombosis history had higher risks of thrombosis events. And IPSET-thrombosis scoring system was applicable in stratifying thrombosis risks of pre-PMF patients. Acknowledgment: The research was supported by the Key R&D Program of Zhejiang, No. 2022C03137; Public Technology Application Research Program of Zhejiang, China, No. LGF21H080003; the Key Project of Jinhua Science and Technology Plan, China, No. 2020-3-011; the 2019-2022 Key Medical Discipline (Hematology) Fund of Jinhua, China. Correspondence to: Dr Jian Huang, Department of Hematology, The First Affiliated Hospital of Zhejiang University School of Medicine. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal