Reichard, Kaaren K.

Abstract Objectives VEXAS syndrome is an adult-onset autoinflammatory disease caused by a somatic pathogenic mutation in the UBA1 (ubiquitin-like modifier activating enzyme 1) gene. Patients present with rheumatologic manifestations and cytopenias and may have an increased predisposition to myelodysplastic syndrome (MDS) and plasma cell neoplasms. Prior studies have reported on the peripheral blood and bone marrow findings in patients with VEXAS syndrome. Due to the protean clinical presentation and lack of specificity of morphologic features (eg, vacuoles in early erythroid and granulocytic precursors), an optimal screening methodology to identify these patients in a timely fashion is desirable. Methods To further evaluate and describe the salient diagnostic morphologic features in VEXAS syndrome, we carried out a comprehensive study of the largest single-institution cohort to date. Diagnostic and follow-up bone marrow biopsy specimens from 52 male patients with molecularly identified VEXAS syndrome underwent central review. Results Cytopenias were common in all cases, primarily macrocytic anemia, monocytopenia, and thrombocytopenia. Bone marrow aspirate and biopsy were often hypercellular, with an increased myeloid/erythroid ratio, granulocytic hyperplasia with left shift, erythroid left shift, and megakaryocyte hyperplasia, which exhibited a range of striking morphologic findings. Distinctly vacuolated myeloid and erythroid precursors were seen in more than 95% of cases. Conclusions Our data reveal potential novel diagnostic features, such as a high incidence of monocytopenia and distinct patterns of atypical megakaryopoiesis, that appear different from dysmegakaryopoiesis typically associated with MDS. In our experience, those findings are suggestive of VEXAS, in the appropriate clinical context.

SummaryAmong 210 patients with myelodysplastic syndromes (MDSs) with del(5q), molecular information was available at diagnosis or at least 3 months before leukaemic transformation in 146 cases. Multivariate analysis identified therapy‐related setting (p = 0.02; HR 2.3) and TP53 variant allele frequency (VAF) ≥22% (p < 0.01; HR 2.8), but not SF3B1 mutation (p = 0.65), as independent risk factors for survival. Median survival was 11.7 versus 4 years (5/10‐year survival 73%/52% vs. 42%/14%) in the absence (N = 112) versus presence (N = 34) of ≥1 risk factors; leukaemia‐free survival was affected by TP53 VAF ≥22% (p < 0.01). Such information might inform treatment decision‐making in MDS‐del(5q) regarding allogeneic stem cell transplant.

SummaryMastocytosis constitutes the neoplastic proliferation of mast cells and is broadly classified into systemic mastocytosis (SM), cutaneous mastocytosis and mast cell sarcoma. SM is further partitioned into advanced (AdvSM) and non‐advanced (SM‐non‐Adv) subcategories. AdvSM includes aggressive SM (ASM), SM with an associated haematological neoplasm (SM‐AHN) and mast cell leukaemia (MCL). In 2022, two separate expert committees representing the 5th edition of the World Health Organization (WHO5) and the International Consensus (ICC) classification systems submitted revised classification criteria for SM, highlighted by the ICC‐proposed incorporation of mast cell cytomorphology in the diagnostic criteria for MCL and myeloid‐lineage restriction for the AHN component in SM‐AHN. Recent developments in SM also include the introduction of KIT‐targeting tyrosine kinase inhibitors (KITi), including midostaurin and avapritinib, both drugs have shown potent activity in reducing mast cell and mutant KIT burden and alleviating mast cell‐associated organopathy and mediator symptoms; however, their overall impact on survival or superiority over pre‐KITi era treatment options (e.g. cladribine) has not been studied in a controlled setting. In the current review, we provide a summary of recent changes in disease classification and an analysis of recent clinical trials and their impact on our current treatment approach in AdvSM.

BACKGROUND: Recent World Health Organization (WHO) classifications (5 th edition), International Consensus Classification (ICC) recognized TP53-mutated ( TP53mut) myeloid neoplasms (MNs) as a distinct subcategory, acknowledging their uniformly poor outcomes. However, there are critical differences between the two guidelines in how TP53 allelic status is assigned. Both WHO-5 and ICC acknowledged the poor outcome of multi-hit TP53mut but excluded single-hit TP53mut. These changes are driven by a pivotal study demonstrating favourable outcome of single-hit TP53mut, however this study predominantly included de novo myelodysplastic syndromes (MDS) cases (Bernard et al. Nat Med 2020). In contrast, we previously reported poor outcome of TP53mut therapy-related myeloid neoplasm (t-MN) irrespective of the allelic status of TP53 (Hiwase et al Blood 2022andShah et al BCJ 2023). Finally, the ICC removed the subcategory of “therapy-related,” substituting it with diagnostic qualifiers instead. The WHO has grouped t-MN with secondary MN and renamed it as “myeloid neoplasm post cytotoxic therapy,” with the assertion that only multi-hit T P53mut MDS occurring post-cytotoxic therapy have poor outcome compared with single-hit. METHODS: We analyzed one of the largest international cohorts of TP53mut MN (n=544) with full characterization of TP53mut allele status utilizing uniform ICC criteria and similar large numbers of de novo and t-MN cases managed at Mayo Clinic Enterprise (USA) and South Australian MDS Registry, Adelaide (Australia) with clinical, integrated genomic profiling, and long-term follow-up. RESULTS: In our cohort of TP53mut MN (n=544), 475 (87.3%) MN had TP53mut variant allele frequency (VAF) of ≥10% and thus satisfied ICC criteria for TP53mut MN. Majority of TP53mut t-MN presented with MDS (bone marrow (BM) blasts 0-9%) compared de novo MN (62% vs. 48%; P=0.0012). While acute myeloid leukemia (AML) (BM blasts ≥20%) were more prevalent in TP53mutde novo MN compared to t-MN (38% vs. 24%; P= 0.001) and there was no difference in frequency of MDS/AML (BM blasts 10-19%) between the two cohorts (14% vs. 14%; P=0.8) ( Figure 1A). Majority of TP53mut were observed in the DNA-binding domain in t-MN and de novo MN (95% vs. 96.2%; P=0.48). However, mutations in Tetramer Domain were more frequent in t-MN (4.3% vs. 0.3%; P=0.002), whereas those in the TAD2 domain were more frequent in de novo TP53mut MN (2.4 vs. 0%; P =0.006). Integrating the mutation, conventional G-banding cytogenetic, copy number variation (CNV), FISH, and SNP array analysis, TP53mut MDS (BM blast 0-9%; n=260) were classified as multi-hit (n=179, 68.9%) or multi-equivalent (n=35, 13.5%) and single-hit (n=46, 17.7%) (Arber et al. Blood 2022). Striking enrichment of multi-hit/multi-hit equivalent was observed in TP53mut t-MDS compared to de novo MDS (91% vs. 72%; P&lt;0.0001). While single-hit TP53mut were prevalent in de novo MDS compared to t-MDS (28% vs. 9%; P&lt;0.0001). Importantly, median overall survival (OS) of single-hit was significantly longer than multi-hit de novo MDS (25.4 vs. 7.2 months; P=0.0002). In contrast to this we did not observe OS difference between single-hit and multi-hit TP53mut t-MDS (11.6 vs. 10.9 months; P =0.73) ( Figure 1B). CONCLUSION: Our study based on a large international cohort of T53mut MN provides compelling evidence that in contrast to de novo MDS, single hit TP53mut t-MDS is associated with poor outcome. Our data supports the consideration of single hit t-MDS as a multi-hit equivalent.

Background: Clonal cytopenia(s) of undetermined significance ( CCUS) is defined as persistent cytopenias arising in the context of myeloid neoplasm ( MN)-associated somatic mutations ( MT) in hematopoietic stem cells. Patients ( PTs) with TET2MT CCUS have a high probability of progression to MN. To date, no FDA-approved therapies exist for CCUS, and PTs often have similar cytopenias/transfusion needs as those with myelodysplastic syndromes ( MDS). TET2 is an ascorbic acid (AA)-dependent dioxygenase that catalyzes the conversion of 5-methylcytosine ( 5mC) to 5-hydroxymethylcytosine ( 5hmC), providing rationale to study high-dose IV (HI)-AA in TET2MT CCUS. Here we report the final results of this pilot trial. Methods: This is an investigator-initiated, single-institutional, phase II trial assessing the safety and efficacy of HI-AA in PTs with TET2MT CCUS (NCT03418038). PTs≥ 18 years with ≥1 TET2MT with or without additional somatic MTs, without prior treatment, and with any of the following laboratory criteria: (1) hemoglobin ( Hb) ≤10g/dL, (2) absolute neutrophil count ( ANC) ≤1(10^9/L), (3) platelet count ( PLT)≤100 (10^9/L) were eligible. HI-AA (1g/kg, maximum 100g) was given 3 times weekly for 12 weeks. The primary endpoint was hematologic response rates determined by MDS IWG 2018 criteria at week 20. (Platzbecker Blood 2018) and reported as hematologic improvement ( HI)- erythropoietic ( HI-E), platelets ( HI-P), neutrophils ( HI-N). Secondary endpoints include safety and adverse events ( AEs), graded by NCI-CTCAE v4.03. Correlative studies include changes in TET2MT variant allele fraction ( VAF), in vitro colony formation and differentiation, global and sequence-specific DNA methylation/hydroxymethylation, and quantitative 5hmC estimated by IHC in bone marrow biopsy specimens. Results: Ten patients were enrolled, with a median age of 71.4 (range: 65, 79) years, 8 (80%) males. The median number of mutations was 3, with 9 (90%) having co-mutations. All PTs but one [-Y] had normal karyotype. Baseline Hb levels were normal in all but one who was red blood transfusion dependent. Five (50%) PTs were thrombocytopenic, and 5 (50%) PTs had ANC&lt;1 (10^9/L). ( Table). HI-AA was well tolerated with the most common AEs being infusion-related polyuria (40%) and polydipsia (40%, 3 Grade 1, 1 Grade 2). One (10%) PT had constipation and headaches (both Grade 1), and 1 (10%) experienced dyspepsia. No treatment-related Grade 3 or 4 AEs' or deaths were reported. The median follow-up duration was 16.8 months (range: 9.4, 24.1). Overall, no PTs met the criteria for HI. There were no significant differences with regards to median Hb, PLT, and ANC values at baseline, week 20, and 1-year (median Hb: 13.3, 12.5, and 12.7 g/dL, p=0.93; PLT: 97 vs. 120 vs. 92.5X10^9/L, p&gt;0.99, and ANC: 0.9 vs. 1.2 vs. 1.3X10^9/L, p=0.99). By CTCAE grading criteria for cytopenias, 3 (30%) PTs had an improvement in severity of cytopenias, while 2 had worsening. One (10%) PT had an improvement in PLT (baseline vs. week 20: 62 vs 192X10^9/L) but was morphologically deemed to meet criteria for CMML-1 at week 20. Four (40%) PTs met criteria for disease progression (PT_4, 7,8,10); CMML-1- 2 and MDS-2. The median time to progression was 5 months (95% CI: 4.9, unreached). Two PTs had mutational clonal evolution, with 1 (PT_8) acquiring a RUNX1 (VAF: 20%) and 1 (PT_9) acquiring 2 CBL mutations (both VAF:4%) at one-year follow-up. There were no significant differences regarding the clinical characteristics or the TET2 variants between PTs who had stable disease vs. progression. There were no significant differences in TET2 VAF at baseline, week 20, and 1-year (median: 39% vs. 43% vs. 42%, p=0.54). We profiled changes in 5-mC and 5-hmC (Ilumina EpicArray) in PTs pre- and post-IV AA treatment and did not observe global changes. However, we were able to highlight site-specific changes in differentially methylated regions, largely resulting in hypomethylation at enhancer sites and affecting actively transcribed states in PTs with an improvement in cytopenia(s) severity by CTACE criteria ( Figure). Conclusion: We report the final safety and efficacy analysis of HI-AA in PTs with TET2 MT CCUS. The trial data suggest that HI-AA is safe and well tolerated. Although there were no significant responses by IWG MDS criteria, alleviation in severity of cytopenias in a subset of PTs (n=3) along with correlative epigenetic changes in enhancer regions, needs further exploration.

Introduction: Clonal dendritic cell (DC) aggregates in chronic myelomonocytic leukemia (CMML) are seen in approximately 20-30% patients at diagnosis. Previously, these were thought to be comprised of CD123+ plasmacytoid DCs but our data has suggested that these are heterogenous and comprise of myeloid DCs, monocytes, and myeloid-derived suppressor cells. Furthermore, these aggregates uniformly express immune checkpoints such as indoleamine 2,3-dioxygenase-1 (IDO), associate with an IDO-specific systemic impact (increased trytophan catabolism), and expanded regulatory T cell population in CMML, suggesting a role in inducing immune tolerance. DC aggregates are also associated with increased risk of acute myeloid leukemia (AML) transformation and resistance to hypomethylating agents, highlighting that they are important for disease progression in CMML (Mangaonkar A et al. Blood Adv 2020). We performed this study to decipher the architecture of a CMML DC aggregate to understand the complex inter-cellular interactions, and assess their impact on the adaptive immune system. Methods: In this study, we used archival fresh-frozen paraffin-embedded (FFPE) bone marrow biopsy specimens obtained from CMML patients, and isolated monocytes (CD14+ magnetic bead sorting) and lymphocytes (CD14-ve fraction) from untreated CMML and age-matched normal control (NC) peripheral blood mononuclear cells (PBMCs). DCs were cultured from monocytes using a commercial protocol. Spatial architecture of the CMML BM microenvironment was interrogated through multiplex imaging of FFPE samples by co-detection by indexing (CODEX) using 30 markers (CCR6, CD107a, CD11c, CD20, CD21, CD31, CD3e, CD4, CD44, CD45, CD45RO CD56, CD68, CD8, E-Cadherin, FoxP3, HLA-DR, CD14, CD16, CD11b, CD33, CD15, CD66b, CD123, CD303, CD34, CD163 and IDO). Single-cell transcriptomic signatures were analyzed using the 10X single cell Fixed RNA profiling platform, and validated using bulk RNA seq (Illumina stranded mRNA prep) from isolated and cultured cells. Impact of DCs on lymphocytes was assessed via autologous mixed DC-T culture experiments. Results: BM FFPE samples from 5 CMML cases (3 with DC aggregates) were included for multiplex imaging via CODEX. Results confirm the significant interactions of CD8+ and CD4+ T cells with CD123+/HLA-DR+ DCs within a CMML DC aggregate (Fig. 1A). Upon quantitative assessment of cell-cell interactions, IDO+/CD8+T ( P=0.006), IDO+/MDSC ( P=0.005), and IDO+/macrophage ( P=0.001) interactions were significantly increased in CMML samples with versus without DC aggregates . Specific findings were validated using single-stain immunohistochemistry and multiplex immunoflourescence staining. Next, in order to assess transcriptomic signatures within BM at the single-cell level, we chose two cases; CMML (BM aspirate blast% 1, WBC 49.6 x 10 9/L, ASXL1/GATA2/SETBP1/SRSF2 mutations) and AML transformed from CMML (BM aspirate blast% 81, WBC 152 x 10 9/L, NPM1/DNMT3A/TET2 mutations). Approximately, 10,000 cells were obtained and analyzed with cell type annotation through human PBMC reference (Azimuth). Results show an expanded regulatory T cell population in AML compared to CMML (UMAP plot, Fig. 1B), suggestive of immune tolerance. In order to validate findings and perform functional experiments, we used sorted monocytes and cutured DCs from CMML and NC. DC phenotype was confirmed both through visual inspection and flow cytometry, and clonality was confirmed through Sanger sequencing (for previously identified mutations). Differential gene expression analysis through bulk RNAseq showed significant differences between CMML and NC lymphocytes with immunoinhibitory receptor, LILRB2 (log2fold change=7.05, adjusted P=0.0002) being one of the top five significantly upregulated genes, further confirming immune tolerance. Finally, mixed autologous DC-lymphocyte reaction (1:4 ratio) experiments demonstrated that DCs induce T cell tolerance in CMML (expansion of exhausted PD1/TIM3/LAG3+ CD8 T cells) when compared to NC. Conclusion: Clonal dendritic cell aggregates within the CMML bone marrow microenviroment interact with T cells and induce immune tolerance. This represents a novel mechanism of disease progression in CMML with potential therapeutic targets (IDO, LILRB2, and/or CD123).

Background: Sodium-glucose co-transporter-2 inhibitors (SGLT-2I) are FDA-approved for diabetes mellitus, heart failure and chronic renal disease. Clinical trials on their anti-diabetic and cardiorenal protective effects showed an association with erythrocytosis. We have previously reported on the benign nature of SGLT-2I associated JAK2-unmutated erythrocytosis ( Blood 2021). Recently, we described the same phenomenon in the setting of a previously unrecognized myeloproliferative neoplasm (MPN), which led to unmasking of underlying MPN and thrombosis in 3 (33%) of 9 informative cases ( AJH 2023). The main objective of the current study was to assess the erythropoietic and thrombotic impact of SGLT-2I therapy in patients with a well-established MPN diagnosis, prior to treatment initiation. Methods: The Mayo Clinic MPN database was queried to identify patients prescribed SGLT2-I after diagnosis of essential thrombocythemia (ET), polycythemia vera (PV) or myelofibrosis (MF). Baseline Hgb/Hct at SGLT-2I initiation, peak Hgb/Hct, absolute change in Hgb/Hct levels, and thrombosis during treatment were recorded. Results: SGLT-2I use in essential thrombocythemia Eleven patients with ET (median age; 72 years, 55% males, 73% JAK2 mutated, 64% with thrombosis history) received SGLT-2I. Active treatments included aspirin ( n=10), cytoreductive therapy ( n=10) and systemic anticoagulation ( n=4). All 11 patients displayed increase in Hgb/Hct following SGLT-2I treatment. Baseline median (range) values for Hgb/Hct were 12.8 g/dL (9.5-14.9)/38.9% (30.3-44.6); median (range) peak Hgb/Hct levels during SGLT-2I therapy, were 14.5 g/dL (11-16.4)/44.4% (34.8-51.1), with median (range) increase in Hgb/Hct of 1.5 g/dL (1-4)/5.1% (2.6-13.8); 2 patients (18%), exceeded the proposed ICC Hgb/Hct diagnostic threshold for PV (Patient 7 and 8) (Figure 1). JAK2 mutation/VAF (p=0.54/ 0.20), or serum erythropoietin (Epo) (p=0.48) did not correlate with changes in Hgb/Hct levels. One patient experienced thrombosis, despite treatment with hydroxyurea and aspirin and Hgb/Hct levels below the threshold of concern (11.4 g/dL/37.7%). SGLT-2I use in polycythemia vera Nine patients with PV (median age 74 years, 66% males, JAK2 median VAF 14%, two patients with thrombosis history) received SGLT-2I. Concomitant treatments included aspirin ( n=9), cytoreductive therapy ( n=8), and systemic anticoagulation ( n=3). Phlebotomy intensity, defined as the number of phlebotomies, six months prior to and after SGLT-2I initiation, ranged from 0-3 and 0-12, respectively. Documented Hgb/Hct levels showed an increase in all 9 cases with baseline median (range) values for Hgb 14.1 g/dL (10.5-15.9) and Hct 42.7% (33.3-49.2) and peak median (range) values for Hgb/Hct, 15.4 g/dL (11.2-19.9)/46.7% (35.9-63.3). JAK2 VAF (p=0.85), and serum Epo (p=0.43) did not correlate with increase in Hct. Figure 2 highlights five patients (56%) with Hct exceeding 45% while on SGLT-2I; two patients (22%) had increased phlebotomy requirements; 6-month pre/post SGLT-2I phlebotomies for patient 5 and 9 were 3 and 6, and 0 and 12, respectively. Patient 9, experienced thrombosis in the context of uncontrolled Hgb/Hct level at 19.9 g/dl/63.3% and absence of cytoreductive therapy. SGLT-2I use in myelofibrosis Eight patients with myelofibrosis (primary 6, post-ET 2; median age 71 years; 50% males; 63% JAK2 mutated; MIPSS-70 2.0 int/high 6; on ruxolitinib 4),received SGLT-2I. Baseline median (range) values for Hgb/Hct were 10.6 g/dL (8.2-14.7)/27.5% (33.8-45.9); two (25%) patients displayed anemia with Hgb 8-10 g/dl. Peak median (range) values for Hgb/Hct were 11.5 g/dL (10.2-16.1)/35.3% (31.8-49.8), with median (range) baseline increase in Hgb/Hct of 1.6 g/dL (0.7-3.8)/4.3% (1-7.5). Patient 2 and 4 with baseline Hgb 8.2 g/dl and 9.4 g/dl, and not receiving ruxolitinib, had Hgb increase of 2 g/dl and 1 g/dl, respectively (Figure 3). One thrombotic event occurred during therapy with Hgb/Hct level of 11.6 g/dl/37.3%. Conclusion: The current study suggests that SGLT-2I therapy in MPN patients leads to increase in Hgb/Hct levels, regardless of disease subtype, and in some cases surpass the PV diagnosis and thrombosis risk thresholds. These observations mandate increased awareness to secure patient safety and diagnostic accuracy. The erythropoietic effect of SGLT-2I in MF suggests its potential use for MF-associated anemia.

Background: Non-transplant treatment options in myelofibrosis (MF) are palliative in scope and include JAK2 inhibitors (JAKi): ruxolitinib, fedratinib, pacritinib, and momelotinib. All four JAKi are effective in reducing spleen size and alleviating symptoms. In addition, pacritinib and momelotinib exhibit erythropoietic effect, attributed to alleviation of ineffective erythropoiesis through inhibition of activin A receptor type-I. In the current study, we sought to identify molecular or clinical predictors of response to pacritinib in MF patients treated outside of clinical trials. Methods: The current study was conducted under an institutional review board approved minimum risk protocol that allowed retrospective collection and analysis of data from patient records (Mayo Clinic, Rochester, MN; Scottsdale, AZ; and Jacksonville, FL). Anemia response assignment was restricted to patients with baseline hemoglobin level of &lt;10 g/dL and required, for transfusion dependent cases, achieving transfusion independence and, for transfusion-independent cases, an increase in hemoglobin of ≥1.5 g/dL, both sustained for a minimum 2 months; spleen response was clinically vetted but was also supported by radiologic imaging when available and required a ≥50% decrease in palpable splenomegaly for spleen size &gt;10 cm or the spleen becoming not palpable for a spleen size 5-10 cm at base line. Symptom response was adjudicated by central review and required a marked or moderate improvement to qualify as response; because of the retrospective nature of the study, symptom burden assessment by formal criteria was not attempted. Leukocyte cutoff level used for response analysis was determined by receiver operating characteristic curve analysis. Conventional statistical methods were applied using JMP Pro 16.0.0 software (SAS Institute, Cary, NC, USA). Results: We identified 36 NGS-annotated patients with MF who received pacritinib therapy between April,2022 and June,2023 (median age 66 years; 56% males). Twenty-five (69%) patients had failed previous JAKi therapy while 11 (31%) were JAKi-naïve. Eighteen (50%) patients were transfusion-dependent while the median (range) counts were 106 x 10(9)/L (14-578) for platelets, 6.6 x 10(9)/L (1.2-112.8) for leukocytes, and 9 x 10(9)/L (5-12.4) for hemoglobin; leukocyte count was ≥15 x 10(9)/L in 11 (31%) patients and hemoglobin &lt;10 g/dL in 29 (81%). Karyotype was unfavorable in 12 (34%) patients. Driver mutation distribution was JAK2 55%, CALR 27%, MPL 11%, and triple-negative 5%. NGS revealed mutations involving ASXL1 36%, U2AF1 13%, IDH1 13%, SF3B1 13%, K/NRAS 5%, SRSF2 8%, and EZH2 5%. Pacritinib was started at 200 mg BID in 9 (25%) patients and at lower doses in the remaining. Median duration of pacritinib therapy was 4 months (range 1-15). Anemia response was documented in 5 (17%) of the 29 patients with baseline hemoglobin &lt;10 g/dL, spleen response in 12 of 28 evaluable (42%) and symptom response in 18 (50%). Improvement in symptoms was always accompanied by both anemia (p=0.008) and spleen (p=0.01) response; by contrast, there was no significant correlation between anemia and spleen responses (p=0.9) (Table 1). Significant correlations with molecular or clinical variables were mostly apparent for spleen response, which was more likely to occur in the absence of ASXL1 mutation (61% vs 10%; p=0.006), with leukocyte count &lt;15 x 10 (9)/L (55% vs 0%; p=0.005), and in JAKi-naïve vs previously-exposed cases (75% vs 30%; p=0.03; Table 1); significance was sustained by all three risk factors during multivariable analysis; the prognostic impact of ASXL1 (p=0.02 vs 0.01) and leukocytosis (p=0.01 vs 0.07) was apparent in both JAKi-naïve vs JAKi-exposed cases. Anemia response was non-significantly (p=0.27) higher in transfusion-independent (27%) vs dependent (11%) cases, at baseline, and was more likely (p=0.04) to occur in patients starting at lower dose levels. Treatment-emergent adverse events included diarrhea 44%, nausea/vomiting 19%, and grade-3/4 thrombocytopenia 38% or neutropenia (11%). Conclusions: ASXL1 mutation and leukocytosis (&gt;15 x 10 9/L) are independently associated with inferior spleen response to pacritinib, in both JAKi-naïve and JAKi-exposed myelofibrosis. These observations are important for individualized treatment selection.

Background: According to the international consensus classification (ICC), diagnosis of myelodysplastic syndromes (MDS) with del(5q) requires presence of i) del5q, alone or associated with no more than one other cytogenetic abnormality, exclusive of -7/del(7q), ii) &lt;5% bone marrow (BM) and &lt;2% circulating blasts, and iii) the absence of “multi-hit” TP53 ( Arber et al. Blood 2022;140: 1200). We have recently reported on TP53 variant allele frequency (VAF) ≥22% being the sole genetic predictor of inferior survival in MDS-del(5q) (Fleti. et al. AJH 2023;98:E76). In the current study, we examined the additional prognostic impact of therapy-related setting, in the context of TP53 VAF and other risk factors. Methods : The current study included patients with MDS-del(5q) who were seen at the Mayo Clinic or Moffitt Cancer Center, USA. Diagnosis of MDS-del(5q) and therapy-related qualification was according to ICC criteria ( Arber et al. Blood 2022;140: 1200), with the exception that patients with biallelic or multi-hit TP53 were included to allow unbiased analysis of impact on prognosis. Patient selection was based on availability of mutation information from next-generation sequencing (NGS). The latter was assessed either at diagnosis or at different time points during the disease course, but at least 3 months before leukemic transformation. Otherwise, all analyses, including estimation of overall and leukemia-free survival, were based on variables collected at time of initial diagnosis of MDS-del(5q). TP53 mutation VAF threshold of 22% was considered based on our previously published report cited above. Age cutoff at 67 years was determined by ROC analysis. Conventional statistical methods were applied using JMP Pro 16.0.0 software (SAS Institute, Cary, NC, USA). Results: A total of 210 patients (median age 72 years; 65% females), meeting the above-stipulated criteria for MDS-del(5q), were considered: 172 primary and 38 therapy-related. Table 1 outlines clinical and laboratory characteristics obtained at the time of diagnosis: 42% were transfusion-dependent, 69% had hemoglobin level &lt;10 g/dL, 30% platelet count &lt;150 x 10(9)/L, and 15% carried additional cytogenetic abnormality. NGS data performed either at diagnosis or at least 3 months prior to leukemic transformation was available in 146 patients; the most frequent mutations were TP53 (21%), SF3B1 (16%), DNMT3A (12%), TET2 (12%), ASXL1 (10%), and JAK2 (7%) (Table 1). At a median follow-up of 3.9 years (range 0.002-18.5) for living patients, 95 (45%) deaths and 34 (16%) leukemic transformations were recorded; treatment undertaken included lenalidomide in 162 (77%) cases. NGS data-exclusive multivariable analysis that included all 210 patients identified therapy-related setting (p&lt;0.01; HR 2.9; Figure 1a), age &gt;67 years (p=0.01; HR 1.8), transfusion-dependency (p=0.04; HR 1.5), and platelet count &lt;150 x 10(9)/L (p=0.03; HR 1.6), as independent predictors of inferior survival. The significant effect of age and platelet count was sustained when analysis was limited to patients with primary MDS-del(5q) but lost when applied to therapy-related disease. NGS data-inclusive multivariable analysis, which was applicable to the subset of 146 patients, identified, as risk factors, therapy-related setting (p&lt;0.01; HR 2.3) and TP53 VAF ≥22% (p&lt;0.01; HR 2.4; Figure 1b) while age &gt;67 years sustained borderline significance (p=0.07; HR 1.8). Figure 1c depicts survival data based on the presence or absence of TP53 VAF ≥22% and therapy-related qualification; low-risk with no risk factors (N=112; median survival 11.7 years; 5/10-year survival 73%/52%) and high-risk with one or both risk factors (N=34; median survival 4 years; 5/10-year survival 42%/14%); leukemia-free survival was affected by TP53 VAF ≥22% (p&lt;0.01) but not therapy-related setting (p=0.13). Conclusions : The current study highlights TP53 VAF and therapy-related setting as the most prominent risk factors for survival in MDS-del(5q) and proposes a simple risk model that distinguishes patients with a projected 10-year survival that exceeds 50%, in the absence of both risk factors, and plummets to 14%, in the presence of one of the two risk factors. Accordingly, allogeneic stem cell transplant might be deferred in the former instance while it should be pursued sooner than later, in the latter instance.