Dunbar, Andrew

Abstract Background Myeloproliferative neoplasms (MPNs) can progress from chronic phase to accelerated or blast phase (MPN-AP/BP), a transition with poor prognosis and limited treatment options. While some abnormalities have been described, the genomic and clonal drivers of transformation remain poorly defined. We characterized mutational and structural alterations in MPN-AP/BP, leveraging paired samples to trace clonal evolution. Methods We profiled 186 samples from 160 individuals, including 159 MPN-AP/BP and 27 chronic-phase MPN samples (26 MF, 1 PV), using targeted sequencing of a 588 gene panel (mean depth: 709x). Paired pre- and post-transformation samples were available for 26 patients (n= 52). A consensus pipeline was used to detect somatic mutations, copy number variations (CNVs), and copy-neutral loss of heterozygosity (cnLOH). Clinical cytogenetic data were integrated when available. Single-cell validation is ongoing. Results Genomic complexity was widespread: 99% of patients harbored ≥1 oncogenic mutation (median 4; range 1–10). In total, 859 mutations were identified, most commonly in JAK2 (68%), ASXL1 (28%), TET2 (26%), SRSF2 (24%), TP53 (22%), and RUNX1 (22%). 684 structural alterations were identified in 86% of patients (median 3; range 1-20), including arm-level (22.5%), segmental (>5 Mb; 60%), and focal (<2 Mb; 9%) alterations. Recurrent events included 9p cnLOH (16%), del(5q) (13%), del(17p) (10%), and focal deletion of 21q22.12 involving RUNX1 (10%). Complex karyotypes (≥3 abnormalities) were observed in 55% of patients. Biallelic mutations accounted for 21% (183/859) of mutations, affecting 58% of patients across 21 genes, most frequently JAK2 V617F (28%), TP53 (17%) and TET2 (14%). Patients with biallelic mutations in these genes had significantly higher blast percentages compared to those with monoallelic mutations (p<0.05), highlighting an association with aggressive disease. In 26 patients with longitudinal samples (chronic phase MPN and MPN-AP/BP), 87% (125/144) of mutations were already detectable during the chronic phase, on average more than 7 years prior to transformation. Only 11% (16/144) of mutations were newly acquired, including additional RUNX1 and TP53 hits, or structural changes that converted pre-existing JAK2 V617F and TP53 R248Q mutations from monoallelic to biallelic via 9p cnLOH or 17p loss, respectively. These findings suggest transformation is often driven by outgrowth of pre-existing high-risk clones, indicating that these clones likely have a competitive advantage and that the mutations within these clones have a cooperative biological interaction which underlies this competitive advantage. To investigate this hypothesis, we examined whether patterns of recurrent co-mutations were identifiable in this cohort. We identified recurrent co-occurring mutations in SRSF2-IDH2, ASXL1-SRSF2, and ASXL1-EZH2, which were enriched at transformation (p < 0.05; Fisher exact test). Importantly, presence of these co-mutations was consistently associated with a higher transformation risk in two independent cohorts of MF patients from Mayo Clinic (n= 405) and University of Florence (n= 518). Clonal architecture analysis revealed that these co-mutations frequently arose within the same malignant clone: 100% for SRSF2-IDH2, 80% for ASXL1-SRSF2, and 85% for ASXL1-EZH2, and are often within the dominant clone. Prior studies have demonstrated mechanistic interplay between SRSF2 and IDH2 (Yoshimi et al.), as well as ASXL1 and SRSF2 (Sui et al.) mutations in myeloid malignancies. We assessed functional synergy of ASXL1-EZH2 using a murine model and identified that dual deletion caused a rapidly fatal myeloid neoplasm with shorter survival than single-gene loss. These results reinforce that functional synergy emerges within a shared cellular context, supporting clonal co-dependence as a driver of progression. Single-cell RNA sequencing of ASXL1-EZH2 mutant cases is ongoing and will be presented. Conclusions Transformation to MPN-AP/BP is driven by gradual clonal remodeling and expansion of pre-existing high-risk abnormalities. Biallelic and multi-hit alterations in TP53, JAK2, TET2, and RUNX1 promote clonal dominance and blast progression. Recurrent co-mutations such as IDH2-SRSF2 and ASXL1-EZH2 arise within the same clone, exhibit functional synergy, and are linked to poor outcomes. These findings support the clinical utility of early clonal and co-mutational profiling to guide risk-adapted intervention.

Mastocytosis is a heterogeneous group of disorders comprising cutaneous mastocytosis, systemic mastocytosis, and mast cell sarcoma. It is associated with a variety of symptoms related to the release of mast cell mediators and mast cell tissue infiltration. Referral to specialized centers with expertise in the management of mastocytosis and multidisciplinary collaboration with subspecialists (eg, allergists for the management of anaphylaxis and drug hypersensitivities, anesthesiologists for invasive procedures or surgery, high-risk obstetrician for pregnancy) is recommended. The NCCN Guidelines for Systemic Mastocytosis provide evidence- and consensus-based recommendations for the diagnosis and comprehensive care of patients with systemic mastocytosis. The multidisciplinary panel of experts convenes at least once a year to review requested changes to the guidelines from both internal and external entities as well as to discuss data on existing and new therapies. These NCCN Guidelines Insights focus on some of the recent updates to the guidelines.

Abstract Enasidenib (ENA) is an inhibitor of isocitrate dehydrogenase 2 (IDH2) approved for the treatment of patients with IDH2-mutant relapsed/refractory acute myeloid leukemia (AML). In this phase 2/1b Beat AML substudy, we applied a risk-adapted approach to assess the efficacy of ENA monotherapy for patients aged ≥60 years with newly diagnosed IDH2-mutant AML in whom genomic profiling demonstrated that mutant IDH2 was in the dominant leukemic clone. Patients for whom ENA monotherapy did not induce a complete remission (CR) or CR with incomplete blood count recovery (CRi) enrolled in a phase 1b cohort with the addition of azacitidine. The phase 2 portion assessing the overall response to ENA alone demonstrated efficacy, with a composite complete response (cCR) rate (CR/CRi) of 46% in 60 evaluable patients. Seventeen patients subsequently transitioned to phase 1b combination therapy, with a cCR rate of 41% and 1 dose-limiting toxicity. Correlative studies highlight mechanisms of clonal elimination with differentiation therapy as well as therapeutic resistance. This study demonstrates both efficacy of ENA monotherapy in the upfront setting and feasibility and applicability of a risk-adapted approach to the upfront treatment of IDH2-mutant AML. This trial is registered at www.clinicaltrials.gov as #NCT03013998.

The classic Philadelphia chromosome–negative myeloproliferative neoplasms (MPN) consist of myelofibrosis, polycythemia vera, and essential thrombocythemia and are a heterogeneous group of clonal blood disorders characterized by an overproduction of blood cells. The NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) for MPN were developed as a result of meetings convened by a multidisciplinary panel with expertise in MPN, with the goal of providing recommendations for the management of MPN in adults. The Guidelines include recommendations for the diagnostic workup, risk stratification, treatment, and supportive care strategies for the management of myelofibrosis, polycythemia vera, and essential thrombocythemia. Assessment of symptoms at baseline and monitoring of symptom status during the course of treatment is recommended for all patients. This article focuses on the recommendations as outlined in the NCCN Guidelines for the diagnosis of MPN and the risk stratification, management, and supportive care relevant to MF.

Introduction: Elevated pro-inflammatory cytokines are a hallmark feature of myeloproliferative neoplasms (MPNs). The pro-inflammatory cytokine interleukin-8 (IL8) is increased in patients with myelofibrosis (MF) and correlates with adverse outcome, including overall survival. Previously, the Levine/Fang labs identified increased IL8 secretion from individual CD34+ stem cells in a subset of MF patients. The role of IL8 and its cognate receptors CXCR1/2 in MF pathogenesis has not been delineated. Methods: Single-cell cytokine assays were performed on isolated CD34+ cells from 60 clinically annotated MPN patients (20 MF, 20 PV, 20 ET) using a previously described micro-chip platform (Kleppe et al, Can Disc 2013). 10 healthy donors (CD34+ cells from hip replacements) were used as controls. Integrated RNA-Seq and Assay for Transposase-Accessible Chromatin followed by next-generation sequencing (ATAC-Seq) was performed on CD34+ cells from MPN patients with and without expanded IL8 secreting clones for gene expression/chromatin accessibility analysis. To model the role of IL8-CXCR2 on fibrosis in vivo, the human MPLW515L transplant model (hMPLW515L) of MF was used. Specifically, wild-type (WT) murine bone marrow (Creneg-Cxcr2f/f; Cxcr2WT) or marrow lacking the CXCR2 receptor (VavCre-Cxcr2f/f; Cxcr2KO)were retrovirally infected with MSCV-hMPLW515L-IRES-GFP and transplanted into lethally irradiated WT recipient mice and monitored for disease. Blood counts, chimerism, and flow cytometry were assayed. Moribund mice were sacrificed and assayed for grade reticulin fibrosis and overall survival. Results: Single-cell cytokine assays confirmed an increased proportion of IL8-secreting CD34+ cells in MF patients (40%) in comparison to other MPN sub-types (10% PV/0% ET) (Figure 1A). MF patients with expanded IL8 secreting clones (defined as >50% of total CD34+ cells) had increased leukocytosis (p<0.0001), larger spleen sizes (p=0.0004), greater prevalence of constitutional symptoms (p=0.0084), and higher-grade reticulin fibrosis in marrow (Figure 1B) in comparison to MF patients without prevalent IL8 clones. IHC confirmed increased IL8 expression in marrow biopsies from 8/15 MF patients in comparison to 0/4 normal controls (Figure 1C), and high IL8 expression was also observed in MF splenic megakaryocytes (MKs) as well as in splenic stromal/endothelial cells not seen in normal spleen (Figure 1D). Integrated RNA-Seq/ATAC-Seq analysis of IL8-high MF patients confirmed up-regulation of IL8-CXCR2 signaling and enrichment in pro-inflammatory pathways (i.e TNFa, NFkB, etc) by GSEA, as well as increased expression/accessibility of pro-inflammatory genes S100A8 and S100A9-previously implicated in fibrosis development. Flow analysis of IL8-high MF CD34+ cells revealed enhanced surface expression of CXCR2 and its analog CXCR1, such that MF was characterized by increased IL8 ligand and receptor expression (Figure 1E) and coincided with enhanced NFkB pathway activity (Figure 1F). Consistent with this, colony forming assays of cultured MF CD34+ cells revealed enhanced colony output when cultured with IL8 compared to WT CD34+ cells-an effect ameliorated by co-treatment with the CXCR1/2 antagonist Reparixin (Figure 1G). In vivo, hMPLW515L adoptive transplant with Cxcr2KO hematopoietic donor cells demonstrated improved leukocytosis, thrombocytosis (Figure 2A) and splenomegaly in comparison to Cxcr2WT hMPLW515L recipient mice. Pathologic analysis revealed a reduction in reticulin fibrosis in bone marrow (Figure 2B) and spleen, translating into an improvement in overall survival (Figure 2C). Notably, a significant reduction in dysplastic MKs-a hallmark feature of MF-was also observed in Cxcr2KO hMPLW515L mice (Figure 2D) supporting a role for CXCR2 signaling in MK proliferation. Conclusion: IL8 secreting clones are associated with increased symptom severity and fibrosis grade in MF. Gene expression of MF CD34+ IL8 secreting clones shows up-regulation of inflammatory genes S100A8/A9, implicated in myofibroblast proliferation. Cxcr2 KO abrogates fibrosis formation and prolongs survival in the hMPLW515L model, and CXCR1/2 inhibition impairs colony forming capacity of MF CD34+ cells. These data suggest pharmacologic inhibition of this pathway should be investigated as potential therapy in MF and in PV/ET patients at high risk of fibrotic transformation. Disclosures Fan: IsoPlexis: Current Employment, Current equity holder in private company; Singleron Biotechnologies: Current Employment, Current equity holder in private company. Levine:Morphosys: Consultancy; Prelude Therapeutics: Research Funding; Qiagen: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria; Loxo: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Amgen: Honoraria; Astellas: Consultancy; Imago: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Isoplexis: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Lilly: Consultancy, Honoraria; Janssen: Consultancy. Hoffman:Protagonist: Consultancy; Abbvie: Membership on an entity's Board of Directors or advisory committees; Dompe: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Forbius: Consultancy.

Eosinophilic disorders and related syndromes represent a heterogeneous group of neoplastic and nonneoplastic conditions, characterized by more eosinophils in the peripheral blood, and may involve eosinophil-induced organ damage. In the WHO classification of myeloid and lymphoid neoplasms, eosinophilic disorders characterized by dysregulated tyrosine kinase (TK) fusion genes are recognized as a new category termed, myeloid/lymphoid neoplasms with eosinophilia and rearrangement of PDGFRA , PDGFRB or FGFR1 or with PCM1-JAK2 . In addition to these aforementioned TK fusion genes, rearrangements involving FLT3 and ABL1 genes have also been described. These new NCCN Guidelines include recommendations for the diagnosis, staging, and treatment of any one of the myeloid/lymphoid neoplasms with eosinophilia (MLN-Eo) and a TK fusion gene included in the 2017 WHO Classification, as well as MLN-Eo and a FLT3 or ABL1 rearrangement.

Background: Pegylated Interferon-alpha (PEG-IFNa) improves hematologic parameters and reduces mutant allele fraction in patients with myeloproliferative neoplasms (MPNs); however, widespread clinical use is limited by frequent discontinuation due to side effects. Identifying biomarkers of response to PEG-IFNa therapy might identify patient subsets most likely to derive benefit from this therapy. Serum pro-inflammatory cytokine levels are increased in MPN patients and associated with adverse clinical outcomes. The effect of PEG-IFNa therapy on cytokine levels in MPN patients and correlation with clinical response has not been elucidated. We hypothesized that identification of cytokine signatures could predict for clinical/molecular response to PEG-IFNa in MPNs. Methods: Pre- and post-treatment serum samples were collected from available patients enrolled on the Myeloproliferative Disorders Research Consortium (MPD-RC) 111/112 trials-multicenter studies assessing efficacy of PEG-IFNa in patients with high-risk polycythemia vera/essential thrombocythemia either up front or in the setting of hydroxyurea failure/intolerance. Serum was prepared using the Millipore MAP Human 41-Cytokine Magnet Bead System (Millipore; Cat:HCYTOMAG-60K) and run on the Luminex FLEXMAP 3D platform. Data was analyzed using Luminex xPonent 4.3 software. Correlative mutational data on 156 genes implicated in myeloid malignancies was also available for all patients. Age-matched serum from healthy individuals (N=6) was obtained from AllCellsTM for use as wild-type (WT) controls. Baseline cytokine samples were compared between groups of interest by use of Wilcoxon rank-sum test. Change from baseline in cytokines for each group were compared to the null hypothesis of zero change. P values <0.05 were considered statistically significant. Results: Pre- and post-treatment serum samples were available on 29 MPN patients from the MPN-RC 111/112 cohorts. 21 patients were classified as complete responders (CRs) vs. 8 non-responders (NRs) using European LeukemiaNet criteria. Baseline cytokine levels were generally increased in both CRs and NRs compared to WT, including significant increases in Eotaxin (p=0.023), IL3 (p=0.048), IP10 (p=0.04), and TNFa (p=0.006) (Table 1), as well as a non-significant trend increase in IL8 (p=0.076). Notably, other cytokines were decreased at baseline in comparison to WT, including EGF (p<0.001), as well as MDC (p=0.040) and PDGFaabb (p=0.02), in line with previous studies. Compared to CRs, NRs displayed significantly lower levels of GRO (p=0.020), MDC (p=0.003), and PDGFaabb (p=0.026) at baseline along with elevated levels of IP10 (p=0.028) suggesting pre-treatment levels of these cytokines might predict response to therapy. Comparison of pre- and post- treatment samples revealed a general increase in median cytokine levels in CRs, with significant changes in GM-CSF (p=0.012), IP10 (p<0.001), MCP1 (p=0.015) and IFNa itself (p=0.010). Notably, median cytokine levels in NRs post-treatment, including IFNa, were relatively unchanged from baseline despite on-going PEG-IFNa therapy. Additional pre-treatment serum samples, as well as correlative analysis of cytokine changes with JAK-STAT driver mutant allele fraction and mutational profiling are underway and will be presented at the meeting. Conclusions: Preliminary findings suggest individual cytokines are affected by PEG-IFNa in MPNs. Furthermore, baseline levels of GRO, MDC, PDGFaabb, and IP10 correlate with response to PEG-IFNa. These data may identify MPN patients most likely to respond to PEG-IFNa therapy and those for whom this therapy may be most appropriate. Further analysis with a larger cohort of treatment-naïve patients is underway to validate these findings and identify additional molecular and cytokine signatures of response to PEG-IFNa therapy. Disclosures Yacoub: Agios: Speakers Bureau; Hylapharm: Equity Ownership; Dynavax: Equity Ownership; Cara: Equity Ownership; Ardelyx: Equity Ownership; Incyte: Consultancy, Honoraria, Speakers Bureau; Seattle Genetics: Honoraria, Speakers Bureau; Novartis: Consultancy, Speakers Bureau. Mascarenhas:Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; Roche: Consultancy, Research Funding; Merck: Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; CTI Biopharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Research Funding; Promedior: Research Funding; Merus: Research Funding; Pharmaessentia: Consultancy, Membership on an entity's Board of Directors or advisory committees. Levine:Lilly: Honoraria; Qiagen: Membership on an entity's Board of Directors or advisory committees; Imago Biosciences: Membership on an entity's Board of Directors or advisory committees; Loxo: Membership on an entity's Board of Directors or advisory committees; Prelude Therapeutics: Research Funding; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Research Funding; Gilead: Consultancy; Amgen: Honoraria; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Novartis: Consultancy. Hoffman:Merus: Research Funding. Rampal:Constellation, Incyte, and Stemline Therapeutics: Research Funding; Agios, Apexx, Blueprint Medicines, Celgene, Constellation, and Jazz: Consultancy.