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•An international panel of 38 experts and clinical trialists in MF participated in the preparation of the current document.•New definitions of transfusion status, gender-specific hemoglobin thresholds, and criteria for major and minor responses are submitted.

Background/aimsOcular manifestations of histiocytosis and their genetic underpinnings are poorly characterised. This study characterises ocular sites of histiocytosis, notate genetic alterations and correlates to histiocytosis clinical features including subtype and sites of disease.MethodsProspective registry-based study of predominantly adult histiocytosis patients at a single-institution tertiary referral centre. 180 eyes of 90 patients (46 males, 44 females) with histiocytosis (Erdheim-Chester disease 34, Rosai-Dorfman 20, xanthogranuloma 7, mixed histiocytosis 13, Langerhans cell histiocytosis (LCH) 15, ALK-positive histiocytosis 1). Ocular findings were categorised by the structure involved. Histiocytosis subtype, sites of disease and genetic status were correlated to ocular findings.ResultsOcular disease was present in more than half the histiocytosis patient cohort and occurred with other disease sites. Ocular findings were statistically significantly different across histiocytic subtypes with LCH subtypes having the lowest proportion of ocular findings (7%) and all other subtypes having rates of ocular findings which were five times that of patients with LCH (p=0.0009). Of patients with ocular findings, 41% of patients reported ocular symptoms and were significantly more in the group with ocular disease present versus those patients without ocular involvement. The presence of ocular findings was not statistically different by BRAFV600E, MAP2K1or RASisoform mutational status.ConclusionsOcular disease is a common feature of histiocytosis with significant visual symptomatology and occurrence in tandem with multisystem sites. Ocular findings vary by histiocytic subtype. The mutational profile of the cohort reflects known mutations in this clinical population, with no specific driver mutation associated with ocular disease.

Myeloid malignancies arise from the acquisition of somatic mutations among various genes implicated in essential functioning of hematopoietic stem cells and progenitor cells. In this second part of our two-part review, we discuss the use of mutation profiling in the diagnosis, prognosis, and treatment of patients with myeloproliferative neoplasms and other myeloid diseases. We also discuss the entity known as clonal hematopoiesis of indeterminate potential, awareness of which is a result of the increasing availability and improved quality of mutation profiling., Myeloproliferative Neoplasms: Diagnostic Implications Mutation analysis can yield valuable diagnostic information in many patients. As demonstrated in Table 1, mutations are highly recurrent across myeloid malignancies. It is important to [...]

The advent of high-throughput gene sequencing has revolutionized our understanding of the genetic mutations that drive myeloid malignancies. While these mutations are of interest pathobiologically, they are increasingly being recognized as clinically meaningful in providing diagnostic, prognostic, and therapeutic information to guide patient care. In this first part of our two-part review, we introduce mutation profiling as a relevant clinical tool for hematologists treating patients with myeloid malignancies. Next, we discuss the diagnostic and prognostic role of mutation profiling in myelodysplastic syndrome and acute myeloid leukemia. Finally, we detail the therapeutic implications of specific mutations in myelodysplastic syndrome and acute myeloid leukemia. In Part 2, we will discuss similar clinical approaches using mutation profiling in myeloproliferative neoplasms and other myeloid malignancies., Introduction Enormous strides have been made in recent years that have furthered our understanding of the underlying genetic alterations driving myeloid malignancies. High-throughput gene sequencing, the technological innovation behind these [...]

•Pelabresib plus ruxolitinib combination has potential for higher clinical efficacy than JAKi monotherapy in patients with MF.•Greater SVR and durable TSS improvement was observed with pelabresib plus ruxolitinib vs JAKi monotherapy.

•MF hematopoietic stem cells aberrantly secrete CXCL8 and exhibit enhanced cell growth/output in response to CXCL8 in vitro.•Genetic deletion or inhibition of Cxcr2 in the hMPLW515L-adoptive transfer model ameliorates fibrosis and improves hematologic parameters.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare hematologic malignancy with historically poor outcomes and no worldwide consensus treatment approach. Unique among most hematologic malignancies for its frequent cutaneous involvement, BPDCN can also invade other extramedullary compartments, including the central nervous system. Generally affecting older adults, many patients are unfit to receive intensive chemotherapy, and although hematopoietic stem cell transplantation is preferred for younger, fit individuals, not all are eligible. One recent therapeutic breakthrough is that all BPDCNs express CD123 (IL3Rα) and that this accessible surface marker can be pharmacologically targeted. The first-in-class agent for BPDCN, tagraxofusp, which targets CD123, was approved in December 2018 in the United States for patients with BPDCN aged ≥2 years. Despite favorable response rates in the frontline setting, many patients still relapse in the setting of monotherapy, and outcomes in patients with relapsed/refractory BPDCN remain dismal. Therefore, novel approaches targeting both CD123 and other targets are actively being investigated. To begin to formally address the state of the field, we formed a new collaborative initiative, the North American BPDCN Consortium (NABC). This group of experts, which includes a multidisciplinary panel of hematologists/oncologists, hematopoietic stem cell transplant physicians, pathologists, dermatologists, and pediatric oncologists, was tasked with defining the current standard of care in the field and identifying the most important research questions and future directions in BPDCN. The position findings of the NABC’s inaugural meetings are presented herein.

Myelofibrosis (MF) is a myeloproliferative disorder that exhibits considerable biological and clinical heterogeneity. At the two ends of the disease spectrum are the myelodepletive or cytopenic phenotype and the myeloproliferative phenotype. The cytopenic phenotype has a high prevalence in primary MF (PMF) and is characterized by low blood counts. The myeloproliferative phenotype is typically associated with secondary MF (SMF), mild anemia, minimal need for transfusion support, and normal to mild thrombocytopenia. Differences in somatic driver mutations and allelic burden, as well as the acquisition of non-driver mutations further influences these phenotypic differences, prognosis, and response to therapies such as JAK2 inhibitors. The outcome of patients with the cytopenic phenotype are comparatively worse and frequently pose a challenge to treat given the inherent exacerbation of cytopenias. Recent data indicate that an innate immune deregulated state that hinges on the myddosome-IRAK-NFκB axis favors the cytopenic myelofibrosis phenotype and offers opportunity for novel treatment approaches. We will review the biological and clinical features of the MF disease spectrum and associated treatment considerations.

The BCR-ABL-negative myeloproliferative neoplasms (MPNs) have a variable risk of progressing to accelerated- or blast-phase MPN (MPN-AP/MPN-BP), defined by the presence of 10% to 19% and more than or equal to 20% myeloid blasts in the peripheral blood or bone marrow, respectively. The molecular processes underlying the progression to MPN-AP/MPN-BP are becoming increasingly understood with the acquisition of additional mutations in epigenetic modifiers (eg, ASXL1, EZH2, TET2), TP53, the Ras pathway, or splicing factors (eg, SRSF2, U2AF1), having been described as important steps in this evolutionary process. At least partially driven by the enrichment of these high-risk molecular features, the prognosis of patients with MPN-BP remains inferior to other patients with acute myeloid leukemia, with a median overall survival of 3 to 6 months. Allogeneic hematopoietic cell transplantation remains the only potentially curative therapeutic modality, but only a minority of patients are eligible. In the absence of curative intent, therapeutic strategies or palliative treatment with hypomethylating agents as monotherapy or in combination with ruxolitinib or venetoclax can be considered. Several novel agents are in various stages of clinical development but are not available for routine use at this point, highlighting the need for ongoing research and the prioritization of clinical trial enrollment when feasible.

We found that in regenerative erythropoiesis, the erythroid progenitor landscape is reshaped, and a previously undescribed progenitor population with colony-forming unit-erythroid (CFU-E) activity (stress CFU-E [sCFU-E]) is expanded markedly to restore the erythron. sCFU-E cells are targets of erythropoietin (Epo), and sCFU-E expansion requires signaling from the Epo receptor (EpoR) cytoplasmic tyrosines. Molecularly, Epo promotes sCFU-E expansion via JAK2- and STAT5-dependent expression of IRS2, thus engaging the progrowth signaling from the IGF1 receptor (IGF1R). Inhibition of IGF1R and IRS2 signaling impairs sCFU-E cell growth, whereas exogenous IRS2 expression rescues cell growth in sCFU-E expressing truncated EpoR-lacking cytoplasmic tyrosines. This sCFU-E pathway is the major pathway involved in erythrocytosis driven by the oncogenic JAK2 mutant JAK2(V617F) in myeloproliferative neoplasm. Inability to expand sCFU-E cells by truncated EpoR protects against JAK2(V617F)-driven erythrocytosis. In samples from patients with myeloproliferative neoplasm, the number of sCFU-E-like cells increases, and inhibition of IGR1R and IRS2 signaling blocks Epo-hypersensitive erythroid cell colony formation. In summary, we identified a new stress-specific erythroid progenitor cell population that links regenerative erythropoiesis to pathogenic erythrocytosis.

To determine the survival benefit of allogeneic hematopoietic cell transplantation (allo-HCT) in chronic myelomonocytic leukemias (CMML), we assembled a retrospective cohort of CMML patients 18-70 years old diagnosed between 2000 and 2014 from an international CMML dataset (n = 730) and the EBMT registry (n = 384). The prognostic impact of allo-HCT was analyzed through univariable and multivariable time-dependent models and with a multistate model, accounting for age, sex, CMML prognostic scoring system (low or intermediate-1 grouped as lower-risk, intermediate-2 or high as higher-risk) at diagnosis, and AML transformation. In univariable analysis, lower-risk CMMLs had a 5-year overall survival (OS) of 20% with allo-HCT vs 42% without allo-HCT (P < .001). In higher-risk patients, 5-year OS was 27% with allo-HCT vs 15% without allo-HCT (P = .13). With multistate models, performing allo-HCT before AML transformation reduced OS in patients with lower-risk CMML, and a survival benefit was predicted for men with higher-risk CMML. In a multivariable analysis of lower-risk patients, performing allo-HCT before transformation to AML significantly increased the risk of death within 2 years of transplantation (hazard ratio [HR], 3.19; P < .001), with no significant change in long-term survival beyond this time point (HR, 0.98; P = .92). In higher-risk patients, allo-HCT significantly increased the risk of death in the first 2 years after transplant (HR 1.46; P = .01) but not beyond (HR, 0.60; P = .09). Performing allo-HCT before AML transformation decreases life expectancy in lower-risk patients but may be considered in higher-risk patients.

Leukemic transformation (LT) of myeloproliferative neoplasm (MPN) has a dismal prognosis and is largely fatal. Mutational inactivation of TP53 is the most common somatic event in LT; however, the mechanisms by which TP53 mutations promote LT remain unresolved. Using an allelic series of mouse models of Jak2/Trp53 mutant MPN, we identify that only biallelic inactivation of Trp53 results in LT (to a pure erythroleukemia [PEL]). This PEL arises from the megakaryocyte-erythroid progenitor population. Importantly, the bone morphogenetic protein 2/SMAD pathway is aberrantly activated during LT and results in abnormal self-renewal of megakaryocyte-erythroid progenitors. Finally, we identify that Jak2/Trp53 mutant PEL is characterized by recurrent copy number alterations and DNA damage. Using a synthetic lethality strategy, by targeting active DNA repair pathways, we show that this PEL is highly sensitive to combination WEE1 and poly(ADP-ribose) polymerase inhibition. These observations yield new mechanistic insights into the process of p53 mutant LT and offer new, clinically translatable therapeutic approaches.

The goal of therapy for patients with essential thrombocythemia (ET) and polycythemia vera (PV) is to reduce thrombotic events by normalizing blood counts. Hydroxyurea (HU) and interferon-α (IFN-α) are the most frequently used cytoreductive options for patients with ET and PV at high risk for vascular complications. Myeloproliferative Disorders Research Consortium 112 was an investigator-initiated, phase 3 trial comparing HU to pegylated IFN-α (PEG) in treatment-naïve, high-risk patients with ET/PV. The primary endpoint was complete response (CR) rate at 12 months. A total of 168 patients were treated for a median of 81.0 weeks. CR for HU was 37% and 35% for PEG (P = .80) at 12 months. At 24 to 36 months, CR was 20% to 17% for HU and 29% to 33% for PEG. PEG led to a greater reduction in JAK2V617F at 24 months, but histopathologic responses were more frequent with HU. Thrombotic events and disease progression were infrequent in both arms, whereas grade 3/4 adverse events were more frequent with PEG (46% vs 28%). At 12 months of treatment, there was no significant difference in CR rates between HU and PEG. This study indicates that PEG and HU are both effective treatments for PV and ET. With longer treatment, PEG was more effective in normalizing blood counts and reducing driver mutation burden, whereas HU produced more histopathologic responses. Despite these differences, both agents did not differ in limiting thrombotic events and disease progression in high-risk patients with ET/PV. This trial was registered at www.clinicaltrials.gov as #NCT01259856.

Myeloproliferative neoplasms (MPNs) transform to myelofibrosis (MF) and highly lethal acute myeloid leukemia (AML), although the actionable mechanisms driving progression remain elusive. Here, we elucidate the role of the high mobility group A1 (HMGA1) chromatin regulator as a novel driver of MPN progression. HMGA1 is upregulated in MPN, with highest levels after transformation to MF or AML. To define HMGA1 function, we disrupted gene expression via CRISPR/Cas9, short hairpin RNA, or genetic deletion in MPN models. HMGA1 depletion in JAK2V617F AML cell lines disrupts proliferation, clonogenicity, and leukemic engraftment. Surprisingly, loss of just a single Hmga1 allele prevents progression to MF in JAK2V617F mice, decreasing erythrocytosis, thrombocytosis, megakaryocyte hyperplasia, and expansion of stem and progenitors, while preventing splenomegaly and fibrosis within the spleen and BM. RNA-sequencing and chromatin immunoprecipitation sequencing revealed HMGA1 transcriptional networks and chromatin occupancy at genes that govern proliferation (E2F, G2M, mitotic spindle) and cell fate, including the GATA2 master regulatory gene. Silencing GATA2 recapitulates most phenotypes observed with HMGA1 depletion, whereas GATA2 re-expression partially rescues leukemogenesis. HMGA1 transactivates GATA2 through sequences near the developmental enhancer (+9.5), increasing chromatin accessibility and recruiting active histone marks. Further, HMGA1 transcriptional networks, including proliferation pathways and GATA2, are activated in human MF and MPN leukemic transformation. Importantly, HMGA1 depletion enhances responses to the JAK2 inhibitor, ruxolitinib, preventing MF and prolonging survival in murine models of JAK2V617F AML. These findings illuminate HMGA1 as a key epigenetic switch involved in MPN transformation and a promising therapeutic target to treat or prevent disease progression.

Allogeneic hematopoietic cell transplant (allo-HCT) remains the only potentially curative therapeutic modality for patients with primary or secondary myelofibrosis (MF). However, many patients are considered ineligible for allo-HCT, and transplant-related mortality can be substantial. Data on the efficacy and safety of allo-HCT are mixed and largely derived from retrospective studies. We aimed to synthesize the available evidence on the safety and efficacy of allo-HCT in MF and to identify patient, disease, and transplant characteristics with prognostic impact on outcomes of patients with MF undergoing allo-HCT. For this systematic review and meta-analysis, Cochrane Library, Google Scholar, Ovid Medline, Ovid Embase, PubMed, Scopus, and Web of Science Core Collection were searched from inception to October 11, 2020, for studies on allo-HCT in MF. Random-effects models were used to pool response rates for the co-primary outcomes of 1-year, 2-year, and 5-year overall survival (OS). Rates of non-relapse mortality and acute and chronic graft-versus-host-disease (GVHD) were studied as secondary endpoints. Subgroup analyses on the effect of conditioning regimen intensity, baseline dynamic international prognostic scoring system (DIPSS) score, and patient age were performed. The study protocol has been registered on PROSPERO (CRD42020188706). Forty-three studies with 8739 patients were identified and included in this meta-analysis. Rates of 1-year, 2-year, and 5-year OS were 66.7% (95% confidence interval [CI], 63.5%-69.8%), 64.4% (95% CI, 57.6%-70.6%), and 55.0% (95% CI, 51.8%-58.3%), respectively. Rates of 1-year, 2-year, and 5-year nonrelapse mortality were 25.9% (95% CI, 23.3%-28.7%), 29.7% (95% CI, 24.5%-35.4%), and 30.5% (95% CI, 25.9%-35.5%), respectively. The combined rate of graft failure was 10.6% (95% CI, 8.9%-12.5%) with primary and secondary graft failure occurring in 7.3% (95% CI, 5.7%-9.4%) and 5.9% (95% CI, 4.3%-8.0%) of patients, respectively. Rates of acute and chronic graft-versus-host disease were 44.0% (95% CI, 39.6%-48.4%; grade III/IV: 15.2%) and 46.5% (95% CI, 42.2%-50.8%; extensive or moderate/severe: 26.1%), respectively. Subgroup analyses did not show any significant difference between conditioning regimen intensity (myeloablative versus reduced-intensity), median patient age, and proportion of DIPSS-intermediate-2/high patients. The quality of the evidence is limited by the absence of randomized clinical trials in the field and the heterogeneity of patient and transplant characteristics across included studies. Given the poor prognosis of patients not receiving transplants and in the absence of curative nontransplantation therapies, our results support consideration of allo-HCT for eligible patients with MF.

Data on the efficacy and safety of interferon (IFN)-α for the treatment of essential thrombocythemia (ET) and polycythemia vera (PV) are inconsistent. We conducted a systematic review and meta-analysis and searched MEDLINE and EMBASE via Ovid, Scopus, COCHRANE registry of clinical trials, and Web of Science from inception through 03/2019 for studies of pegylated IFN (peg-IFN) and non-pegylated IFN (non-peg-IFN) in PV and ET patients. Random-effects models were used to pool response rates for the primary outcome of overall response rate (ORR) defined as a composite of complete response, partial response, complete hematologic response (CHR) and partial hematologic response. Peg-IFN and non-peg-IFN were compared by meta-regression analyses. In total, 44 studies with 1359 patients (730 ET, 629 PV) were included. ORR were 80.6% (95% confidence interval: 76.6–84.1%, CHR: 59.0% [51.5%–66.1%]) and 76.7% (67.4–84.0%; CHR: 48.5% [37.8–59.4%]) for ET and PV patients, respectively. In meta-regression analyses results did not differ significantly for non-peg-IFN vs. peg-IFN. Annualized rates of thromboembolic complications and treatment discontinuation due to adverse events were low at 1.2% and 8.8% for ET and 0.5% and 6.5% for PV patients, respectively. Both peg-IFN and non-peg-IFN can be effective and safe long-term treatments for ET and PV.

Background: Histiocytic neoplasms (HN) are clonal hematopoietic disorders characterized by diverse mutations in the mitogen-activated protein kinase (MAPK) pathway. BRAF inhibition is highly effective for patients with HN harboring the BRAFV600E mutation, and implementation of BRAF inhibitors transformed management of BRAFV600E-mutated Erdheim-Chester disease (ECD) and Langerhans cell histiocytosis (LCH). No standard or approved therapies existed, however, for patients with HN lacking the BRAFV600E mutation. Efficacy of mitogen-activated protein kinase (MEK) inhibition for HN harboring various MAPK pathway mutations has been observed in case reports and series. We present here results from 26 patients treated in a phase 2 trial of single-agent cobimetinib for adults with HN (NCT02649972). The data were submitted to the Food and Drug Administration (FDA) in a supplemental new drug application for cobimetinib, and FDA approval was granted in October, 2022.

Accelerated and blast phase myeloproliferative neoplasms are advanced stages of the disease with historically a poor prognosis and little improvement in outcomes thus far. The lack of responses to standard treatments likely results from the more aggressive biology reflected by the higher incidence of complex karyotype and high-risk somatic mutations, which are enriched at the time of transformation. Treatment options include induction chemotherapy (7 + 3) as that used on de novoacute myeloid leukemia or hypomethylating agent–based therapy, which has shown similar outcomes. Allogeneic stem cell transplantation remains the only potential for cure.

Plasmacytoid dendritic cells (pDCs) are the principal natural type I interferon–producing dendritic cells. Neoplastic expansion of pDCs and pDC precursors leads to blastic plasmacytoid dendritic cell neoplasm (BPDCN), and clonal expansion of mature pDCs has been described in chronic myelomonocytic leukemia. The role of pDC expansion in acute myeloid leukemia (AML) is poorly studied. Here, we characterize patients with AML with pDC expansion (pDC-AML), which we observe in ∼5% of AML cases. pDC-AMLs often possess cross-lineage antigen expression and have adverse risk stratification with poor outcome. RUNX1 mutations are the most common somatic alterations in pDC-AML (>70%) and are much more common than in AML without pDC expansion and BPDCN. We demonstrate that pDCs are clonally related to, as well as originate from, leukemic blasts in pDC-AML. We further demonstrate that leukemic blasts from RUNX1-mutated AML upregulate a pDC transcriptional program, poising the cells toward pDC differentiation and expansion. Finally, tagraxofusp, a targeted therapy directed to CD123, reduces leukemic burden and eliminates pDCs in a patient-derived xenograft model. In conclusion, pDC-AML is characterized by a high frequency of RUNX1 mutations and increased expression of a pDC transcriptional program. CD123 targeting represents a potential treatment approach for pDC-AML.

PAC203 is a randomized dose-finding study of pacritinib, an oral JAK2/IRAK1 inhibitor, in patients with advanced myelofibrosis who are intolerant of or resistant to ruxolitinib. Patients were randomized 1:1:1 to pacritinib 100 mg once per day, 100 mg twice per day, or 200 mg twice per day. Enhanced eligibility criteria, monitoring, and dose modifications were implemented to mitigate risk of cardiac and hemorrhagic events. Efficacy was based on ≥35% spleen volume response (SVR) and ≥50% reduction in the 7-component total symptom score (TSS) through week 24. Of 161 patients, 73% were intolerant of and 76% had become resistant to ruxolitinib; 50% met criteria for both. Severe thrombocytopenia (platelet count <50 × 103/μL) was present in 44%. SVR rates were highest with 200 mg twice per day (100 mg once per day, 0%; 100 mg twice per day, 1.8%; 200 mg twice per day, 9.3%), particularly among patients with baseline platelet counts <50 × 103/μL (17%; 4 of 24). Although TSS response rate was similar across doses (100 mg once per day, 7.7%; 100 mg twice per day, 7.3%; 200 mg twice per day, 7.4%), median percent reduction in TSS suggested a dose-response relationship (–3%, −16%, and −27%, respectively). Pharmacokinetic and pharmacodynamic modeling based on all available data showed greatest SVR and TSS reduction at 200 mg twice per day compared with lower doses. Common adverse events were gastrointestinal events, thrombocytopenia, and anemia. There was no excess of grade ≥3 hemorrhagic or cardiac events at 200 mg twice per day. Pacritinib 200 mg twice per day demonstrated clinical activity and an acceptable safety profile and was selected as the recommended dose for a pivotal phase 3 study in patients with myelofibrosis and severe thrombocytopenia. This trial was registered at www.clinicaltrials.gov as #NCT03165734.

Activation of the P53 pathway through inhibition of MDM2 using nutlins has shown clinical promise in the treatment of solid tumors and hematologic malignancies. There is concern, however, that nutlin therapy might stimulate the emergence or expansion of TP53-mutated subclones. We recently published the results of a phase 1 trial of idasanutlin in patients with polycythemia vera (PV) that revealed tolerability and clinical activity. Here, we present data indicating that idasanutlin therapy is associated with expansion of TP53 mutant subclones. End-of-study sequencing of patients found that 5 patients in this trial harbored 12 TP53 mutations; however, only 1 patient had been previously identified as having a TP53 mutation at baseline. To identify the origin of these mutations, further analysis of raw sequencing data of baseline samples was performed and revealed that a subset of these mutations was present at baseline and expanded during treatment with idasanutlin. Follow-up samples were obtained from 4 of 5 patients in this cohort, and we observed that after cessation of idasanutlin, the variant allele frequency (VAF) of 8 of 9 TP53 mutations decreased. Furthermore, disease progression to myelofibrosis or myeloproliferative neoplasm blast phase was not observed in any of these patients after 19- to 32-month observation. These data suggest that idasanutlin treatment may promote transient TP53 mutant clonal expansion. A larger study geared toward high-resolution detection of low VAF mutations is required to explore whether patients acquire de novo TP53 mutations after idasanutlin therapy.

Myeloid malignancies, including acute myeloid leukaemia (AML), arise from the expansion of haematopoietic stem and progenitor cells that acquire somatic mutations. Bulk molecular profiling has suggested that mutations are acquired in a stepwise fashion: mutant genes with high variant allele frequencies appear early in leukaemogenesis, and mutations with lower variant allele frequencies are thought to be acquired later1–3. Although bulk sequencing can provide information about leukaemia biology and prognosis, it cannot distinguish which mutations occur in the same clone(s), accurately measure clonal complexity, or definitively elucidate the order of mutations. To delineate the clonal framework of myeloid malignancies, we performed single-cell mutational profiling on 146 samples from 123 patients. Here we show that AML is dominated by a small number of clones, which frequently harbour co-occurring mutations in epigenetic regulators. Conversely, mutations in signalling genes often occur more than once in distinct subclones, consistent with increasing clonal diversity. We mapped clonal trajectories for each sample and uncovered combinations of mutations that synergized to promote clonal expansion and dominance. Finally, we combined protein expression with mutational analysis to map somatic genotype and clonal architecture with immunophenotype. Our findings provide insights into the pathogenesis of myeloid transformation and how clonal complexity evolves with disease progression.

Myeloproliferative neoplasms (MPN) that have evolved into accelerated or blast phase disease (MPN-AP/BP) have poor outcomes with limited treatment options and therefore represent an urgent unmet need. We have previously demonstrated in a multicenter, phase 1 trial conducted through the Myeloproliferative Neoplasms Research Consortium that the combination of ruxolitinib and decitabine is safe and tolerable and is associated with a favorable overall survival (OS). In this phase 2 trial, 25 patients with MPN-AP/BP were treated at the recommended phase 2 dose of ruxolitinib 25 mg twice daily for the induction cycle followed by 10 mg twice daily for subsequent cycles in combination with decitabine 20 mg/m2 for 5 consecutive days in a 28-day cycle. Nineteen patients died during the study follow-up. The median OS for all patients on study was 9.5 months (95% confidence interval, 4.3-12.0). Overall response rate (complete remission + incomplete platelet recovery + partial remission) was 11/25 (44%) and response was not associated with improved survival. We conclude that the combination of decitabine and ruxolitinib was well tolerated, demonstrated favorable OS, and represents a therapeutic option for this high-risk patient population. This trial was registered at www.clinicaltrials.gov as #NCT02076191.

The ongoing COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 is a global public health crisis. Multiple observations indicate poorer post-infection outcomes for patients with cancer than for the general population. Herein, we highlight the challenges in caring for patients with acute leukaemias and myeloid neoplasms amid the COVID-19 pandemic. We summarise key changes related to service allocation, clinical and supportive care, clinical trial participation, and ethical considerations regarding the use of lifesaving measures for these patients. We recognise that these recommendations might be more applicable to high-income countries and might not be generalisable because of regional differences in health-care infrastructure, individual circumstances, and a complex and highly fluid health-care environment. Despite these limitations, we aim to provide a general framework for the care of patients with acute leukaemias and myeloid neoplasms during the COVID-19 pandemic on the basis of recommendations from international experts.

The median overall survival of patients with high-risk myelodysplastic syndromes refractory to hypomethylating agents is less than 6 months. Currently, no standard therapy for such patients exists. Preclinical studies have shown that inhibition of the nuclear export protein exportin 1 (XPO1) causes nuclear accumulation of p53 and disruption of NF-κB signalling, both relevant targets for myelodysplastic syndromes. We therefore aimed to assess the safety and activity of selinexor in patients with myelodysplastic syndromes or oligoblastic acute myeloid leukaemia refractory to hypomethylating agents.

Secondary acute myeloid leukemias (AMLs) evolving from an antecedent myeloproliferative neoplasm (MPN) are characterized by a unique set of cytogenetic and molecular features distinct from de novo AML. Given the high frequency of poor-risk cytogenetic and molecular features, malignant clones are frequently insensitive to traditional AML chemotherapeutic agents. Allogeneic stem cell transplant, the only treatment modality shown to have any beneficial long-term outcome, is often not possible given the advanced age of patients at time of diagnosis and frequent presence of competing comorbidities. Even in this setting, relapse rates remain high. As a result, outcomes are generally poor and there remains a significant unmet need for novel therapeutic strategies. Although advances in cancer genomics have dramatically enhanced our understanding of the molecular events governing clonal evolution in MPNs, the cell-intrinsic and -extrinsic mechanisms driving leukemic transformation at this level remain poorly understood. Here, we review known risk factors for the development of leukemic transformation in MPNs, recent progress made in our understanding of the molecular features associated with leukemic transformation, current treatment strategies, and emerging therapeutic options for this high-risk myeloid malignancy.

Allogeneic hematopoietic cell transplantation (HCT) is the only curative therapy for myelofibrosis (MF). In this large multicenter retrospective study, overall survival (OS) in MF patients treated with allogeneic HCT (551 patients) and without HCT (non-HCT) (1377 patients) was analyzed with Cox proportional hazards model. Survival analysis stratified by the Dynamic International Prognostic Scoring System (DIPSS) revealed that the first year of treatment arm assignment, due to upfront risk of transplant-related mortality (TRM), HCT was associated with inferior OS compared with non-HCT (non-HCT vs HCT: DIPSS intermediate 1 [Int-1]: hazard ratio [HR] = 0.26, P < .0001; DIPSS-Int-2 and higher: HR, 0.39, P < .0001). Similarly, in the DIPSS low-risk MF group, due to upfront TRM risk, OS was superior with non-HCT therapies compared with HCT in the first-year post treatment arm assignment (HR, 0.16, P = .006). However, after 1 year, OS was not significantly different (HR, 1.38, P = .451). Beyond 1 year of treatment arm assignment, an OS advantage with HCT therapy in Int-1 and higher DIPSS score patients was observed (non-HCT vs HCT: DIPSS-Int-1: HR, 2.64, P < .0001; DIPSS-Int-2 and higher: HR, 2.55, P < .0001). In conclusion, long-term OS advantage with HCT was observed for patients with Int-1 or higher risk MF, but at the cost of early TRM. The magnitude of OS benefit with HCT increased as DIPSS risk score increased and became apparent with longer follow-up.

Acute myeloid leukemia (AML) is a highly heterogeneous disease arising from acquired genetic and epigenetic aberrations which stifle normal development and differentiation of hematopoietic precursors. Despite the complex and varied biological underpinnings, induction therapy for AML has remained fairly uniform over 4 decades and outcomes remain poor for most patients. Recently, enhanced understanding of the leukemic epigenome has resulted in the translational investigation of a number of epigenetic modifying agents currently in various stages of clinical development. These novel therapies are based on mechanistic rationale and offer the potential to improve AML patient outcomes. In light of many recent advances in this field, we provide an updated, clinically oriented review of the evolving landscape of epigenetic modifying agents for the treatment of AML.

RAS-pathway mutations are recurrent events in myeloid malignancies. However, there is limited data on the significance of RAS-pathway mutations in patients with myelofibrosis (MF). We analyzed next-generation sequencing data of 16 genes, including RAS-pathway genes, from 723 patients with primary and secondary MF across three international centers and evaluated their significance. N/KRASvariants were present in 6% of patients and were typically sub-clonal (median VAF = 20%) relative to other genes variants. RASvariants were associated with advanced MF features including leukocytosis (p= 0.02), high somatic mutation burden (p< 0.01) and the presence of established “molecular high-risk” (MHR) mutations. MF patients with N/KRASmutations had shorter 3-year overall survival (OS) (34% vs 58%, p< 0.001) and higher incidence of acute myeloid leukemia at 3 years (18% vs 11%, p= 0.03). In a multivariate Cox model, RASmutations were associated with decreased OS (HR 1.93, p< 0.001). We created a novel score to predict OS incorporating RASmutations, and it predicted OS across training and validation cohorts. Patients with intermediate risk/high-risk DIPSS with RASmutations who received ruxolitinib had a nonsignificant longer 2-year OS relative to those who did not receive ruxolitinib. These data demonstrate the importance of identifying RASmutations in MF patients.

•Parsaclisib, a PI3Kδ inhibitor, reduced spleen volume and improved symptom scores when added to ruxolitinib for patients with myelofibrosis.•The safety and tolerability of the combination was acceptable, and daily parsaclisib dosing may provide the greatest benefit.

To determine the survival benefit of allogeneic hematopoietic cell transplantation (allo-HCT) in chronic myelomonocytic leukemias (CMML), we assembled a retrospective cohort of CMML patients aged 18-70 years diagnosed between 2000 and 2014 from an International CMML Dataset (ICD, n=730) and from the EBMT registry (n=384). The prognostic impact of allo-HCT was analyzed through univariable and multivariable time-dependent models and with a multi-state model, accounting for age, sex, CMML prognostic scoring system (CPSS low and intermediate-1: lower-risk, intermediate-2 and high: higher-risk) at diagnosis, and AML transformation. In univariable analysis, lower-risk CMMLs had a 5-year OS of 20% (95%CI 12-33%) with allo-HCT versus 42% (95%CI 35-49%) without allo-HCT (P<0.001). In higher-risk patients, 5-year OS was 27% (95%CI 21-34%) with allo-HCT versus 15% (95%CI 11-22%) without allo-HCT (P=0.13). With multi-state models, performing allo-HCT before AML transformation reduced overall survival in patients with lower risk CMML while a survival benefit was predicted for men with higher risk CMML. In a multivariable analysis of lower-risk patients, performing allo-HCT before transformation to AML significantly increased the risk of death within two years of transplantation (HR=3.19, 95%CI 2.30-4.42, P<0.001), with no significant change in long-term survival beyond this time point (HR=0.98, 95%CI 0.58-1.64, P=0.92). In higher risk patients, allo-HCT significantly increased the risk of death in the first two years after transplant (HR=1.46, 95%CI 1.09-1.96, P=0.01), but not beyond (HR=0.60, 95%CI 0.34-1.08, P=0.09). Performing allo-HCT before AML transformation decreases life expectancy in lower risk patients but may be considered in higher risk patients.

1)Median OS in MPN-AP/BP is 0.86 years in a modern cohort without significant difference based on frontline treatment choice2)Median OS in those that underwent allo-HCT is 2.3 years from time of allo-HCT; response prior to allo-HCT did not impact survival

Prior studies have reported high response rates with recombinant interferon-a (rIFN-a) therapy in patients with essential thrombocythemia (ET) and polycythemia vera (PV). To further define the role of rIFN-a, we investigated the outcomes of pegylated-rIFN-a2a (PEG) therapy in ET and PV patients previously treated with hydroxyurea (HU). The Myeloproliferative Disorders Research Consortium (MPD-RC)-111 study was an investigator-initiated, international, multicenter, phase 2 trial evaluating the ability of PEG therapy to induce complete (CR) and partial (PR) hematologic responses in patients with high-risk ET or PV who were either refractory or intolerant to HU. The study included 65 patients with ET and 50 patients with PV. The overall response rates (ORRs; CR/PR) at 12 months were 69.2% (43.1% and 26.2%) in ET patients and 60% (22% and 38%) in PV patients. CR rates were higher in CALR-mutated ET patients (56.5% vs 28.0%; P = .01), compared with those in subjects lacking a CALR mutation. The median absolute reduction in JAK2V617F variant allele fraction was -6% (range, -84% to 47%) in patients achieving a CR vs +4% (range, -18% to 56%) in patients with PR or nonresponse (NR). Therapy was associated with a significant rate of adverse events (AEs); most were manageable, and PEG discontinuation related to AEs occurred in only 13.9% of subjects. We conclude that PEG is an effective therapy for patients with ET or PV who were previously refractory and/or intolerant of HU. This trial was registered at www.clinicaltrials.gov as #NCT01259856.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an uncommon hematologic malignancy with poor outcomes. Existing data on the clinical behavior of BPDCN are limited because reported outcomes are from small retrospective series, and standardized treatment guidelines are lacking. The interleukin-3 cytotoxin conjugate tagraxofusp was recently tested in phase 1/2 trials that led to US Food and Drug Administration approval, the first ever for BPDCN. However, because there was no matched internal comparator in this or any clinical study to date, results of BPDCN trials testing new drugs are difficult to compare with alternative therapies. We therefore sought to define the clinical characteristics and outcomes of a group of patients with BPDCN treated at 3 US cancer centers in the modern era but before tagraxofusp was available. In 59 studied patients with BPDCN, the median overall survival from diagnosis was 24 months, and outcomes were similar in patients with “skin only” or with systemic disease at presentation. Intensive first-line therapy and “lymphoid-type” chemotherapy regimens were associated with better outcomes. Only 55% of patients received intensive chemotherapy, and 42% of patients underwent stem cell transplantation. Clinical characteristics at diagnosis associated with poorer outcomes included age >60 years, abnormal karyotype, and terminal deoxynucleotidyltransferase (TdT) negativity in the BPDCN cells. We also identified disease responses to pralatrexate and enasidenib in some patients. This study highlights poor outcomes for patients with BPDCN in the modern era and the need for new treatments. Outcomes from ongoing clinical trials for BPDCN can be evaluated relative to this contemporary cohort.

A limited number of drugs are available to treat patients with polycythemia vera (PV) and essential thrombocythemia (ET). We attempted to identify alternative agents that may target abnormalities within malignant hematopoietic stem (HSCs) and progenitor cells (HPCs). Previously, MDM2 protein levels were shown to be upregulated in PV/ET CD34+ cells, and exposure to a nutlin, an MDM2 antagonist, induced activation of the TP53 pathway and selective depletion of PV HPCs/HSCs. This anticlonal activity was mediated by upregulation of p53 and potentiated by the addition of interferon-a2a (IFN-a2a). Therefore, we performed an investigator-initiated phase 1 trial of the oral MDM2 antagonist idasanutlin (RG7388; Roche) in patients with high-risk PV/ET for whom at least 1 prior therapy had failed. Patients not attaining at least a partial response by European LeukemiaNet criteria after 6 cycles were then allowed to receive combination therapy with low-dose pegylated IFN-a2a. Thirteen patients with JAK2 V617F+ PV/ET were enrolled, and 12 (PV, n = 11; ET, n = 1) were treated with idasanutlin at 100 and 150 mg daily, respectively, for 5 consecutive days of a 28-day cycle. Idasanutlin was well tolerated; no dose-limiting toxicity was observed, but low-grade gastrointestinal toxicity was common. Overall response rate after 6 cycles was 58% (7 of 12) with idasanutlin monotherapy and 50% (2 of 4) with combination therapy. Median duration of response was 16.8 months (range, 3.5-26.7). Hematologic, symptomatic, pathologic, and molecular responses were observed. These data indicate that idasanutlin is a promising novel agent for PV; it is currently being evaluated in a global phase 2 trial. This trial was registered at www.clinicaltrials.gov as #NCT02407080.

Erdheim-Chester disease (ECD) is an ultra-rare hematologic neoplasm characterized by somatic mutations of the MAPK pathway and by accumulation of lesional histiocytes within tissues. Clinical phenotypes and sites of disease involvement are heterogenous in ECD, and no tool exists for systematic and comprehensive assessment of ECD symptomatology. We describe a collaborative effort among ECD specialists, patient-reported outcome (PRO) methodologists, and ECD patients to develop the Erdheim-Chester Disease Symptom Scale (ECD-SS): a symptom inventory for clinical ECD care and evaluation of ECD therapies. Methodologically rigorous focus groups led to the identification of 63 ECD symptoms in 6 categories, incorporated into the ECD-SS with respect to both severity and frequency. Among 50 ECD patients participating in a prospective registry study completing the ECD-SS, 46 (92%) reported neurological/psychological symptoms, 29 (58%) reported pain, and at least one-half reported mood symptoms, memory problems, or fatigue. Symptoms were highly frequent or almost constant regardless of their severity. The ECD-SS is a rigorously developed, patient-centered tool that demonstrates the wide and previously unappreciated burden of symptomatology experienced by ECD patients. Further studies will refine the symptom inventory and define its psychometric properties and role in clinical care and investigation in the context of ECD.

Histiocytic neoplasms are a heterogeneous group of clonal haematopoietic disorders that are marked by diverse mutations in the mitogen-activated protein kinase (MAPK) pathway1,2. For the 50% of patients with histiocytosis who have BRAFV600mutations3–5, RAF inhibition is highly efficacious and has markedly altered the natural history of the disease6,7. However, no standard therapy exists for the remaining 50% of patients who lack BRAFV600mutations. Although ERK dependence has been hypothesized to be a consistent feature across histiocytic neoplasms, this remains clinically unproven and many of the kinase mutations that are found in patients who lack BRAFV600mutations have not previously been biologically characterized. Here we show ERK dependency in histiocytoses through a proof-of-concept clinical trial of cobimetinib, an oral inhibitor of MEK1 and MEK2, in patients with histiocytoses. Patients were enrolled regardless of their tumour genotype. In parallel, MAPK alterations that were identified in treated patients were characterized for their ability to activate ERK. In the 18 patients that we treated, the overall response rate was 89% (90% confidence interval of 73–100). Responses were durable, with no acquired resistance to date. At one year, 100% of responses were ongoing and 94% of patients remained progression-free. Cobimetinib treatment was efficacious regardless of genotype, and responses were observed in patients with ARAF, BRAF, RAF1, NRAS, KRAS, MEK1(also known as MAP2K1) and MEK2(also known as MAP2K2) mutations. Consistent with the observed responses, the characterization of the mutations that we identified in these patients confirmed that the MAPK-pathway mutations were activating. Collectively, these data demonstrate that histiocytic neoplasms are characterized by a notable dependence on MAPK signalling—and that they are consequently responsive to MEK inhibition. These results extend the benefits of molecularly targeted therapy to the entire spectrum of patients with histiocytosis.

Myeloproliferative neoplasms (MPNs) are a group of blood cancers that arise following the sequential acquisition of genetic lesions in hematopoietic stem and progenitor cells (HSPCs). We identify mutational cooperation between Jak2V617F expression and Dnmt3a loss that drives progression from early-stage polycythemia vera to advanced myelofibrosis. Using in vivo, clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR-associated protein 9 (Cas9) disruption of Dnmt3a in Jak2V617F knockin HSPC, we show that Dnmt3a loss blocks the accumulation of erythroid elements and causes fibrotic infiltration within the bone marrow and spleen. Transcriptional analysis and integration with human data sets identified a core DNMT3A-driven gene-expression program shared across multiple models and contexts of Dnmt3a loss. Aberrant self-renewal and inflammatory signaling were seen in Dnmt3a−/− Jak2V617F HSPC, driven by increased chromatin accessibility at enhancer elements. These findings identify oncogenic cooperativity between Jak2V617F-driven MPN and Dnmt3a loss, leading to activation of HSPC enhancer–driven inflammatory signaling.

Myeloproliferative neoplasms (MPN), including polycythemia vera, essential thrombocythemia, and primary myelofibrosis, have a propensity to evolve into accelerated and blast-phase disease (MPN-AP/BP), carrying a dismal prognosis. Conventional antileukemia therapy has limited efficacy in this setting. Thus, MPN-AP/BP is an urgent unmet clinical need. Modest responses to hypomethylating agents and single-agent ruxolitinib have been reported. More recently, combination of ruxolitinib and decitabine has demonstrated synergistic in vitro activity in human and murine systems. These observations led us to conduct a phase 1 study to explore the safety of combined decitabine and dose-escalated ruxolitinib in patients with MPN-AP/BP. A total of 21 patients were accrued to this multicenter study. Ruxolitinib was administered at doses of 10, 15, 25, or 50 mg twice daily in combination with decitabine (20 mg/m2per day for 5 days) in 28-day cycles. The maximum tolerated dose was not reached. The most common reasons for study discontinuation were toxicity/adverse events (37%) and disease progression (21%). Fourteen patients died during study treatment period or follow-up. The median overall survival for patients on study was 7.9 months (95% confidence interval, 4.1-not reached). Among evaluable patients, the overall response rate by protocol-defined criteria (complete remission with incomplete count recovery + partial remission) was 9/17 (53%) and by intention-to-treat analysis was 9/21 (42.9%). The combination of decitabine and ruxolitinib was generally well tolerated by patients with MPN-AP/BP and demonstrates potentially promising clinical activity. A phase 2 trial evaluating the efficacy of this combination regimen is ongoing within the Myeloproliferative Disorder Research Consortium.