Oral decitabine-cedazuridine versus intravenous decitabine for myelodysplastic syndromes and chronic myelomonocytic leukaemia (ASCERTAIN): a registrational, randomised, crossover, pharmacokinetics, phase 3 study.

BACKGROUND: The DNA methyltransferase inhibitors azacitidine and decitabine for individuals with myelodysplastic syndromes or chronic myelomonocytic leukaemia are available in parenteral form. Oral therapy with similar exposure for these diseases would offer potential treatment benefits. We aimed to compare the safety and pharmacokinetics of oral decitabine plus the cytidine deaminase inhibitor cedazuridine versus intravenous decitabine. METHODS: We did a registrational, multicentre, open-label, crossover, phase 3 trial of individuals with myelodysplastic syndromes or chronic myelomonocytic leukaemia and individuals with acute myeloid leukaemia, enrolled as separate cohorts; results for only participants with myelodysplastic syndromes or chronic myelomonocytic leukaemia are reported here. In 37 academic and community-based clinics in Canada and the USA, we enrolled individuals aged 18 years or older who were candidates to receive intravenous decitabine, with Eastern Cooperative Oncology Group performance status 0 or 1 and a life expectancy of at least 3 months. Participants were randomly assigned (1:1) to receive 5 days of oral decitabine-cedazuridine (one tablet once daily containing 35 mg decitabine and 100 mg cedazuridine as a fixed-dose combination) or intravenous decitabine (20 mg/m2 per day by continuous 1-h intravenous infusion) in a 28-day treatment cycle, followed by 5 days of the other formulation in the next treatment cycle. Thereafter, all participants received oral decitabine-cedazuridine from the third cycle on until treatment discontinuation. The primary endpoint was total decitabine exposure over 5 days with oral decitabine-cedazuridine versus intravenous decitabine for cycles 1 and 2, measured as area under the curve in participants who received the full treatment dose in cycles 1 and 2 and had decitabine daily AUC0-24 for both oral decitabine-cedazuridine and intravenous decitabine (ie, paired cycles). On completion of the study, all patients were rolled over to a maintenance study. This study is registered with ClinicalTrials.gov, NCT03306264. FINDINGS: Between Feb 8, 2018, and June 7, 2021, 173 individuals were screened, 138 (80%) participants were randomly assigned to a treatment sequence, and 133 (96%) participants (87 [65%] men and 46 [35%] women; 121 [91%] White, four [3%] Black or African-American, three [2%] Asian, and five [4%] not reported) received treatment. Median follow-up was 966 days (IQR 917-1050). Primary endpoint of total exposure of oral decitabine-cedazuridine versus intravenous decitabine was 98·93% (90% CI 92·66-105·60), indicating equivalent pharmacokinetic exposure on the basis of area under the curve. The safety profiles of oral decitabine-cedazuridine and intravenous decitabine were similar. The most frequent adverse events of grade 3 or worse were thrombocytopenia (81 [61%] of 133 participants), neutropenia (76 [57%] participants), and anaemia (67 [50%] participants). The incidence of serious adverse events in cycles 1-2 was 31% (40 of 130 participants) with oral decitabine-cedazuridine and 18% (24 of 132 participants) with intravenous decitabine. There were five treatment-related deaths; two deemed related to oral therapy (sepsis and pneumonia) and three to intravenous treatment (septic shock [n=2] and pneumonia [n=1]). INTERPRETATION: Oral decitabine-cedazuridine was pharmacologically and pharmacodynamically equivalent to intravenous decitabine. The results support use of oral decitabine-cedazuridine as a safe and effective alternative to intravenous decitabine for treatment of individuals with myelodysplastic syndromes or chronic myelomonocytic leukaemia. FUNDING: Astex Pharmaceuticals.

Integrative analysis of drug response and clinical outcome in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a cancer of myeloid-lineage cells with limited therapeutic options. We previously combined ex vivo drug sensitivity with genomic, transcriptomic, and clinical annotations for a large cohort of AML patients, which facilitated discovery of functional genomic correlates. Here, we present a dataset that has been harmonized with our initial report to yield a cumulative cohort of 805 patients (942 specimens). We show strong cross-cohort concordance and identify features of drug response. Further, deconvoluting transcriptomic data shows that drug sensitivity is governed broadly by AML cell differentiation state, sometimes conditionally affecting other correlates of response. Finally, modeling of clinical outcome reveals a single gene, PEAR1, to be among the strongest predictors of patient survival, especially for young patients. Collectively, this report expands a large functional genomic resource, offers avenues for mechanistic exploration and drug development, and reveals tools for predicting outcome in AML.

A case study of 10 patients administered HBOC-201 in high doses over a prolonged period: outcomes during severe anemia when transfusion is not an option.

BACKGROUND: Hemoglobin-Based Oxygen Carriers (HBOCs) can act as an "oxygen bridge" in acute severe anemia when transfusion is indicated, but not possible. We present data on 10 Expanded Access (EA) patients treated with high cumulative doses of Hemopure (HBOC-201), to assess the ability of HBOC-201 to safely treat life threatening anemia in situations where high volumes of product were administered over an extended period of time. STUDY DESIGN AND METHODS: Inclusion in this study required that the patient receive at least 10 units of HBOC-201 between 2014 and 2017 under the FDA-sanctioned EA program. Depending on a patient's geographical location, treatment with HBOC-201 was obtained through either a single patient emergency Investigational New Drug (IND) application, or an intermediate size population IND. Of the 41 patients who were treated during this period, 10 patients received 10 or more units of the product. Data were obtained from medical records. RESULTS: Treatments with HBOC-201 started within 24 hours of signing consent and were administered at an average rate of 1.99 (SD 0.17) units per day over a mean of 8.2 days (SD 2.9), during which patients received on average 16.2 units (SD 5.7 units) of HBOC-201. The median pre-treatment nadir corpuscular hemoglobin (Hb) concentration was 3.3 (SD 0.9) g/dL and post-treatment Hemoglobin was 7.3 (SD 1.7) g/dL. Common side effects included methemoglobinemia, gastrointestinal symptoms, and hypertension. However, no product-related serious adverse events (SAEs) were noted. All patients survived. CONCLUSIONS: Administration of HBOC-201 over an extended period is a feasible and safe oxygen bridge for severely anemic patients who cannot be transfused with RBC.

Efficacy of Ruxolitinib in Patients With Chronic Neutrophilic Leukemia and Atypical Chronic Myeloid Leukemia.

PURPOSE: Colony-stimulating factor-3 receptor (CSF3R)-T618I is a recurrent activating mutation in chronic neutrophilic leukemia (CNL) and to a lesser extent in atypical chronic myeloid leukemia (aCML) resulting in constitutive JAK-STAT signaling. We sought to evaluate safety and efficacy of the JAK1/2 inhibitor ruxolitinib in patients with CNL and aCML, irrespective of CSF3R mutation status. METHODS: We conducted a phase II study of ruxolitinib in 44 patients (21 CNL and 23 aCML). The primary end point was overall hematologic response rate (ORR) by the end of 6 continuous 28-day cycles for the first 25 patients enrolled. We considered a response as either partial (PR) or complete response (CR). We expanded accrual to 44 patients to increase our ability to evaluate secondary end points, including grade ≥ 3 adverse events, spleen volume, symptom assessment, genetic correlates of response, and 2-year survival. RESULTS: ORR was 32% for the first 25 enrolled patients (8 PR [7 CNL and 1 aCML]). In the larger cohort of 44 patients, 35% had a response (11 PR [9 CNL and 2 aCML] and 4 CR [CNL]), and 50% had oncogenic CSF3R mutations. The mean absolute allele burden reduction of CSF3R-T618I after 6 cycles was greatest in the CR group, compared with the PR and no response groups. The most common cause of death is due to disease progression. Grade ≥ 3 anemia and thrombocytopenia were observed in 34% and 14% of patients, respectively. No serious adverse events attributed to ruxolitinib were observed. CONCLUSION: Ruxolitinib was well tolerated and demonstrated an estimated response rate of 32%. Patients with a diagnosis of CNL and/or harboring CSF3R-T618I were most likely to respond.

Genomic landscape of neutrophilic leukemias of ambiguous diagnosis.

Chronic neutrophilic leukemia (CNL), atypical chronic myeloid leukemia (aCML), and myelodysplastic/myeloproliferative neoplasms, unclassifiable (MDS/MPN-U) are a group of rare and heterogeneous myeloid disorders. There is strong morphologic resemblance among these distinct diagnostic entities as well as a lack of specific molecular markers and limited understanding of disease pathogenesis, which has made diagnosis challenging in certain cases. The treatment has remained empirical, resulting in dismal outcomes. We, therefore, performed whole-exome and RNA sequencing of these rare hematologic malignancies and present the most complete survey of the genomic landscape of these diseases to date. We observed a diversity of combinatorial mutational patterns that generally do not cluster within any one diagnosis. Gene expression analysis reveals enrichment, but not cosegregation, of clinical and genetic disease features with transcriptional clusters. In conclusion, these groups of diseases represent a continuum of related diseases rather than discrete diagnostic entities.

Allogeneic transplantation outcomes amongst a contemporary cohort of high-risk myelodysplastic syndrome and acute myeloid leukemia patients aged ≥70 years.

Allogeneic hematopoietic stem cell transplantation (HSCT) is an integral therapy for patients with hematological malignancies, myelodysplasia, and bone marrow failure. Its use has been increasing over the past decade, as understanding of the treatment and its related toxicities has led to changes in patient selection, conditioning regimens, and post-transplant care. Older (age ≥65 years) patients are often considered unfit for transplantation; however, more recent data suggest that older patients, when selected appropriately, tolerate transplantation well. We report our institutional experience with HSCT in patients aged ≥70 years. A cohort of 22 patients underwent HSCT. Median overall survival was 5.16 years [95% confidence interval (CI): 1.5-8.7 years], and median post-transplant survival was 2.2 years (myelodysplastic syndrome: median 1.3 years, 95% CI: 4.7 months-2.2 years; acute myeloid leukemia: median not reached). Thirty-day mortality following HSCT was 9.5% (n = 2). These data provide further support for the use of HSCT in selected older patients, and highlight the impact of HSCT on overall survival among a patient cohort primarily of acute myeloid leukemia and myelodysplasia.

Two myeloid leukemia cases with rare FLT3 fusions.

Genetic rearrangements involving FLT3 are rare and only recently have been detected in myeloid/lymphoid neoplasms associated with eosinophilia (MLN-eos) and chronic myeloproliferative disorders. Here we report two cases with FLT3 fusions in patients demonstrating mixed features of myelodysplastic/myeloproliferative neoplasms. In the first case, FLT3 was fused with a new fusion partner MYO18A in a patient with marrow features most consistent with atypical chronic myeloid leukemia; the second case involving ETV6-FLT3 fusion was observed in a case with bone marrow features most consistent with chronic myelomonocytic leukemia. Notably, we observed that samples from both patients demonstrated FLT3 inhibitor (quizartinib and sorafenib) sensitivity in ex vivo drug screening assay.

Functional genomic landscape of acute myeloid leukaemia.

The implementation of targeted therapies for acute myeloid leukaemia (AML) has been challenging because of the complex mutational patterns within and across patients as well as a dearth of pharmacologic agents for most mutational events. Here we report initial findings from the Beat AML programme on a cohort of 672 tumour specimens collected from 562 patients. We assessed these specimens using whole-exome sequencing, RNA sequencing and analyses of ex vivo drug sensitivity. Our data reveal mutational events that have not previously been detected in AML. We show that the response to drugs is associated with mutational status, including instances of drug sensitivity that are specific to combinatorial mutational events. Integration with RNA sequencing also revealed gene expression signatures, which predict a role for specific gene networks in the drug response. Collectively, we have generated a dataset-accessible through the Beat AML data viewer (Vizome)-that can be leveraged to address clinical, genomic, transcriptomic and functional analyses of the biology of AML.

Recent Progress in Chronic Neutrophilic Leukemia and Atypical Chronic Myeloid Leukemia.

PURPOSE OF REVIEW: We reviewed recent diagnostic and therapeutic progress in chronic neutrophilic leukemia (CNL) and atypical chronic myeloid leukemia (aCML). We summarized recent genetic data that may guide future efforts towards implementing risk-adapted therapy based on mutational profile and improving disease control and survival of affected patients. RECENT FINDINGS: Recent genetic data in CNL and aCML prompted modifications to the World Health Organization (WHO) diagnostic criteria, which have improved our understanding of how CNL and aCML are different diseases despite sharing common findings of peripheral granulocytosis and marrow myeloid hyperplasia. The overlap of recurrently mutated genes between aCML and CMML support considering CSF3R-T618I mutated cases as a distinct entity, either as CNL or CNL with dysplasia. Ongoing preclinical and clinical studies will help to further inform the therapeutic approach to these diseases. Our understanding of CNL and aCML has greatly advanced over the last few years. This will improve clarity for the diagnosis of these diseases, provide a strategy for risk stratification, and guide risk-adapted therapy.

Myelodysplastic Syndromes: Updates and Nuances.

Myelodysplastic syndrome (MDS) is a heterogeneous, clonal stem cell disorder of the blood and marrow typically diagnosed based on the presence of persistent cytopenia(s), dysplastic cells, and genetic markers. Common issues that arise in the clinical management include difficulty confirming MDS diagnosis, lack of a standard approach with novel agents in MDS, and few prospective long-term, randomized controlled MDS clinical studies to guide allogeneic blood and marrow transplant. With the recent genetic characterization of MDS, certain aspects of these issues will be better addressed by integrating genetic data into clinical study design and clinical practice.

Polycythemia Vera Management and Challenges in the Community Health Setting.

Patients with polycythemia vera (PV) experience shortened survival, increased risk of thromboembolic and hemorrhagic events, and burdensome symptoms. For all patients with PV, treatment with aspirin and hematocrit control with phlebotomy are recommended. In addition, patients with high-risk status or poor hematocrit control benefit from cytoreductive therapy with hydroxyurea, although approximately 1 in 4 patients develops resistance or intolerance. For patients who are resistant to or intolerant of hydroxyurea, studies have shown that ruxolitinib, a Janus kinase 1/2 inhibitor, provides hematocrit control, reduces spleen size, normalizes blood counts, and improves PV-related symptoms. For many patients, PV is managed in a community health setting, and it is important that community hematologists, oncologists, and internists are familiar with the contemporary management of PV to improve patient outcomes, including management for patients who present with unique health-care needs. This review provides an overview of current treatment options for patients with PV and discusses challenging circumstances encountered by community providers in the management of PV, including symptom assessment, identification of hydroxyurea resistance/intolerance, pregnancy, elective surgeries, concomitant immunosuppressants, and managing patients in areas with limited access to specialized hematologic care.

Clonal hematopoiesis as determined by the HUMARA assay is a marker for acquired mutations in epigenetic regulators in older women.

Recent large cohort studies revealed that healthy older individuals harbor somatic mutations that increase their risk for hematologic malignancy and all-cause cardiovascular deaths. The majority of these mutations are in chromatin and epigenetic regulatory genes (CERGs). CERGs play a key role in regulation of DNA methylation (DNMT3A and TET2) and histone function (ASXL1) and in clonal proliferation of hematopoietic stem cells. We hypothesize that older women manifesting clonal hematopoiesis, defined here as a functional phenomenon in which a hematopoietic stem cell has acquired a survival and proliferative advantage, harbor a higher frequency of somatic mutations in CERGs. The human androgen receptor gene (HUMARA) assay was used in our study to detect the presence of nonrandom X inactivation in women, a marker for clonal hematopoiesis. In our pilot study, we tested 127 blood samples from women ≥65 years old without a history of invasive cancer or hematologic malignancies. Applying stringent qualitative criteria, we found that 26% displayed clonal hematopoiesis; 52.8% displayed polyclonal hematopoiesis; and 21.3% had indeterminate patterns (too close to call by qualitative assessment). Using Illumina MiSeq next-generation sequencing, we identified somatic mutations in CERGs in 15.2% of subjects displaying clonal hematopoiesis (three ASXL1 and two DNMT3A mutations with an average variant allele frequency of 15.7%, range: 6.3%-23.3%). In a more limited sequencing analysis, we evaluated the frequency of ASXL1 mutations by Sanger sequencing and found mutations in 9.7% of the clonal samples and 0% of the polyclonal samples. By comparing several recent studies (with some caveats as described), we determined the fold enrichment of detecting CERG mutations by using the HUMARA assay as a functional screen for clonal hematopoiesis. We conclude that a functional assay of clonal hematopoiesis is enriching for older women with somatic mutations in CERGs, particularly for ASXL1 and TET2 mutations and less so for DNMT3A mutations.

Mutant calreticulin-expressing cells induce monocyte hyperreactivity through a paracrine mechanism.

Mutations in the calreticulin gene (CALR) were recently identified in approximately 70-80% of patients with JAK2-V617F-negative essential thrombocytosis and primary myelofibrosis. All frameshift mutations generate a recurring novel C-terminus. Here we provide evidence that mutant calreticulin does not accumulate efficiently in cells and is abnormally enriched in the nucleus and extracellular space compared to wildtype calreticulin. The main determinant of these findings is the loss of the calcium-binding and KDEL domains. Expression of type I mutant CALR in Ba/F3 cells confers minimal IL-3-independent growth. Interestingly, expression of type I and type II mutant CALR in a nonhematopoietic cell line does not directly activate JAK/STAT signaling compared to wildtype CALR and JAK2-V617F expression. These results led us to investigate paracrine mechanisms of JAK/STAT activation. Here we show that conditioned media from cells expressing type I mutant CALR exaggerate cytokine production from normal monocytes with or without treatment with a toll-like receptor agonist. These effects are not dependent on the novel C-terminus. These studies offer novel insights into the mechanism of JAK/STAT activation in patients with JAK2-V617F-negative essential thrombocytosis and primary myelofibrosis.

What's different about atypical CML and chronic neutrophilic leukemia?

Atypical chronic myeloid leukemia (aCML) and chronic neutrophilic leukemia (CNL) are rare myeloid neoplasms defined largely by morphologic criteria. The discovery of CSF3R mutations in aCML and CNL have prompted a more comprehensive genetic profiling of these disorders. These studies have revealed aCML to be a genetically more heterogeneous disease than CNL, however, several groups have reported that SETBP1 and ASXL1 mutations occur at a high frequency and carry prognostic value in both diseases. We also report a novel finding-our study reveals a high frequency of U2AF1 mutations at codon Q157 associated with CSF3R mutant myeloid neoplasms. Collectively, these findings will refine the WHO diagnostic criteria of aCML and CNL and help us understand the genetic lesions and dysregulated signaling pathways contributing to disease development. Novel therapies that emerge from these genetic findings will need to be investigated in the setting of a clinical trial to determine the safety and efficacy of targeting various oncogenic drivers, such as JAK1/2 inhibition in CSF3R-T618I-positive aCML and CNL. In summary, recent advances in the genetic characterization of CNL and aCML are instrumental toward the development of new lines of therapy for these rare leukemias that lack an established standard of care and are historically associated with a poor prognosis.

Durable Disease Control with MEK Inhibition in a Patient with NRAS-mutated Atypical Chronic Myeloid Leukemia.

Atypical chronic myeloid leukemia (aCML) and chronic neutrophilic leukemia (CNL) are rare hematologic neoplasms characterized by leukocytosis and a hypercellular bone marrow. Although recurrent mutations in the colony-stimulating factor 3 receptor (CSF3R) are frequently observed in patients with (CNL), the mutational landscape in (aCML) is less well-defined. In this report, we describe an 81-year-old male who was diagnosed with aCML. He presented with leukocytosis and anemia but no significant clinical symptoms. Standard laboratory studies revealed the absence of the Philadelphia chromosome. Massively parallel sequencing demonstrated no mutations in CSF3R, but the presence of a heterozygous NRAS-G12D variant (47% allele frequency). The patient was started on treatment with trametinib, an MEK1/2 inhibitor with Food and Drug Administration approval for malignant melanoma. Therapy with trametinib resulted in exceptional improvements in his blood counts and continued disease control with 14 months of follow-up. This case highlights the need for clinical trials evaluating the safety and efficacy of MEK1/2 as a therapeutic target for the treatment of patients with NRAS-mutated aCML/CNL.

Significant clinical response to JAK1/2 inhibition in a patient with CSF3R-T618I-positive atypical chronic myeloid leukemia.

Mutations in CSF3R (colony-stimulating factor 3 receptor) are frequent oncogenic drivers in chronic neutrophilic leukemia (CNL) and atypical chronic myeloid leukemia (aCML). Here we describe a 75 year old man who was diagnosed with CSF3R-T618I-positive atypical CML. He presented with leukocytosis, anemia, and thrombocytopenia and developed massive splenomegaly and severe constitutional symptoms. Hydroxyurea was given over a 6 month period but failed to provide any measureable clinical benefit. Eventually, he was treated with ruxolitinib, an FDA-approved JAK1/2 inhibitor, which resulted in dramatic improvement of his blood counts. He also had significant reduction of spleen volume and constitutional symptoms. This case highlights the need for a clinical trial to interrogate JAK1/2 as a potential molecular target in CNL and aCML in patients with or without CSF3R mutation. A clinical trial evaluating the safety and efficacy of ruxolitinib for this patient population is registered at ClinicalTrials.gov (NCT02092324).

The PI3K/Akt1 pathway enhances steady-state levels of FANCL.

Fanconi anemia hematopoietic stem cells display poor self-renewal capacity when subjected to a variety of cellular stress. This phenotype raises the question of whether the Fanconi anemia proteins are stabilized or recruited as part of a stress response and protect against stem cell loss. Here we provide evidence that FANCL, the E3 ubiquitin ligase of the Fanconi anemia pathway, is constitutively targeted for degradation by the proteasome. We confirm biochemically that FANCL is polyubiquitinated with Lys-48-linked chains. Evaluation of a series of N-terminal-deletion mutants showed that FANCL's E2-like fold may direct ubiquitination. In addition, our studies showed that FANCL is stabilized in a complex with axin1 when glycogen synthase kinase-3ß is overexpressed. This result leads us to investigate the potential regulation of FANCL by upstream signaling pathways known to regulate glycogen synthase kinase-3ß. We report that constitutively active, myristoylated-Akt increases FANCL protein level by reducing polyubiquitination of FANCL. Two-dimensional PAGE analysis shows that acidic forms of FANCL, some of which are phospho-FANCL, are not subject to polyubiquitination. These results indicate that a signal transduction pathway involved in self-renewal and survival of hematopoietic stem cells also functions to stabilize FANCL and suggests that FANCL participates directly in support of stem cell function.

Next-generation medicine: combining BCR-ABL and Hedgehog-targeted therapies.

Disease relapse remains a major cause of death in patients with BCR-ABL-positive leukemia despite advances in treatment with kinase inhibitors. Significant efforts are underway to target pathways that maintain leukemia stem cells. Targeting these pathways holds promise for definitive leukemia eradication or improvement of the effectiveness of currently available therapies.