Ali Tabarroki

Impact of molecular mutations on treatment response to DNMT inhibitors in myelodysplasia and related neoplasms

We hypothesized that specific molecular mutations are important biomarkers for response to DNA methyltransferase inhibitors (DNMT inhibitors) and may have prognostic value in patients with myelodysplastic syndromes (MDS). Mutational analysis was performed in 92 patients with MDS and related disorders who received 5-azacytidine (n=55), decitabine (n=26) or both (n=11). Mutational status was correlated with overall response rate (ORR), progression-free survival (PFS) and overall survival (OS) by univariate and multivariate analysis. Risk stratification models were created. TET2, DNMT3A, IDH1/IDH2, ASXL1, CBL, RAS and SF3B1 mutations were found in 18, 9, 8, 26, 3, 2 and 13% of patients, respectively. In multivariate analysis, TET2MUT and/or DNMT3AMUT (P=0.03), platelets⩾100 × 109/l (P=0.007) and WBC<3.0 × 109/l (P=0.03) were independent predictors of better response. TET2MUT and/or DNMT3AMUT (P=0.04) status was also independently prognostic for improved PFS, as were good or intermediate cytogenetic risk (P<0.0001), age<60 (P=0.0001), treatment with both 5-azacytidine and decitabine (P=0.02) and hemoglobin⩾10 g/dl (P=0.01). Better OS was associated with ASXL1WT (P=0.008) and SF3B1MUT (P=0.01), and, similar to PFS, cytogenetic risk (P=0.0002), age (P=0.02) and hemoglobin (P=0.04). These data support the role of molecular mutations as predictive biomarkers for response and survival in MDS patients treated with DNMT inhibitors.

Phase 2 study of the lenalidomide and azacitidine combination in patients with higher-risk myelodysplastic syndromes

Lenalidomide and azacitidine each have activity in myelodysplastic syndromes (MDS) patients, where both microenvironment and cell-regulatory mechanisms contribute to disease pathogenesis. The objective of this multicenter, phase 2 expansion trial was to determine the efficacy and safety of combination therapy with azacitidine (75 mg/m(2)/d for 5 days) and lenalidomide (10 mg/d for 21 days; 28-day cycle) in patients with higher-risk MDS. Among 36 patients enrolled (18 phase 1, 18 phase 2), median age was 68 years (range, 47-78 years) and follow-up was 12 months (range, 3-55 years). IPSS categories included intermediate-1 (n = 5 patients with excess blasts), intermediate-2 (20), and high (11). Common grade 3/4 nonhematologic adverse events included febrile neutropenia (22% of patients), other infection (11%), pulmonary (11%), cardiac (11%), constitutional (11%), and dermatologic (11%). The overall response rate (per modified MDS International Working Group criteria) was 72%: 16 patients (44%) achieved a complete response (CR), and 10 (28%) had hematologic improvement. Median CR duration was 17+ months (range, 3-39+); median overall survival was 37+ months (range, 7-55+) for CR patients, and 13.6 months for the entire cohort (range, 3-55). TET2/DNMT3A/IDH1/2 mutational status was associated with response in a limited number of patients. The lenalidomide/azacitidine combination is well-tolerated and highly active in treating greater-risk MDS.

Increased CDA Expression/Activity in Males Contributes to Decreased Cytidine Analog Half-Life and Likely Contributes to Worse Outcomes with 5-Azacytidine or Decitabine Therapy

Purpose: The cytidine analogs 5-azacytidine and decitabine, used to treat myelodysplastic syndromes (MDS), produce a molecular epigenetic effect, depletion of DNA-methyltransferase 1 (DNMT1). This action is S-phase dependent. Hence, genetic factors that decrease the half-lives of these drugs could impact efficacy. Documentation of such impact, and elucidation of underlying mechanisms, could lead to improved clinical application. Experimental design: Cytidine deaminase (CDA) rapidly inactivates 5-azacytidine/decitabine. The effect of CDA SNP A79C and gender on CDA expression, enzyme activity, and drug pharmacokinetics/pharmacodynamics was examined in mice and humans, and the impact on overall survival (OS) was evaluated in 5-azacytidine/decitabine-treated patients with MDS (n = 90) and cytarabine-treated patients with acute myeloid leukemia (AML) (n = 76). Results: By high-performance liquid chromatography (HPLC), plasma CDA activity was decreased as expected in individuals with the SNP A79C. Interestingly and significantly, there was an even larger decrease in females than in males. Explaining this decrease, liver CDA expression was significantly lower in female versus male mice. As expected, decitabine plasma levels, measured by mass spectrometry, were significantly higher in females. In mathematical modeling, the detrimental impact of shorter drug half-life (e.g., in males) was greater in low compared with high S-phase fraction disease (e.g., MDS vs. AML), because in high S-phase fraction disease, even a short exposure treats a major portion of cells. Accordingly, in multivariate analysis, OS was significantly worse in male versus female patients with MDS treated with 5-azacytidine/decitabine. Conclusions: Increased CDA expression/activity in males contributes to decreased cytidine analog half-life and likely contributes to worse outcomes with 5-azacytidine or decitabine therapy. Clin Cancer Res; 19(4); 938–48. ©2012 AACR.

Distinct iron architecture in SF3B1 -mutant myelodysplastic syndrome patients is linked to an SLC25A37 splice variant with a retained intron

Perturbation in iron homeostasis is a hallmark of some hematologic diseases. Abnormal sideroblasts with accumulation of iron in the mitochondria are named ring sideroblasts (RS). RS is a cardinal feature of refractory anemia with RS (RARS) and RARS with marked thrombocytosis (RARS/-T). Mutations in SF3B1, a member of the RNA splicing machinery are frequent in RARS/-T and defects of this gene were linked to RS formation. Here we showcase the differences in iron architecture of SF3B1-mutant and wild-type (WT) RARS/-T and provide new mechanistic insights by which SF3B1 mutations lead to differences in iron. We found higher iron levels in SF3B1 mutant vs WT RARS/-T by transmission electron microscopy/spectroscopy/flow cytometry. SF3B1 mutations led to increased iron without changing the valence as shown by the presence of Fe2+ in mutant and WT. Reactive oxygen species and DNA damage were not increased in SF3B1-mutant patients. RNA-sequencing and Reverse transcriptase PCR showed higher expression of a specific isoform of SLC25A37 in SF3B1-mutant patients, a crucial importer of Fe2+ into the mitochondria. Our studies suggest that SF3B1 mutations contribute to cellular iron overload in RARS/-T by deregulating SLC25A37.

Ruxolitinib leads to improvement of pulmonary hypertension in patients with myelofibrosis

Pulmonary hypertension (PH) is a frequently under recognized complication of myelofibrosis (MF). The pathophysiology of PH in MF is unknown and no definitive therapies have been established. We studied 15 patients with MF-associated PH and compared their echocardiographic and PH relevant biomarkers (nitric oxide (NO), N-terminal pro-hormone of brain natriuretic peptide (NT-pro BNP), von Willebrand antigen (vWB), ristocetin-cofactor activity (RCA) and uric acid (UA)) pre- and post-ruxolitinib treatment. Ruxolitinib decreased the plasma levels of NT-pro BNP (73%; P=0.043), UA (60%), vWB (86%) and RCA (73%; P=0.036). Improvements in echocardiographic findings were also seen in 66% of patients (P=0.022). Furthermore, marked increase in NO compared with baseline (69.75 vs 40.1 picomolar (pM); P=0.001) was observed post-ruxolitinib therapy, whereas no changes were noted with conventional therapies. Treatment with ruxolitinib also resulted in the reduction of key cytokines (tumor necrosis factor alpha, interleukin-4 (IL-4), IL-6 and IL-8) and induction of interferon-gamma. Animal studies further supported the role of ruxolitinib in the induction of NO levels. In conclusion, aberrant Janus kinase (JAK)-signal transducer and activator of transcription signaling in MF may mediate PH through dysregulation of NO and cytokine levels, which can be restored by therapy with JAK inhibitors suggesting that inhibition of this pathway is a novel target for the management of patients with PH.

Successful use of very low dose subcutaneous decitabine to treat high-risk myelofibrosis with Sweet syndrome that was refractory to 5-azacitidine.

1 Department of Translational Hematology and Oncology Research, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH, USA, 2 Department of Anatomic Pathology, Cleveland Clinic, Cleveland, OH USA, 3 Department of Clinical Pathology, Cleveland Clinic, Cleveland, OH USA and 4 Leukemia Program, Department of Hematologic Oncology and Blood Disorders, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH, USA

Familial systemic mastocytosis with germline KIT K509I mutation is sensitive to treatment with imatinib, dasatinib and PKC412.

Mastocytosis are myeloproliferative neoplasms commonly related to gain-of-function mutations involving the tyrosine kinase domain of KIT. We herein report a case of familial systemic mastocytosis with the rare KIT K509I germ line mutation affecting two family members: mother and daughter. In vitro treatment with imatinib, dasatinib and PKC412 reduced cell viability of primary mast cells harboring KIT K509I mutation. However, imatinib was more effective in inducing apoptosis of neoplastic mast cells. Both patients with familial systemic mastocytosis had remarkable hematological and skin improvement after three months of imatinib treatment, suggesting that it may be an effective front line therapy for patients harboring KIT K509I mutation.

Immunomodulatory agents in myelofibrosis

Introduction: The treatment options for patients with myelofibrosis (MF) remain limited. Anemia, thrombocytopenia, extramedullary hematopoiesis, constitutional symptoms, and disease progression are the primary causes of morbidity and mortality. Traditional non-transplant therapies remain non-curative. Moreover, in the JAK2 inhibitor era, no single pharmacologic agent has been shown to improve all MF-related clinical manifestations. Immunomodulatory agents (IMiDs), such as thalidomide and lenalidomide, have been useful in the treatment of some MF patients while newer IMiDs such as pomalidomide are showing promise in MF. Areas covered: This review focuses on the biologic rationales of IMiDs and the clinical results supporting their use in MF. It includes data on the new IMiD, pomalidomide and also explores the possible utility of combining IMiDs with other agents. A PubMed search of articles related to IMiDs and myelofibrosis were conducted. Relevant studies and clinical studies with sample size of > 15 were included. Expert opinion: In the JAK2 inhibitor era, IMiDs are alternative treatments in managing splenomegaly and constitutional symptoms. They remain useful in the treatment of cytopenias. Pomalidomides good anemia response may lead to its inclusion as one of the frontline anemia therapies in MF. Molecular biomarkers may allow us to identify patients who will respond to IMiDs.

Ruxolitinib in combination with DNA methyltransferase inhibitors: clinical responses in patients with symptomatic myelofibrosis with cytopenias and elevated blast(s) counts

* Dr. Brady L. Stein and Dr. Ramon V. Tiu share equal senior authorship for this article. Correspondence: Brady L. Stein, MD, Assistant Professor of Medicine, Division of Hematology and Oncology, Department of Medicine, Northwestern Feinberg University School of Medicine, Chicago, IL, USA. Tel: 312-695-6832. Fax: 312-695-7814. E-mail: bstein@nmff .org. Ramon V. Tiu, MD, Assistant Professor of Molecular Medicine, Department of Translational Hematology and Oncology Research, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH, USA. Tel: 216-444-6730. Fax: 216-636-2498. E-mail: tiur@ccf.org

SF3B1 mutations are infrequently found in non-myelodysplastic bone marrow failure syndromes and mast cell diseases but, if present, are associated with the ring sideroblast phenotype

The bone marrow failure syndromes (BMFS) are hematologic disorders characterized by impaired production and sometimes function of peripheral blood (PB) cells. While they can be either acquired or inherited, the majority are acquired and include myelodysplastic syndromes (MDS), aplastic anemia (AA),