Paula Rodriguez-Otero

Epigenetic Silencing of the Tumor Suppressor MicroRNA Hsa-miR-124a Regulates CDK6 Expression and Confers a Poor Prognosis in Acute Lymphoblastic Leukemia

Whereas transcriptional silencing of genes due to epigenetic mechanisms is one of the most important alterations in acute lymphoblastic leukemia (ALL), some recent studies indicate that DNA methylation contributes to down-regulation of miRNAs during tumorigenesis. To explore the epigenetic alterations of miRNAs in ALL, we analyzed the methylation and chromatin status of the miR-124a loci in ALL. Expression of miR-124a was down-regulated in ALL by hypermethylation of the promoter and histone modifications including decreased levels of 3mk4H3 and AcH3 and increased levels of 2mK9H3, 3mK9H3, and 3mK27H3. Epigenetic down-regulation of miR-124a induced an up-regulation of its target, CDK6, and phosphorylation of retinoblastoma (Rb) and contributed to the abnormal proliferation of ALL cells both in vitro and in vivo. Cyclin-dependent kinase 6 (CDK6) inhibition by sodium butyrate or PD-0332991 decreased ALL cell growth in vitro, whereas overexpression of pre-miR124a led to decreased tumorigenicity in a xenogeneic in vivo Rag2(-/-)gammac(-/-) mouse model. The clinical implications of these findings were analyzed in a group of 353 patients diagnosed with ALL. Methylation of hsa-miR-124a was observed in 59% of the patients, which correlated with down-regulation of miR-124a (P < 0.001). Furthermore, hypermethylation of hsa-miR-124a was associated with higher relapse rate (P = 0.001) and mortality rate (P < 0.001), being an independent prognostic factor for disease-free survival (P < 0.001) and overall survival (P = 0.005) in the multivariate analysis. These results provide the grounds for new therapeutic strategies in ALL either targeting the epigenetic regulation of microRNAs and/or directly targeting the CDK6-Rb pathway.

MicroRNA expression profiling in Imatinib-resistant Chronic Myeloid Leukemia patients without clinically significant ABL1-mutations.

The development of Imatinib Mesylate (IM), the first specific inhibitor of BCR-ABL1, has had a major impact in patients with Chronic Myeloid Leukemia (CML), establishing IM as the standard therapy for CML. Despite the clinical success obtained with the use of IM, primary resistance to IM and molecular evidence of persistent disease has been observed in 20-25% of IM treated patients. The existence of second generation TK inhibitors, which are effective in patients with IM resistance, makes identification of predictors of resistance to IM an important goal in CML. In this study, we have identified a group of 19 miRNAs that may predict clinical resistance to IM in patients with newly diagnosed CML.

Assessing complement blockade in patients with paroxysmal nocturnal hemoglobinuria receiving eculizumab

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by intravascular hemolysis, which is effectively controlled with eculizumab, a humanized monoclonal antibody that binds complement protein 5 (C5). The residual functional activity of C5 can be screened using a 50% hemolytic complement (CH50) assay, which is sensitive to the reduction, absence, and/or inactivity of any components of the classical and terminal complement pathway. Little data exist on complement blockade during treatment. From 2010 to 2012, clinical data, hemolysis biomarkers, complement assessment, and free eculizumab circulating levels were systematically measured immediately before every injection given to 22 patients with hemolytic PNH while receiving eculizumab therapy. During the study, 6 patients received ≥1 red blood cell transfusion. Lack of detectable CH50 activity (defined by CH50 ≤ 10% of normal values) was found in 184 samples (51%) and was significantly associated with lower lactate dehydrogenase levels (P = .002). Low levels of circulating free eculizumab (<50 µg/mL) correlated with detectable CH50 activity (CH50 > 10%; P = .004), elevated bilirubin levels (P < .0001), and the need for transfusions (P = .034). This study suggests that both CH50 activity and circulating free eculizumab levels may help physicians to manage PNH patients receiving eculizumab.

Frequent and Simultaneous Epigenetic Inactivation of TP53 Pathway Genes in Acute Lymphoblastic Leukemia

Aberrant DNA methylation is one of the most frequent alterations in patients with Acute Lymphoblastic Leukemia (ALL). Using methylation bead arrays we analyzed the methylation status of 807 genes implicated in cancer in a group of ALL samples at diagnosis (n = 48). We found that 154 genes were methylated in more than 10% of ALL samples. Interestingly, the expression of 13 genes implicated in the TP53 pathway was downregulated by hypermethylation. Direct or indirect activation of TP53 pathway with 5-aza-2′-deoxycitidine, Curcumin or Nutlin-3 induced an increase in apoptosis of ALL cells. The results obtained with the initial group of 48 patients was validated retrospectively in a second cohort of 200 newly diagnosed ALL patients. Methylation of at least 1 of the 13 genes implicated in the TP53 pathway was observed in 78% of the patients, which significantly correlated with a higher relapse (p = 0.001) and mortality (p<0.001) rate being an independent prognostic factor for disease-free survival (DFS) (p = 0.006) and overall survival (OS) (p = 0.005) in the multivariate analysis. All these findings indicate that TP53 pathway is altered by epigenetic mechanisms in the majority of ALL patients and correlates with prognosis. Treatments with compounds that may reverse the epigenetic abnormalities or activate directly the p53 pathway represent a new therapeutic alternative for patients with ALL.

Deregulation of FGFR1 and CDK6 oncogenic pathways in acute lymphoblastic leukaemia harbouring epigenetic modifications of the MIR9 family.

The role of epigenetic mechanisms in the regulation of microRNAs (miRNAs) with a tumour‐suppressor function in human neoplasms has recently been established. Several miRNAs have been found to be inappropriately regulated by DNA methylation in patients with acute lymphoblastic leukaemia (ALL). We analysed the methylation status of the three members of the MIR9 family (MIR9‐1, MIR9‐2 and MIR9‐3) in a uniformly treated cohort of 200 newly diagnosed ALLs. MIR9 was methylated in 54% of the patients and was associated with downregulation of MIR9 (P < 0·01). Hypermethylation of MIR9 was an independent prognostic factor for disease‐free survival, overall survival and event‐free survival in a multivariate analysis (P < 0·01). Epigenetic downregulation of MIR9 induced upregulation of its targets, FGFR1 and CDK6, while treatment of ALL cells with FGFR1 (PD‐173074) and CDK6 (PD‐0332991) inhibitors induced a decrease in cell proliferation and an increase in apoptosis of ALL cells. Our results indicate that the MIR9 family is involved in the pathogenesis and clinical behaviour of ALL and provide the basis for new therapeutic strategies in the treatment of ALL, targeting the epigenetic regulation of miRNAs and/or the FGFR1 or CDK6‐RB pathway directly.

Epigenetic regulation of the non‐canonical Wnt pathway in acute myeloid leukemia

Wnt5a is a member of the Wnt family of proteins that signals through the non‐canonical Wnt/Ca2+pathway to suppress cyclin D1. Deregulation of this pathway has been found in animal models suggesting that it acts as tumour suppressor in acute myeloid leukemia (AML). Although DNA methylation is the main mechanism of regulation of the canonical Wnt pathway in AML, the role of WNT5A abnormalities has never been evaluated in this clinical setting. The methylation status of WNT5A promoter–exon 1 was analyzed by methylation‐specific PCR and sequencing in eleven AML‐derived cell lines and 252 AML patients. We observed WNT5A hypermethylation in seven cell lines and in 43% (107/252) of AML patients. WNT5A methylation was associated with decreased WNT5A expression (P < 0.001) that was restored after exposure to 5‐Aza‐2’‐deoxycytidine. Moreover, WNT5A hypermethylation correlated with upregulation of CYCLIN D1 expression (P < 0.001). Relapse (15%vs 37%, P < 0.001) and mortality (61%vs 79%, P = 0.004) rates were lower for patients in the non‐methylated group. Disease‐free survival and overall survival at 6 and 7 years, respectively, were 60% and 27% for unmethylated patients and 20% and 0% for hypermethylated patients (P = 0.0001 and P = 0.04, respectively). Interestingly, significant differences were also observed when the analysis was carried out according to cytogenetic risk groups. We demonstrate that WNT5A, a putative tumor suppressor gene in AML, is silenced by methylation in this disease and that this epigenetic event is associated with upregulation of CYCLIN D1 expression and confers poor prognosis in patients with AML. (Cancer Sci 2009)

Long-term follow up after allogeneic stem cell transplantation in patients with severe aplastic anemia after cyclophosphamide plus antithymocyte globulin conditioning

Background Due to increased rates of secondary solid organ cancer in patients with severe aplastic anemia who received an irradiation-based conditioning regimen, we decided some years ago to use the combination of cyclophosphamide and antithymocyte globulin. We report the long-term follow up of patients who underwent hematopoietic stem cell transplantation from an HLA-matched sibling donor after this conditioning regimen. Design and Methods We analyzed 61 consecutive patients transplanted from June 1991 to February 2010, following conditioning with cyclophosphamide (200 mg/kg) and antithymocyte globulin (2.5 mg/kg/day × 5 days). Results Median age was 21 years (range 4–43); 41 of the 61 patients were adults. Median duration of the disease before hematopoietic stem cell transplantation was 93 days. All but 2 patients received bone marrow as the source of stem cells and all but 2 engrafted. Cumulative incidence of acute grade II–IV graft-versus-host disease was 23% (95%CI 13–34) and 18 developed chronic graft-versus-host disease (cumulative incidence 32% at 72 months, 95% CI 20–46). In multivariate analysis, a higher number of infused CD3 cells was associated with an increased risk of developing chronic graft-versus-host disease (P=0.017). With a median follow up of 73 months (range 8–233), the estimated 6-year overall survival was 87% (95% CI 78–97). At 72 months, the cumulative incidence of avascular necrosis was 21% and 12 patients presented with endocrine dysfunction (cumulative incidence of 19%). Only one patient developed a secondary malignancy (Hodgkin’s lymphoma) during follow up. Conclusions Cyclophosphamide and antithymocyte globulin is an effective conditioning regimen for patients with severe aplastic anemia and is associated with low treatment-related mortality. Long-term complications include avascular necrosis and endocrine dysfunction.

Defining and treating high-risk multiple myeloma

Multiple myeloma (MM) is more recently being recognized as a heterogeneous group of disease with variability in outcomes based on specific clinical and biologic predictors. MM patients can be broadly categorized into standard, intermediate and high risk for disease relapse, morbidity and mortality. The high-risk features include patient-specific factors such as old age, poor performance status and comorbidities; clinical features such as primary plasma cell leukemia and extramedullary disease; disease-specific biologic features such as deletion 17p, t(4;14) and high-risk gene expression profiling signatures. The current paper reviews the available data on best therapeutic approaches for high-risk MM.

Parvovirus B19 acute infection and a reactivation of cytomegalovirus and herpesvirus 6 in a chronic myeloid leukemia patient during treatment with dasatinib (BMS-354825).

T lymphocyte activation is controlled by the T cell antigen receptor (TCR) in combination with additional signals triggered by accessory molecules present on the surface of the antigen-presenting cells (APC). Several studies have reported that treatment with imatinib can induce inhibition of the T-cell mediated immune response [1,2]. Imatinib inhibits T cell receptor-mediated T cell proliferation and activation in a dose-dependent manner and reduces tyrosine phosphorylation of ZAP-70 and LAT in response to activation through TCR [1]. Studies conducted in a mouse model indicate that imatinib treatment also leads to a selective inhibition of memory CTLs without affecting primary T or B cells responses [2]. Recent reports describing the development of severe viral infections such as varicella-zoster [3] and fatal hepatitis B virus reactivation in patients with CML treated with imatinib along with in vitro data suggest that the potential for immune suppression and viral reactivation in patients treated with abl and src kinase inhibitors should be considered [4]. We describe the development of a cytomegalovirus and herpes 6 virus associated with treatment with dasatinib (BMS354825) in a patient with diagnosis of CML. A 53-year-old woman was diagnosed with CML in November 2003. Treatment with imatinib was initiated at doses of 400 mg/day and further increased to 800 mg/day due to the persistence of BCR-ABL chromosome after 1 year. Treatment with imatinib was discontinued 2 years after diagnosis (September 2005) due to the development of grade III/IV cutaneous and gastrointestinal complications. In March 2006 treatment with dasatinib (BMS354825) was started at doses of 140 mg/day. One month after initiation of treatment with dasatinib, the patient was admitted to the hospital with atypical pneumonia (bilateral infiltrates in chest radiography, fever, dry cough, dyspnea and pleuritic pain). Despite extensive work-up, only three positive citomegalovirus cells were identified by the rapid viral culture (shell vial method) from peripheral blood. The patient was treated with antiviral (Ganciclovir) and antibiotic therapy (TeicoplaninMeropenem) with good response and was discharged after 15 days. In May 2006, the patient presented with a generalized cutaneous rash with positive serological test for parvovirus B19 (IgG titer 512 and IgM titer 40) and herpesvirus 6 (IgG titer 80). This pattern is compatible with an acute infection from parvovirus B19 because detectable low levels of B19 specific IgM can be found within 7 – 10 days of the exposure. We excluded the possibility to herpesvirus 6 infection because the patient had low levels of specific herpesvirus 6 IgG and a percentage of healthy adults are IgM positive at any time, making

Splenic marginal zone lymphoma with Evans’ syndrome, autoimmunity, and peripheral gamma/delta T cells

Dear Editor, Several theories have tried to explain the occurrence of autoantibodies in lymphoid malignancies. Autoimmune phenomena like Evans’ syndrome, lupus anticoagulants, autoimmune thrombocytopenia, and autoimmune hemolytic anemias are frequently associated with lymphomas [4, 5]. Recently, systemic lupus erythematosus (SLE) was associated with an increased risk of diffuse large B cell and marginal zone lymphomas [3]. Splenic marginal zone lymphoma (SMZL) is an indolent B cell lymphoma, which generally presents splenomegaly and involvement of both bone marrow and peripheral blood. In this report, we describe a patient with autoimmunity and bone marrow infiltration that probably broke the mechanisms of central tolerance in bone marrow producing the emergency of new auto-reactive clones and autoantibodies. A 60-year-old man had a 12-month history of B symptoms. Physical examination was unremarkable, except for the presence of massive splenomegaly. A computerized tomograhpy (CT) scan of the abdomen confirmed splenomegaly and visualized enlarged lymph nodes in retroperitoneum (Fig. 1). Blood smear and peripheral blood immunophenotype suggest SMZL with villous lymphocytes. The patient underwent laparotomy for splenectomy, lymph node, and bone marrow biopsies. Histology corresponds to SMZL. Follow-up studies detected the persistence of lymphoid neoplastic cells (1.4%) and a γδ T cell population (TCR γδ CD2+, CD3+, CD5 low+, CD7+, CD8 low+/−, CD16 −/+ CD56+, CD57 +/−) that represented 12% of the cells in peripheral blood. Five months after diagnosis, the patient presented with abdominal discomfort. On a CT scan, abdominal adenopatic progression was observed and the two populations (3.4% of SMZL and 4.9% of γδ T cells) persisted in peripheral blood. The patient received eight cycles of chemotherapy with cyclophosphamide–doxorubicin–vincristin–prednisone regime, resulting in radiological remission. At this moment, flow cytometry of peripheral blood revealed 0.0026% of tumor cells and 13.75% of γδ T cells. Four months after chemotherapy, the patient was admitted to the hospital with a recurrence of SZML. He showed, besides, respiratory insufficiency and Evans’ syndrome, associated with high titers of antiphospholipid antibodies, lupus anticoagulant and prolonged partial tromboplastin time, high levels of immune complexes, and low levels of both immunoglobulins and C3 and C4 fractions of complement. Antinuclear, antineutrophil cytoplasmic, anticardiolipin, antiplatelets, anti-HLA I, and antiEPCR antibodies were also detected. CT scan ascertained the presence of pulmonary emphysema and hepatomegaly, without associated enlarged lymph nodes. Peripheral blood flow-cytometry indicated an increase in the neoplastic B cell population (50%) and a decrease of γδ T cells (5%). The bone marrow biopsy confirmed lymphoid infiltration (SMZL 35% and γδ T cells 4%) by immunophenotype. Ann Hematol (2009) 88:177–178 DOI 10.1007/s00277-008-0555-z