Carlos E. Bueso-Ramos

MicroRNAs in body fluids--the mix of hormones and biomarkers.

Since the discovery of microRNAs (miRNAs), the study of these small noncoding RNAs has steadily increased and more than 10,000 papers have already been published. The great interest in miRNAs reflects their central role in gene-expression regulation and the implication of miRNA-specific aberrant expression in the pathogenesis of cancer, cardiac, immune-related and other diseases. Another avenue of current research is the study of circulating miRNAs in serum, plasma, and other body fluids—miRNAs may act not only within cells, but also at other sites within the body. The presence of miRNAs in body fluids may represent a gold mine of noninvasive biomarkers in cancer. Since deregulated miRNA expression is an early event in tumorigenesis, measuring circulating miRNA levels may also be useful for early cancer detection, which can contribute greatly to the success of treatment. In this Review, we discuss the role of fluid-expressed miRNAs as reliable cancer biomarkers and treatment-response predictors as well as potential new patient selection criteria for clinical trials. In addition, we explore the concept that miRNAs could function as hormones.

Curcumin Suppresses the Paclitaxel-Induced Nuclear Factor-κB Pathway in Breast Cancer Cells and Inhibits Lung Metastasis of Human Breast Cancer in Nude Mice

Currently, there is no effective therapy for metastatic breast cancer after surgery, radiation, and chemotherapy have been used against the primary tumor. Because curcumin suppresses nuclear factor-κB (NF-κB) activation and most chemotherapeutic agents activate NF-κB that mediates cell survival, proliferation, invasion, and metastasis, we hypothesized that curcumin would potentiate the effect of chemotherapy in advanced breast cancer and inhibit lung metastasis. We tested this hypothesis using paclitaxel (Taxol)-resistant breast cancer cells and a human breast cancer xenograft model. As examined by electrophoretic mobility gel shift assay, paclitaxel activated NF-κB in breast cancer cells and curcumin inhibited it; this inhibition was mediated through inhibition of IκBα kinase activation and IκBα phosphorylation and degradation. Curcumin also suppressed the paclitaxel-induced expression of antiapoptotic (XIAP, IAP-1, IAP-2, Bcl-2, and Bcl-xL), proliferative (cyclooxygenase 2, c-Myc, and cyclin D1), and metastatic proteins (vascular endothelial growth factor, matrix metalloproteinase-9, and intercellular adhesion molecule-1). It also enhanced apoptosis. In a human breast cancer xenograft model, dietary administration of curcumin significantly decreased the incidence of breast cancer metastasis to the lung and suppressed the expression of NF-κB, cyclooxygenase 2, and matrix metalloproteinase-9. Overall, our results indicate that curcumin, which is a pharmacologically safe compound, has a therapeutic potential in preventing breast cancer metastasis possibly through suppression of NF-κB and NF-κB–regulated gene products.

Chemoimmunotherapy with hyper-CVAD plus rituximab for the treatment of adult Burkitt and Burkitt-type lymphoma or acute lymphoblastic leukemia

Adult Burkitt‐type lymphoma (BL) and acute lymphoblastic leukemia (B‐ALL) are rare entities composing 1% to 5% of non‐Hodgkin lymphomas NHL) or ALL. Prognosis of BL and B‐ALL has been poor with conventional NHL or ALL regimens, but has improved with dose‐intensive regimens.

Long‐term follow‐up results of hyperfractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone (Hyper‐CVAD), a dose‐intensive regimen, in adult acute lymphocytic leukemia

Modern intensive chemotherapy regimens have improved the prognosis for patients with adult acute lymphocytic leukemia (ALL). With these regimens, the complete response rates are now reported to be > 80%, and the long‐term survival rates range from 30% to 45%. The current analysis updated the long‐term results with the original hyperfractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone (Hyper‐CVAD) program, with a median follow‐up time of 63 months.

Curcumin downregulates cell survival mechanisms in human prostate cancer cell lines

While the role of nuclear transcription factor activator protein-1 (AP-1) in cell proliferation, and of nuclear factor-κB (NF-κB) in the suppression of apoptosis are known, their role in survival of prostate cancer cells is not well understood. We investigated the role of NF-κB and AP-1 in the survival of human androgen-independent (DU145) and -dependent (LNCaP) prostate cancer cell lines. Our results show that the faster rate of proliferation of DU145 cells when compared to LNCaP cells correlated with the constitutive expression of activated NF-κB and AP-1 in DU-145 cells. The lack of constitutive expression of NF-κB and AP-1 in LNCaP cells also correlated with their sensitivity to the antiproliferative effects of tumor necrosis factor (TNF). TNF induced NF-κB activation but not AP-1 activation in LNCaP cells. In DU145 cells both c-Fos and c-Jun were expressed and treatment with TNF activated c-Jun NH2-terminal kinase (JNK), needed for AP-1 activation. In LNCaP cells, however, only low levels of c-Jun was expressed and treatment with TNF minimally activated JNK. Treatment of cells with curcumin, a chemopreventive agent, suppressed both constitutive (DU145) and inducible (LNCaP) NF-κB activation, and potentiated TNF-induced apoptosis. Curcumin alone induced apoptosis in both cell types, which correlated with the downregulation of the expression of Bcl-2 and Bcl-xL and the activation of procaspase-3 and procaspase-8. Overall, our results suggest that NF-κB and AP-1 may play a role in the survival of prostate cancer cells, and curcumin abrogates their survival mechanisms.

Genetic Analysis of Transforming Events That Convert Chronic Myeloproliferative Neoplasms to Leukemias

The oncogenetic events that transform chronic myeloproliferative neoplasms (MPN) to acute myeloid leukemias (AML) are not well characterized. We investigated the role of several genes implicated in leukemic transformation by mutational analysis of 63 patients with AML secondary to a preexisting MPN (sAML). Frequent mutations were identified in TET2 (26.3%), ASXL1 (19.3%), IDH1 (9.5%), and JAK2 (36.8%) mutations in sAML, and all possible mutational combinations of these genes were also observed. Analysis of 14 patients for which paired samples from MPN and sAML were available showed that TET2 mutations were frequently acquired at leukemic transformation [6 of 14 (43%)]. In contrast, ASXL1 mutations were almost always detected in both the MPN and AML clones from individual patients. One case was also observed where TET2 and ASXL1 mutations were found before the patient acquired a JAK2 mutation or developed clinical evidence of MPN. We conclude that mutations in TET2, ASXL1, and IDH1 are common in sAML derived from a preexisting MPN. Although TET2/ASXL1 mutations may precede acquisition of JAK2 mutations by the MPN clone, mutations in TET2, but not ASXL1, are commonly acquired at the time of leukemic transformation. Our findings argue that the mutational order of events in MPN and sAML varies in different patients, and that TET2 and ASXL1 mutations have distinct roles in MPN pathogenesis and leukemic transformation. Given the presence of sAML that have no preexisting JAK2/TET2/ASXL1/IDH1 mutations, our work indicates the existence of other mutations yet to be identified that are necessary for leukemic transformation.

Chemoimmunotherapy With a Modified Hyper-CVAD and Rituximab Regimen Improves Outcome in De Novo Philadelphia Chromosome–Negative Precursor B-Lineage Acute Lymphoblastic Leukemia

PURPOSE The adverse prognosis of CD20 expression in adults with de novo precursor B-lineage acute lymphoblastic leukemia (ALL) prompted incorporation of monoclonal antibody therapy with rituximab into the intensive chemotherapy regimen hyper-CVAD (fractionated cyclophosphamide, vincristine, doxorubicin, dexamethasone). Other modifications (irrespective of CD20 expression) included early anthracycline intensification, alterations in number of risk-adapted intrathecal chemotherapy treatments for CNS prophylaxis, additional early and late intensifications, and extension of maintenance phase chemotherapy by 6 months. PATIENTS AND METHODS Two hundred eighty-two adolescents and adults with de novo Philadelphia chromosome (Ph)-negative precursor B-lineage ALL were treated with standard or modified hyper-CVAD regimens. The latter incorporated standard-dose rituximab if CD20 expression > or = 20%. RESULTS The complete remission (CR) rate was 95% with 3-year rates of CR duration (CRD) and survival (OS) of 60% and 50%, respectively. In the younger (age < 60 years) CD20-positive subset, rates of CRD and OS were superior with the modified hyper-CVAD and rituximab regimens compared with standard hyper-CVAD (70% v 38%; P < .001% and 75% v 47%, P = .003). In contrast, rates of CRD and OS for CD20-negative counterparts treated with modified versus standard hyper-CVAD regimens were similar (72% v 68%, P = not significant [NS] and 64% v 65%, P = NS, respectively). Older patients with CD20-positive ALL did not benefit from rituximab-based chemoimmunotherapy (rates of CRD 45% v 50%, P = NS and OS 28% v 32%, P = NS, respectively), related in part to deaths in CR. CONCLUSION The incorporation of rituximab into the hyper-CVAD regimen appears to improve outcome for younger patients with CD20-positive Ph-negative precursor B-lineage ALL.

Stimulation of Megakaryocyte and Platelet Production by a Single Dose of Recombinant Human Thrombopoietin in Patients with Cancer

Thrombocytopenia is an important clinical problem in the management of patients in hematology and oncology practices. In the United States, the use of platelet transfusions to manage severe thrombocytopenia has steadily increased: Approximately 4 million units were transfused in 1982, and more than 8 million units were transfused in 1992 [1, 2]. This marked increase in the need for platelets has paralleled advances in organ transplantation, bone marrow transplantation, cardiac surgery, and the use of dose-intensive therapy in the treatment of chemosensitive malignant conditions. Although platelet transfusions may decrease the risk for fatal bleeding complications, repeated transfusions increase the risk for transmission of bacterial and viral pathogens, transfusion reactions, and transfusion-associated graft-versus-host disease. These transfusions also contribute to increasing health care costs and inconvenience to patients [3]. Thus, an agent that can increase platelet production and prevent or attenuate thrombocytopenia would be an important advance. Thrombopoietin, the ligand for the c-Mpl receptor (found on platelets and megakaryocyte progenitors), was recently cloned by several investigators and was shown to be a primary regulator of platelet production in vivo [4-8]. Thrombopoietin promotes both the proliferation of megakaryocyte progenitors and their maturation into platelet-producing megakaryocytes. In preclinical studies done in normal mice and nonhuman primates, thrombopoietin increased platelet counts to a level higher than those previously achieved with other thrombopoietic cytokines [9, 10]. Moreover, in a murine model for myelosuppression, recombinant thrombopoietin given as a single dose decreased the nadir and accelerated platelet recovery in mice that had been rendered pancytopenic by sublethal radiation and chemotherapy [11]. In these studies, more prolonged treatment (for as long as 8 days) provided no additional benefit and was associated with marked thrombocytosis during the recovery phase. On the basis of these observations, we initiated a phase I and II clinical and laboratory investigation of recombinant human thrombopoietin in patients with cancer who were at high risk for severe chemotherapy-induced thrombocytopenia. This trial was divided into two parts: Part I studied thrombopoietin given before chemotherapy, and part II studied thrombopoietin given after chemotherapy. The objective of part I, the results of which are reported here, was to assess the hematopoietic effects, pharmacodynamics, and clinical tolerance of this novel agent in patients who had normal hematopoietic function before chemotherapy. Methods Patients Patients with sarcoma who had never had chemotherapy, were suitable candidates for subsequent chemotherapy, and did not have rapidly progressive disease were eligible for this trial. Patients were required to have a Karnofsky performance status score of 80 or more, adequate bone marrow (absolute neutrophil count 1.5 109/L; platelet count 150 109/L and 450 109/L), adequate renal function (serum creatinine level 120 mol/L), and adequate hepatic function (alanine aminotransferase level < 3 times normal; bilirubin level < 1.5 times normal). Patients with a history of thromboembolic or bleeding disorders, significant cardiac disease, or previous pelvic radiation were excluded. Written informed consent was obtained from all patients before study entry in accordance with institutional guidelines. Design During the phase I dose-ranging portion of this clinical cohort study, thrombopoietin was administered as a single intravenous dose 3 weeks before chemotherapy. At study entry, three patients were assigned to each of four dose levels (0.3, 0.6, 1.2, and 2.4 g/kg of body weight). Patients who had no dose-limiting toxicity and did not develop neutralizing antibodies to thrombopoietin were eligible to receive thrombopoietin at the same doses after chemotherapy. Recombinant Human Thrombopoietin The thrombopoietin used in this study was provided by Genentech, Inc. (South San Francisco, California). Thrombopoietin is a full-length glycosylated molecule produced in a genetically modified mammalian cell line and purified by standard techniques. It was mixed with preservative-free normal saline as a diluent for injections. Clinical and Laboratory Monitoring Before and during the clinical trial, patients were monitored by complete histories; physical examinations; and laboratory tests, including a complete blood cell count with differential counts, serum chemistry, coagulation profile, urinalysis, assessment of thrombopoietin antibody formation, chest radiography, and electrocardiography. Blood counts were obtained daily for the first 5 days and then at least three times per week. Peripheral smears were examined serially for platelet morphology. Platelet counts and the average size of platelets (mean platelet volume) were derived from 64-channel platelet histograms. Bone marrow aspiration and biopsy were done before and 1 week after thrombopoietin treatment. The bone marrow specimens were initially fixed in 10% neutral formalin, embedded in paraffin, cut into sections 5 m thick, and stained with hematoxylin-eosin for morphologic analysis and with Masson trichrome for analysis of collagen fiber content. Fresh, air-dried smears of bone marrow were stained with Wright-Giemsa. Bone marrow samples were examined for overall cellularity and morphology in a blinded manner. Megakaryocyte counts were measured by choosing 10 high-power (40x) fields in areas without artifactual zones or trabecula. The relative size of the megakaryocyte was assessed by examining bone marrow aspirate smears using the Magiscan Image Analysis System (Compix, Cranberry, Pennsylvania). Bone marrow aspirates were also assayed for hematopoietic progenitor cell number and cycle status, for content of CD34+ and CD41+ cell subsets (by flow cytometry), and for megakaryocyte ploidy (by flow cytometry). Blood samples were assayed for hematopoietic progenitor cell number and for platelet function. Pharmacokinetics Profiles Serum samples were collected before and at 2, 5, 10, 60, and 90 minutes and 2, 4, 6, 8, 10, 12, 24, 48, 72, 96, and 120 hours after thrombopoietin administration. Concentration-time profile at each dose level was evaluated by using standard pharmacokinetics methods. Serum thrombopoietin levels were quantitated by enzyme-linked immunosorbent assay for thrombopoietin [12]. Hematopoietic Progenitor Cell Assays Assays for colony-forming unit-granulocyte-macrophage (CFU-GM); burst-forming unit-erythroid (BFU-E); and colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM) using low-density bone marrow [13] and peripheral blood cells [14] were done with methyl cellulose assays. The percentage of bone marrow CFU-GM and BFU-E in DNA synthesis (S-phase of cell cycle) was measured by a high-specific-activity tritiated thymidine suicide technique [15]. Assays for colony-forming unit-megakaryocyte (CFU-MK) and burst-forming unit-megakaryocyte (BFU-MK) were done using a fibrin clot assay [16]. Ploidy Analysis Megakaryocyte-enriched cell fractions were prepared from bone marrow cell suspensions by using a Percoll gradient technique. Ploidy was determined by flow cytometric measurement of the relative DNA content after staining with propidium iodide in hypotonic citrate solution [17]. Cells were also stained with anti-CD41b (8D9)-FITC (SyStemix, Palo Alto, California) to allow gating on CD41b+ megakaryocytes. At least 3000 CD41+ events were collected for each sample. The percentage of CD41+ cells in ploidy class was determined from the fluorescence-activated cell-sorting dot plots. Platelet Function Platelet aggregation was measured in response to three agonists: adenosine diphosphate (final concentration, 20 g/mL), collagen (6 g/mL), and thrombin (5 g/mL). Standard methods were used [18]. The concentrations of agonists were chosen on the basis of previous in vitro studies done on blood from normal controls. The instruments used for the assays were the Bio/Data Pap 4A (Horsham, Pennsylvania) and the Crono-log 560CA (Havertown, Pennsylvania). Immunophenotypic Analysis Immunophenotypic analysis was done using anti-CD34 (Becton Dickinson, San Jose, California) and anti-CD41 monoclonal antibodies (Immunotech, Westbrook, Maine) by a standard dual-color flow cytometry technique [19]. Statistical Analysis Continuous variables were compared by using the Wilcoxon matched-pairs signed-rank test. Trends for possible dose-response relation were evaluated using the Spearman rank correlation coefficients (rS) between dose and outcome. Industry Role Thrombopoietin and partial funding for the study were provided by Genentech, Inc. The study was a collaborative effort between the principal investigator and the industrial sponsor. Data collection, data analysis, the writing of the manuscript, and the decision to publish the manuscript were under the control of the principal investigator. The manuscript was reviewed by the industrial sponsor before submission. Results Twelve chemotherapy-naive patients (7 men and 5 women) with sarcoma of diverse histologic sub-types were entered into the dose-ranging portion of this phase I trial, which studied thrombopoietin before chemotherapy. All patients were considered evaluable for clinical tolerance and response to thrombopoietin. The median age of these patients was 42 years (range, 16 to 63 years), and the median Karnofsky performance status score was 90 (range, 80 to 100). Four patients had previously received radiation therapy, and eight had previously had surgery. Peripheral Blood Counts Treatment with a single dose of thrombopoietin was associated with increases (1.3-fold to 3.6-fold) in platelet counts (baseline mean, 264 109/L; maximal mean, 592 109/L) (P = 0.002). The increase in platelet count was seen at all dose levels (Figure 1) in all patients. The peak response i

MicroRNA fingerprints identify miR-150 as a plasma prognostic marker in patients with sepsis

BACKGROUND The physiopathology of sepsis continues to be poorly understood, and despite recent advances in its management, sepsis is still a life-threatening condition with a poor outcome. If new diagnostic markers related to sepsis pathogenesis will be identified, new specific therapies might be developed and mortality reduced. Small regulatory non-coding RNAs, microRNAs (miRNAs), were recently linked to various diseases; the aim of our prospective study was to identify miRNAs that can differentiate patients with early-stage sepsis from healthy controls and to determine if miRNA levels correlate with the severity assessed by the Sequential Organ Failure Assessment (SOFA) score. METHODOLOGY/PRINCIPAL FINDINGS By using genome-wide miRNA profiling by microarray in peripheral blood leukocytes, we found that miR-150, miR-182, miR-342-5p, and miR-486 expression profiles differentiated sepsis patients from healthy controls. We also proved by quantitative reverse transcription-polymerase chain reaction that miR-150 levels were significantly reduced in plasma samples of sepsis patients and correlated with the level of disease severity measured by the SOFA score, but were independent of the white blood counts (WBC). We found that plasma levels of tumor necrosis factor alpha, interleukin-10, and interleukin-18, all genes with sequence complementarity to miR-150, were negatively correlated with the plasma levels of this miRNA. Furthermore, we identified that the plasma levels ratio for miR-150/interleukin-18 can be used for assessing the severity of the sepsis. CONCLUSIONS/SIGNIFICANCE We propose that miR-150 levels in both leukocytes and plasma correlate with the aggressiveness of sepsis and can be used as a marker of early sepsis. Furthermore, we envision miR-150 restoration as a future therapeutic option in sepsis patients.

Mutations in CBL occur frequently in juvenile myelomonocytic leukemia

Juvenile myelomonocytic leukemia is an aggressive myeloproliferative disorder characterized by malignant transformation in the hematopoietic stem cell compartment with proliferation of differentiated progeny. Seventy-five percent of patients harbor mutations in the NF1, NRAS, KRAS, or PTPN11 genes, which encode components of Ras signaling networks. Using single nucleotide polymorphism arrays, we identified a region of 11q isodisomy that contains the CBL gene in several JMML samples, and subsequently identified CBL mutations in 27 of 159 JMML samples. Thirteen of these mutations alter codon Y371. In this report, we also demonstrate that CBL and RAS/PTPN11 mutations were mutually exclusive in these patients. Moreover, the exclusivity of CBL mutations with respect to other Ras pathway-associated mutations indicates that CBL may have a role in deregulating this key pathway in JMML.