Leandro S. Thiago

Overweight as a Prognostic Factor in Children With Acute Lymphoblastic Leukemia

Our purpose was to investigate the prognostic impact of overweight/obesity in 5‐year event‐free survival (EFS) in a cohort of children with acute lymphoblastic leukemia (ALL). We retrospectively analyzed 181 newly diagnosed ALL children enrolled between 1990 and 2009 and treated with Berlin‐Frankfurt‐Munich (BFM) protocols. The majority of children in our cohort were <10 years‐old. Our data clearly indicated that overweight/obesity is an independent predictor of relapse risk, mainly in the intermediate‐ and high‐risk groups (HR) of children. These results could be explained by changes in the chemotherapy pharmacokinetics in overweight/obese patients and by the antiapoptotic effects in leukemic cells caused by adipocytes.

Contribution of Multiparameter Flow Cytometry Immunophenotyping to the Diagnostic Screening and Classification of Pediatric Cancer

Pediatric cancer is a relatively rare and heterogeneous group of hematological and non-hematological malignancies which require multiple procedures for its diagnostic screening and classification. Until now, flow cytometry (FC) has not been systematically applied to the diagnostic work-up of such malignancies, particularly for solid tumors. Here we evaluated a FC panel of markers for the diagnostic screening of pediatric cancer and further classification of pediatric solid tumors. The proposed strategy aims at the differential diagnosis between tumoral vs. reactive samples, and hematological vs. non-hematological malignancies, and the subclassification of solid tumors. In total, 52 samples from 40 patients suspicious of containing tumor cells were analyzed by FC in parallel to conventional diagnostic procedures. The overall concordance rate between both approaches was of 96% (50/52 diagnostic samples), with 100% agreement for all reactive/inflammatory and non-infiltrated samples as well as for those corresponding to solid tumors (n = 35), with only two false negative cases diagnosed with Hodgkin lymphoma and anaplastic lymphoma, respectively. Moreover, clear discrimination between samples infiltrated by hematopoietic vs. non-hematopoietic tumor cells was systematically achieved. Distinct subtypes of solid tumors showed different protein expression profiles, allowing for the differential diagnosis of neuroblastoma (CD56hi/GD2+/CD81hi), primitive neuroectodermal tumors (CD271hi/CD99+), Wilms tumors (>1 cell population), rhabdomyosarcoma (nuMYOD1+/numyogenin+), carcinomas (CD45−/EpCAM+), germ cell tumors (CD56+/CD45−/NG2+/CD10+) and eventually also hemangiopericytomas (CD45−/CD34+). In summary, our results show that multiparameter FC provides fast and useful complementary data to routine histopathology for the diagnostic screening and classification of pediatric cancer.

Circulating clonotypic B cells in multiple myeloma and monoclonal gammopathy of undetermined significance

The B-cell compartment in which multiple myeloma stem cells reside remains unclear. We investigated the potential presence of mature, surface-membrane immunoglobulin-positive B lymphocytes clonally related to the tumor bone marrow plasma cells among different subsets of peripheral blood B cells from ten patients (7 with multiple myeloma and 3 with monoclonal gammopathies of undetermined significance). The presence of clonotypic immunoglobulin heavy chain gene rearrangements was determined in multiple highly-purified fractions of peripheral blood B-lymphocytes including surface-membrane IgM+ CD27− naïve B-lymphocytes, plus surface-membrane IgG+, IgA+ and IgM+ memory CD27+ B cells, and normal circulating plasma cells, in addition to (mono)clonal plasma cells, by a highly-specific and sensitive allele-specific oligonucleotide polymerase chain reaction directed to the CDR3 sequence of the rearranged IGH gene of tumor plasma cells from individual patients. Our results showed systematic absence of clonotypic rearrangements in all the different B-cell subsets analyzed, including M-component isotype-matched memory B-lymphocytes, at frequencies <0.03 cells/μL (range: 0.0003–0.08 cells/μL); the only exception were the myeloma plasma cells detected and purified from the peripheral blood of four of the seven myeloma patients. These results indicate that circulating B cells from patients with multiple myeloma and monoclonal gammopathies of undetermined significance are usually devoid of clonotypic B cells while the presence of immunophenotypically aberrant myeloma plasma cells in peripheral blood of myeloma patients is a relatively frequent finding.

Cholesterol depletion by methyl-β-cyclodextrin enhances cell proliferation and increases the number of desmin-positive cells in myoblast cultures.

Skeletal myogenesis comprises myoblast replication and differentiation into striated multinucleated myotubes. Agents that interfere with myoblast replication are important tools for the understanding of myogenesis. Recently, we showed that cholesterol depletion by methyl-β-cyclodextrin (MCD) enhances the differentiation step in chick-cultured myogenic cells, involving the activation of the Wnt/β-catenin signaling pathway. However, the effects of cholesterol depletion on myoblast replication have not been carefully studied. Here we show that MCD treatment increases cell proliferation in primary chick myogenic cell cultures. Treatment of myogenic cells with the anti-mitotic reagent cytosine arabinoside, immediately following cholesterol depletion, blocks the MCD-induced effects on proliferation. Cholesterol depletion induced an increase in the number of desmin-positive mononucleated cells, and an increase in desmin expression. MCD induces an increase in the expression of the cell cycle regulator p53 and the master switch gene MyoD1. Treatment with BIO, a specific inhibitor of GSK3β, induced effects similar to MCD on cell proliferation; while treatment with Dkk1, a specific inhibitor of the Wnt/β-catenin pathway, neutralized the effects of MCD. These findings indicate that rapid changes in the cholesterol content in cell membranes of myoblasts can induce cell proliferation, possibly by the activation of the Wnt/β-catenin signaling pathway.

The Wnt signaling pathway regulates Nalm-16 b-cell precursor acute lymphoblastic leukemic cell line survival and etoposide resistance.

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most common malignancy in children. The Wnt signaling pathway has been found to be extensively involved in cancer onset and progression but its role in BCP-ALL remains controversial. We evaluate the role of the Wnt pathway in maintenance of BCP-ALL cells and resistance to chemotherapy. Gene expression profile revealed that BCP-ALL cells are potentially sensitive to modulation of Wnt pathway. Nalm-16 and Nalm-6 cell lines displayed low levels of canonical activation, as reflected by the virtually complete absence of total beta-catenin in Nalm-6 and the beta-catenin cell membrane distribution in Nalm-16 cell line. Canonical activation with Wnt3a induced nuclear beta-catenin translocation and led to BCP-ALL cell death. Lithium chloride (LiCl) also induced a cytotoxic effect on leukemic cells. In contrast, both Wnt5a and Dkk-1 increased Nalm-16 cell survival. Also, Wnt3a enhanced the in vitro sensitivity of Nalm-16 to etoposide (VP-16) while treatment with canonical antagonists protected leukemic cells from chemotherapy-induced cell death. Overall, our results suggest that canonical activation of the Wnt pathway may exerts a tumor suppressive effect, thus its inhibition may support BCP-ALL cell survival.

First proposed panels on acute leukemia for four‐color immunophenotyping by flow cytometry from the Brazilian group of flow cytometry‐GBCFLUX

Multiparameter flow cytometry is a highly sensitive, fast, and specific diagnostic technology with a wide range of applicability in hematology. Although well‐established eight‐color immunophenotyping panels are already available, most Brazilian clinical laboratories are equipped with four‐color flow cytometer facilities. Based on this fact, the Brazilian Group of Flow Cytometry (Grupo Brasileiro de Citometria de Fluxo, GBCFLUX) for standardization of clinical flow cytometry has proposed an antibody panel designed to allow precise diagnosis and characterization of acute leukemia (AL) within resource‐restricted areas. Morphological analysis of bone marrow smears, together with the screening panel, is mandatory for the primary identification of AL. The disease‐oriented panels proposed here are divided into three levels of recommendations (mandatory, recommendable, and optional) in order to provide an accurate final diagnosis, as well as allow some degree of flexibility based on available local resources and patient‐specific needs. The proposed panels will be subsequently validated in an interlaboratory study to evaluate its effectiveness on the diagnosis and classification of AL. (Assoc editor comm. 2).

Racemic Etodolac is cytotoxic and cytostatic for B-cell precursor acute lymphoblastic leukemia cells☆

Several epidemiological studies have provided evidence that administration of nonsteroidal anti-inflammatory drugs (NSAIDs) could have a prophylactic effect against some cancers such as sporadic colorectal cancer and leukemia. Indeed, various NSAIDs have been shown to induce apoptosis in malignant cells. We evaluated the effect of racemic Etodolac on proliferation and cell survival in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cells. Etodolac decreased survival of Nalm-16 and Nalm-6 BCP-ALL cell lines and also decreased cell proliferation in Nalm-16 cell line. Ours findings indicate, for the first time to our knowledge, that Etodolac is cytotoxic and cytostatic for BCP-ALL cells.

An uncommon case of childhood biphenotypic precursor-B/T acute lymphoblastic leukemia†

documented previously only as a single case report in a child [9]. Although, T lymphoblastic lymphomawith orbital involvement has been reported [10,11] but our case here with T-NHL was of nonlymphoblastic type. This series highlights the varied spectrum, relatively higher incidence of childhood ocular/orbital lymphomas in our center and overall favorable outcome. Sameer Bakhshi, MD* Talvir Sidhu Department of Medical Oncology Dr. B.R.A. Institute Rotary Cancer Hospital All India Institute of Medical Sciences, New Delhi, India

Flow cytometry as a diagnostic support tool in juvenile myelomonocytic leukemia.

Th e diagnosis of juvenile myelomonocytic leukemia (JMML) follows a diffi cult question in pediatric hematology [1]. No consistently recurring cytogenetic abnormalities are reported in JMML, and normal karyotypes are found in 40 – 70% of patients. Monosomy 7, del(7q) or other abnormalities of chromosome 7 have been reported in ∼ 25% of cases [2,3]. Mutations in NRAS, NF1 and PTPN11 genes are found in ∼ 20 – 35% of JMML patients and are mutually exclusive; thus, overall, nearly 75% of patients with JMML have one of these abnormalities [3,4]. Although these molecular abnormalities are of help in the diagnostic workup of JMML, they are not pathognomonic, since these abnormalities are also found in acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) and others myelodysplastic syndrome/myeloproliferative disorders (MDS/ MPL) [3]. Moreover, these genetic tests are not widely available for clinical use. In addition, no specifi c phenotypic abnormalities have been reported in JMML to date [3]. Based on this, new diagnostic tools are urgently required to improve patient care. For several decades now, fl ow cytometry (FCM) immunophenotyping has been shown to be essential for rapid diagnosis, classifi cation and monitoring of most hematological malignancies [5,6] and also holds promise for the diagnosis of pediatric solid tumors [7]. However, the role of FCM in the diagnosis of JMML has been restricted to blast cell compartment analysis. Our aim was to identify abnormalities in the relative distribution and phenotypic characteristics of the diff erent compartments of hematopoietic cells that could contribute to the diagnosis of JMML. Here, we describe recurrent immunophenotypic abnormalities in three children ( � 22 months of age) with JMML that were referred to our hospital to investigate intermittent fever due to recurrent episodes of severe infections. Th e study was approved by the Ethical Committee from IPPMG/UFRJ and is in accordance with the Helsinki Declaration. Clinical and laboratory fi ndings are summarized in Table I. In all cases, fi nal diagnosis of JMML was established based on World Health Organization (WHO) 2008 criteria. Th e identifi cation, quantifi cation and characterization of hematopoietic cells were done by FCM on bone marrows (BM, all cases) and also on peripheral blood (PB) from case#1 at diagnosis and evolution. Briefl y, all samples were stained with the following three-color combinations of antibodies (all from BD Biosciences) conjugated with fl uorescein isothiocyanate (FITC)/phycoerythrin (PE)/perid inclorophyll proteincyanine 5.5 (PerCP-Cy5.5): CD19/CD10/CD45; CD34/ HLA-DR/CD45; CD15/CD34/CD45; CD7/CD34/CD45; CD7/CD117/CD45; CD13/CD11b/CD45; Cy MPO/CD13/ CD45; CD36/CD64/CD45; CD14/CD33/CD45; CD14/ HLA-DR/CD45; CD4/CD33/CD45, CD4/CD13/CD45. Th e samples (50 μ l per tube) were incubated for 15 min at room temperature in the dark, in the presence of saturating amounts of each of the above-mentioned monoclonal antibodies (MAb). Afterward, 2 ml of FACS lysing solution (BD) diluted 1:10 (v/v) in distilled water was added and the samples were incubated for another 10 min under the same conditions as those mentioned above. Th en, cells were centrifuged (5 min at 540 g) and the cell pellet was washed with 2 ml of PBS BSA 0.5%. Finally, cells were resuspended in 0.4 ml of PBS BSA 0.5%. Stained cells were acquired at low speed in a FACSCalibur fl ow cytometer (BD) using the