Edgar G. Rizzatti

Gene expression profiling of mantle cell lymphoma cells reveals aberrant expression of genes from the PI3K-AKT, WNT and TGFbeta signalling pathways.

Microarray studies have revealed the differential expression of several genes in mantle cell lymphoma (MCL), but it is unknown which of these differences are dependent on the transformed MCL cell itself or on the tumour microenvironment. To investigate which genes and signalling pathways are aberrantly expressed in MCL cells we used oligonucleotide microarrays to perform gene expression profiling of both purified leukaemic MCL cells and their normal counterparts, the naive B cells. A total of 106 genes were differentially expressed at least threefold in MCL cells compared with naive B cells; 63 upregulated and 43 downregulated. To validate the microarray results in a larger set of samples, we selected 10 differentially expressed genes and quantified their expression by real‐time polymerase chain reaction in peripheral blood of MCL patients (n = 21), purified MCL cells (n = 6) and naive B cells (n = 4), obtaining fully concordant results. A computer‐assisted approach was used to procure specific molecular signalling pathways that were aberrantly expressed in MCL cells. Several genes related to apoptosis and to the PI3K/AKT, WNT and tumour growth factor β signalling pathways were altered in MCL cells when compared with naive B cells. These pathways may play a significant role in the pathogenesis of MCL and deserve further investigation as candidates for new therapeutic targets.

A texture approach to leukocyte recognition

Millions of white blood cells are manually classified in laboratories using microscopes, a painstaking and subjective task. A trained medical technician takes about 15min to evaluate and count 100 cells for each blood slide, a time consuming and susceptible to error procedure. Leukocyte shape is usually insufficient to differentiate even among normal types since it varies widely. The current paper addresses the pattern recognition problem of blood image analysis and how textural information can improve differentiation among leukocytes. Cooccurrence probabilities can be used as a measure of gray scale image texture, a statistical method for characterizing the spatial organization of the gray-tones. We calculate five textural attributes based on gray level cooccurrence matrices (GLCM) as energy, entropy, inertia and local homogeneity, testing these features in leukocyte recognition. Several parameters must be estimated for obtaining GLCM, therefore we implement datamining algorithms for estimating suitable scales. Feature selection methods are also applied to define the most discriminative attributes for describing the cellular patterns. Experimental results show that texture parameters are essential to differentiate among the five types of normal leukocytes and chronic lymphocytic leukemia, evidencing the importance of biological aspects regarded by hematologists as nuclear chromatin and cytoplasmical granularity.

Noxa mediates bortezomib induced apoptosis in both sensitive and intrinsically resistant mantle cell lymphoma cells and this effect is independent of constitutive activity of the AKT and NF-kappaB pathways

Bortezomib is more active against mantle cell lymphoma (MCL) than against most other lymphoma subtypes. Nevertheless, up to half of patients with MCL have bortezomib resistant disease. Factors contributing to intrinsic resistance to bortezomib have not been determined. Here we used a panel of eight bortezomib sensitive (median IC50 5.9 nM) and three relatively bortezomib resistant cell lines (median IC50 12.9 nM) to investigate differences in tumor biology that could determine sensitivity to bortezomib. Bortezomib effectively inhibited high baseline proteasome activity and induced a comparable degree of proteasome inhibition in both sensitive and resistant cells. At 10 nM, bortezomib induced the proapoptotic BH3-only protein Noxa in sensitive but not resistant cells. At higher concentrations of bortezomib, however, Noxa was also upregulated in resistant cells and this effect was sufficient to induce apoptosis. Silencing of Noxa with siRNA rescued these cells from apoptosis, arguing against a defect in Noxa regulation or function as the basis of bortezomib resistance. Bortezomib was equally effective against cells with high and low constitutive NF-κB signaling. Also, sensitive and resistant MCL cell lines showed comparable activation of the AKT pathway. We conclude that bortezomib can overcome classic mechanisms of resistance to apoptosis and that determinants of bortezomib sensitivity in MCL are due to differences in signaling or stress pathways upstream of Noxa.

Treatment-Induced Oxidative Stress and Cellular Antioxidant Capacity Determine Response to Bortezomib in Mantle Cell Lymphoma

Purpose: Proteasome inhibition disrupts protein homeostasis and induces apoptosis. Up to 50% of patients with relapsed mantle cell lymphoma (MCL) respond to bortezomib. We used gene expression profiling to investigate the connection between proteasome inhibition, cellular response, and clinical efficacy. Experimental Design: We assessed transcriptional changes in primary tumor cells from five patients during treatment with bortezomib in vivo, and in 10 MCL cell lines exposed to bortezomib in vitro, on Affymetrix microarrays. Key findings were confirmed by western blotting. Results: MCL cell lines exposed to bortezomib in vitro showed upregulation of endoplasmic reticulum and oxidative stress response pathways. Gene expression changes were strongest in bortezomib-sensitive cells and these cells were also more sensitive to oxidative stress induced by H2O2. Purified tumor cells obtained at several timepoints during bortezomib treatment in 5 previously untreated patients with leukemic MCL showed strong activation of the antioxidant response controlled by NRF2. Unexpectedly, activation of this homeostatic program was significantly stronger in tumors with the best clinical response. Consistent with its proapoptotic function, we found upregulation of NOXA in circulating tumor cells of responding patients. In resistant cells, gene expression changes in response to bortezomib were limited and upregulation of NOXA was absent. Interestingly, at baseline, bortezomib-resistant cells displayed a relatively higher expression of the NRF2 gene-expression signature than sensitive cells (P < 0.001). Conclusion: Bortezomib triggers an oxidative stress response in vitro and in vivo. High cellular antioxidant capacity contributes to bortezomib resistance. Clin Cancer Res; 17(15); 5101–12. ©2011 AACR.

CD200 has an important role in the differential diagnosis of mature B-cell neoplasms by multiparameter flow cytometry.

Multiparameter flow cytometry is a useful tool for the diagnostic evaluation of mature B‐cell neoplasms (MBN). Recently, it has been shown that CD200 may improve the distinction between chronic lymphocytic leukemia (CLL; CD200+) and mantle cell lymphoma (MCL; CD200−), but the role of CD200 expression in atypical CLL and other MBN remains to be established.

Expression of CD117 and CD11b in Bone Marrow Can Differentiate Acute Promyelocytic Leukemia From Recovering Benign Myeloid Proliferation

The morphologic characteristics of bone marrow aspirates from patients recovering from acute agranulocytosis may be closely similar to the pattern observed in cases of acute promyelocytic leukemia (APL). The clinical manifestation also can be ambiguous in a substantial number of cases. The immunophenotypic features of bone marrow from 5 patients recovering from acute agranulocytosis, showing an increase in the percentage of promyelocytes (26%-66%), were compared with the immunophenotype of 31 consecutive patients with APL whose diagnosis was confirmed by PML-RAR alpha gene rearrangement. All markers were similarly expressed, except for CD117 and CD11b. CD117 was positive in 24 (77%) of the APL cases and in none of the acute agranulocytosis cases. On the other hand, CD11b was positive in 5 (100%) of the acute agranulocytosis cases and in only 2 (6%) of the APL cases. Thus, the CD117-CD11b+ phenotype was detected in all patients recovering from agranulocytosis and in only 1 (3%) of 31 APL cases. Therefore, we suggest that the combination of both markers is helpful in the differentiation of APL from recovering benign myeloid proliferation.

Características hematológicas e perfil de expressão de antígenos mielóides de pacientes com leucemia promielocítica aguda: análise de fatores prognósticos para o desenvolvimento da síndrome do ácido retinóico

OBJETIVO: A leucemia promielocitica aguda (LPA) apresenta uma boa resposta ao tratamento com o acido all trans retinoico (ATRA). Entretanto, alguns pacientes desenvolvem uma complicacao grave chamada sindrome do acido retinoico (SAR). O objetivo deste estudo foi comparar as caracteristicas hematologicas e imunofenotipicas de pacientes com LPA que desenvolveram a SAR com as daqueles que nao a desenvolveram. METODOS: Foram analisados retrospectivamente os prontuarios, exames radiologicos, lâminas de esfregaco de sangue e medula ossea de 71 pacientes com LPA, dos quais a analise imunofenotipica havia sido realizada em 56 casos. Foram identificados oito casos de SAR que, do ponto de vista clinico, caracterizaram-se por insuficiencia respiratoria (n=8), insuficiencia renal (n=2), febre (n=5), ganho ponderal (n=3), edema periferico (n=3) e derrame pleural (n=5). As seguintes variaveis foram comparadas entre pacientes com e sem SAR: dosagem de hemoglobina, contagens de leucocitos e plaquetas no sangue periferico, distribuicao dos subtipos hipergranular e variante, percentagens de blastos CD33+, CD13+, CD117+ na medula ossea, intensidade e variacao dos valores de fluorescencia destes antigenos nas celulas leucemicas, expressas atraves dos canais medianos (CMFs) e dos coeficientes de variacao (CVs) de fluorescencia, respectivamente. RESULTADOS: A incidencia da SAR foi de 11,26% e o tempo medio para seu desenvolvimento 11,5 dias do inicio do tratamento. Todos os pacientes apresentaram desconforto respiratorio agudo, por vezes associado a febre, ganho de peso, edema e insuficiencia renal. Os achados radiologicos mais comuns foram: opacidades em vidro fosco, derrame pleural, espessamento peribronquico e aumento da trama vascular pulmonar. Nenhuma das variaveis laboratoriais analisadas correlacionou-se significativamente ao risco de desenvolvimento da SAR, entretanto as Odd Ratios para CMF para o CD117 > 30 ua e CV para o CD33 < 50 foram de 7,14 (P=0,08) e de 7,86 (P=0,06), respectivamente. CONCLUSAO: A incidencia e as caracteristicas da SAR neste grupo de pacientes brasileiros foi semelhante a descrita na literatura. Nenhum dos parâmetros estudados correlacionou-se significativamente a um maior risco de desenvolvimento desta complicacao.

Molecular genetic tests for JAK2V617F, Exon12_JAK2 and MPLW515K/L are highly informative in the evaluation of patients suspected to have BCR-ABL1-negative myeloproliferative neoplasms

Polycythaemia vera (PV), essential thrombocythemia (ET) and idiopathic myelofibrosis (MF), are the most common myeloproliferative neoplasms (MPN) in patients without the BCR-ABL1 gene rearrangement. They are caused by clonal expansion of haematopoietic stem cells and share, as a diagnostic criterion, the identification of JAK2V617F mutation. Classically, when other clinical criteria are present, a JAK2V617F negative case requires the analysis of Exon12_JAK2 for the diagnosis of PV, and of MPL515K/L mutations for the diagnosis of ET and MF. Here, we evaluated 78 samples from Brazilian patients suspected to have MPN, without stratification for PV, ET or MF. We found that 28 (35.9%) are JAK2V617F carriers; from the 50 remaining samples, one (2%) showed an Exon12_JAK2 mutation, and another (2%) was positive for MPLW515L mutation. In summary, the investigation of JAK2V617F, Exon12_JAK2 and MPLW515K/L was relevant for the diagnosis of 38.4% of patients suspected to have BCR-ABL1-negative MPN, suggesting that molecular genetic tests are useful for a quick and unequivocal diagnosis of MPN.

Adhesion molecule profiles of B-cell non-Hodgkin's lymphomas in the leukemic phase

We evaluated the expression of 10 adhesion molecules on peripheral blood tumor cells of 17 patients with chronic lymphocytic leukemia, 17 with mantle-cell lymphoma, and 13 with nodal or splenic marginal B-cell lymphoma, all in the leukemic phase and before the beginning of any therapy. The diagnosis of B-cell non-Hodgkins lymphomas was based on cytological, histological, immunophenotypic, and molecular biology methods. The mean fluorescence intensity of the adhesion molecules in tumor cells was measured by flow cytometry of CD19-positive cells and differed amongst the types of lymphomas. Comparison of chronic lymphocytic leukemia and mantle-cell lymphoma showed that the former presented a higher expression of CD11c and CD49c, and a lower expression of CD11b and CD49d adhesion molecules. Comparison of chronic lymphocytic leukemia and marginal B-cell lymphoma showed that the former presented a higher expression of CD49c and a lower expression of CD11a, CD11b, CD18, CD49d, CD29, and CD54. Finally, comparison of mantle-cell lymphoma and marginal B-cell lymphoma showed that marginal B-cell lymphoma had a higher expression of CD11a, CD11c, CD18, CD29, and CD54. Thus, the CD49c/CD49d pair consistently demonstrated a distinct pattern of expression in chronic lymphocytic leukemia compared with mantle-cell lymphoma and marginal B-cell lymphoma, which could be helpful for the differential diagnosis. Moreover, the distinct profiles of adhesion molecules in these diseases may be responsible for their different capacities to invade the blood stream.

Leukocyte detection using nucleus contour propagation

We propose a new technique for medical image segmentation, focused on front propagation in blood smear images to fully automate leukocyte detection. The current approach also incorporates contextual information, which it is especially important in direct general algorithms to the applied problem. A Bayesian classification of pixels is used to estimate cytoplasm color and is embedded in the speed function to accomplish cytoplasm boundary estimation. We report encouraging results, with evaluations considering difficult situations as cell adjacency and filamentous cytoplasmic projections.