Low ABCB1 and high OCT1 levels play a favorable role in the molecular response to imatinib in CML patients in the community clinical practice.

Despite the favorable clinical evolution of patients with chronic myeloid leukemia (CML), resistance or intolerance to imatinib is present in approximately 35% of patients. Sokal score is a widely used risk factor, however efflux and influx transporters are provisional risk factors implicated in imatinib resistance. This study analyzed Sokal score, ABCB1, ABCG2 and OCT1 mRNA transporter expression levels as well as P-glycoprotein expression and efflux transporters activity to seek a possible correlation between these factors and the molecular response at 12 months from imatinib start as well as 8-year overall survival (OS). Low plus intermediate Sokal score correlated to optimal imatinib responses, as well as OS at 8-years, thus confirming the established role of Sokal score as a prognostic factor in CML patients. Low ABCB1 and high OCT1 mRNA levels were associated with an optimal molecular response, while the inverse levels were associated with non-responders (warning and failure) patients. Our results suggest that ABCB1 and OCT1 mRNA expressions may present biological relevance to identify responder and non-responder patients to imatinib treatment.

The significance of major and stable molecular responses in chronic myeloid leukemia in the tyrosine kinase inhibitor era

Tyrosine kinase inhibitors have changed the management and outcomes of chronic myeloid leukemia patients. Quantitative polymerase chain reaction is used to monitor molecular responses to tyrosine kinase inhibitors. Molecular monitoring represents the most sensitive tool to judge chronic myeloid leukemia disease course and allows early detection of relapse. Evidence of achieving molecular response is important for several reasons: 1. early molecular response is associated with major molecular response rates at 18-24 months; 2. patients achieving major molecular response are less likely to lose their complete cytogenetic response; 3. a durable, stable major molecular response is associated with increased progression-free survival. However, standardization of molecular techniques is still challenging.

Rapid and sensitive allele-specific (AS)-RT-PCR assay for detection of T315I mutation in chronic myeloid leukemia patients treated with tyrosine-kinase inhibitors.

Point mutations in the kinase domain of BCR-ABL were described in 40-90% of patients with chronic myeloid leukemia (CML) resistant to Imatinib. We herein describe the development of a rapid allele-specific (AS)-RT-PCR assay to identify the T315I mutation, which confers full resistance to all available tyrosine-kinase inhibitors (TKI). The mutation status of 65 patients with resistant CML was evaluated, and the T315I was detected in 3/65 (4.6%). Comparisons between sequencing and AS-RT-PCR results, as well as serial dilutions experiments proved that the method is specific and reproducible, with maximum sensitivity of 1 × 10(-3). The developed assay is a convenient and easy tool to be used in research of CML resistance for rapid mutation screening and, together with sequencing, may be included in efficient strategies for early detection of TKI resistance in patients with CML.

The significance of major and stable molecular responses in chronic myeloid leukemia in the tyrosine kinase inhibitor era.

Tyrosine kinase inhibitors have changed the management and outcomes of chronic myeloid leukemia patients. Quantitative polymerase chain reaction is used to monitor molecular responses to tyrosine kinase inhibitors. Molecular monitoring represents the most sensitive tool to judge chronic myeloid leukemia disease course and allows early detection of relapse. Evidence of achieving molecular response is important for several reasons: 1. early molecular response is associated with major molecular response rates at 18-24 months; 2. patients achieving major molecular response are less likely to lose their complete cytogenetic response; 3. a durable, stable major molecular response is associated with increased progression-free survival. However, standardization of molecular techniques is still challenging.

Prognostic relevance of KIT and PDGFRA mutations in gastrointestinal stromal tumors.

BACKGROUND: Prediction of biological behavior is crucial for selection of new therapeutic modalities in GIST. Here, we aimed to assess whether KIT and PDGFRA mutations have survival impact in gastrointestinal stromal tumors (GIST). PATIENTS AND METHODS: Fifty-five Brazilian patients with completely resected GIST were examined for KIT and PDGFRA mutations. The 5-year disease-free survival (DFS) was analyzed. RESULTS: KIT and PDGFRA mutations were identified in 74.5% and 7.3% of patients, respectively. The 5-year DFS rate for all patients was 52.8%. The 5-year DFS rate was lower in patients with tumors having in-frame deletions or concomitant in-frame deletions and insertions affecting codons 557-558 than in patients with tumors having other exon 11 KIT mutations (p=0.023). Conversely, when the patients with concomitant deletion-insertion mutations affecting codons 557-558 were excluded from the analysis, deletions involving codons 557-558 had no influence on 5-year DFS rates. CONCLUSION: Our findings indicate that a specific KIT mutation may be associated with unfavorable behavior in GIST. This finding may have implications on selecting patients for adjuvant therapy.

Methylation status of nine tumor suppressor genes in multiple myeloma.

Aberrant methylation in promoter-associated CpG islands has been recognized as a major mechanism for tumor suppressor gene silencing in several malignancies. We determined the methylation status of nine tumor suppressor genes in 68 newly diagnosed MM patients by methylation-specific PCR. The frequency of promoter hypermethylation for individual genes was: CDH1, 50%; p16 INK4a, 42.8%; p15 INK4b, 16.2%; SHP1, 14.7%; ER and BNIP3, 13.2%; RAR beta, 11.8%; DAPK 5.9%; and MGMT 0%. Overall, 79% of patients presented at least one hypermethylated gene. By univariate analysis, hypermethylation of DAPK (P < 0.001) and RAR beta (P = 0.01) genes were identified as adverse prognostic features. Median OS of patients with hypermethylation in DAPK (4 months) and RAR beta (34 months) was significantly lower than in patients without hypermethylation (median survival not reached), with values of P < 0.001 and P = 0.01, respectively. Our data suggest that DAPK and RAR beta hypermethylation are adverse prognostic factors in MM. The relevance of these findings as poor prognosis indicators requires confirmation in a larger sample with longer follow-ups.

Pediatric Hodgkin lymphoma in 2 South American series: a distinctive epidemiologic pattern and lack of association of Epstein-Barr virus with clinical outcome.

Hodgkin lymphoma (HL) shows a bimodal distribution with a first peak in developing countries during childhood. The causative role and prognostic significance of Epstein-Barr virus (EBV) association in patients with HL is controversial. Our aim was to perform a comparative study of EBV association in 2 Latin American pediatric HL series, and to correlate it with patient's survival. Epstein-Barr encoded RNAs in situ hybridization and latent membrane protein 1 immunohistochemistry were performed on formalin-fixed, paraffin-embedded HL biopsies from 176 pediatric patients from 2 public institutions from Argentina and Southeast Brazil. Mixed cellularity subtype was prevalent in Argentine HL (Arg HL) (52%) and nodular sclerosis subtype in Brazilian HL (BR HL) (83%). EBV expression was detected in 52% of cases, namely 54% Arg HL and 48% Br HL. EBV was significantly associated with mixed cellularity subtype in both populations. In Arg HL, EBV positivity was significantly higher in patients

Burkitt lymphoma/leukaemia transformed from a precursor B cell: clinical and molecular aspects.

Burkitt lymphoma/leukaemia (BL/L) is a heterogeneous disease with respect to epidemiological patterns and cell origin. The occurrence of BL/L with an immature phenotype raises the question whether this phenotype might be a consequence of early B-cell transformation or, alternatively, a secondary feature of transformed, mature B cells. It also poses important clinical questions regarding diagnosis and therapeutic procedures. Here we describe the case of a 4-yr-old child with BL/L and FAB L3 morphology, with phenotypic and genotypic characteristics of a CD10+ precursor B-cell acute lymphoid leukaemia (ALL) associated with t(8;14)(q24;q32). Molecular analysis showed expression of RAG1 and RAG2 and an unmutated VDJCmu immunoglobulin rearrangement coinciding with a lack of AICDA expression, indicating an immature B-cell origin. His clinical response suggested that FAB L3 ALL with MYC rearrangement and an aberrant precursor B-cell phenotype is clinically similar to BL/L. Moreover, short, intensive chemotherapeutic protocols seemed to be beneficial. This case also allowed us to refine the description of cellular and molecular variants of BL/L regarding the cell origin and pathogenesis of this biologically heterogeneous disease.

Structural variability of the carboxy-terminus of Epstein-Barr virus encoded latent membrane protein 1 gene in Hodgkin's lymphomas.

Epstein-Barr virus (EBV) is implicated in the pathogenesis of several lymphoid and epithelial neoplasms. Latent membrane protein 1 (LMP1) is the major viral oncogene and it is controversial whether tumor LMP1 variants reflect their geographical predominance or are associated with enhanced oncogenic properties. This study aimed to analyze LMP1 molecular variability of 62 EBV+ Hodgkin's lymphomas and 22 non-neoplastic controls from Brazil and Argentina. EBV association was characterized by EBER-ISH, LMP1 immunohistochemistry and PCR assays for EBNA2 and 3C (typing), LMP1 30 bp deletion (del30) and number of 33 bp tandem repeats. LMP1 C-terminal sequencing was performed in 42 cases. EBV1 was the predominant strain in both geographical Hodgkin's lymphoma groups (average 82%). A higher frequency of del30 variant was observed in lymphomas (41/63) than in non-neoplastic controls (6/22) (OR 4.97, CI 95% 1.53-16.79; P = 0.005, chi(2) test). A large number (5-7) of 33 bp repeat units was characteristic of del30 LMP1 variants (P < 0.0001, Fisher's exact test). Sequence analysis showed a similar mutation spectrum to that described worldwide but none of the current classification schemes could be applied completely. A distinct structural pattern was observed in del30 variants, characterized by a large number of 33 bp repeat units and the presence of a 15 bp insertion encoding the JAK3 Box-1a motif (3/15 wt vs. 16/20 del30; P = 0.001, chi(2) test). The results suggest a pathogenic role for LMP1 del30 variants in Hodgkin's lymphoma from South America and point to particular virus-host molecular mechanisms, such as genomic instability in LMP1 carboxy-terminus, leading to enhanced production and selection of these deletion variants.

Mutations within the 5' region of FAS/CD95 gene in nodal diffuse large B-cell lymphoma.

CD95 is a cell-surface receptor that mediates apoptosis. A possible association between CD95 mutations and extranodal diffuse large B-cell lymphomas (DLBCL) has been reported. To further elucidate this question, a mutation analysis within the 5' region and exon 9 of CD95 was performed in a series of 66 DLBCL patients, by polymerase chain reaction, single-strand conformational polymorphism, and sequencing in all cases. Four mutations, all within the 5' region, were detected in three cases of primary nodal DLBCL (6.3% of primary DLBCL), probably originated as by-products of the somatic hypermutation process. No CD95 mutations in the two analyzed regions were detected in primary extranodal DLBCL, mediastinal large B-cell lymphoma (MLBCL), and DLBCL arising from indolent low-grade lymphomas. Because of our results, a review of published data with respect to the site of mutations was performed, which suggested a different distribution of mutations in nodal and extranodal DLBCL.

Burkitt-like lymphoma in an infant: a case report.

Childhood non-Hodgkin's lymphomas, including Burkitt and Burkitt-like, are rarely diagnosed in infants. A case of B-cell lymphoma in a 13-month-old girl with extensive abdominal disease, ascites, pleural effusion, and tumor lysis syndrome is reported. Phenotypic analysis showed a germinal center B-cell phenotype, and a B-cell clonality was confirmed by polymerase chain reaction. There was no evidence of Epstein-Barr and HIV infection. The case herein reported emphasizes the need for considering the diagnosis of lymphoma even in very young children.

Burkitt-like lymphoma in an infant: a case report

Childhood non-Hodgkin's lymphomas, including Burkitt and Burkitt-like, are rarely diagnosed in infants. A case of B-cell lymphoma in a 13-month-old girl with extensive abdominal disease, ascites, pleural effusion, and tumor lysis syndrome is reported. Phenotypic analysis showed a germinal center B-cell phenotype, and a B-cell clonality was confirmed by polymerase chain reaction. There was no evidence of Epstein-Barr and HIV infection. The case herein reported emphasizes the need for considering the diagnosis of lymphoma even in very young children

Biologia e perfil imunocelular das leucemias: rearranjo gênico em desordens linfoproliferativas

Estudos multidisciplinares de pacientes com leucemia podem determinar a origem celular, clonalidade das células envolvidas e alterções gênicas de valor diagnóstico e prognóstico, contribuindo para a melhoria destes, e para a detecção de genes, envolvidos no processo de transformação maligna. Estes estudos, aqui apresentados, tem sido úteis para o estabelecimento de uma classificação das leucemias linfociticas e linfobláticas. Um estudo da distribuição da doença foi feito em um grupo padrão de LeucemiasLinfóides Agudas (LLA) da infância.A análise imunológica de fluxo foi realizada em 152 pacientes. A análise molecular dos genes das Imunoglobinas (Ig) e do Receptor de Células T (RCT) foi usado para determinar a origem celular das Leucemias Linfóides Agudas T (LLA T) mais indiferenciadas, leucemias bifenotípicas e Leucemias Agudas de origem Indeterminada (LAI), definidas pela imunofenotipagem. Estes estudos foram úteis para aferir a oriegem celular dos casos indeterminados e redefinir o grupo de leucemias bifenotípicas em leucemias de origem mielóide ou multipotente. A percentagem de rearranjos inapropriados dos genes da Ig e do RCT nos casos de LLA de origem T e B respectivamente sugerem o uso de rotina da abordagem molecular para a classificação e entendimento biológico das LLAs.A utilização da reação em cadeia pela polimerase (PCR) para determinação da clonalidae nas leucemias T e B de acordo com a imunofenotipagem mostrou resultados consistentes. A técnica de PCR deve ser utilizada preferencialmente à imunofenotipagem nos casos de desordens linfoproliferativas T. Uma vez implantado, nosso protocolo possibilitou a avaliação de doença residual mínima e recaída.Finalmente, o estudo dos aspectos biológicos das leucemias em geral e da regulação dos genes da Ig e do RCT em particular, à partir do estudo do desenvolvimento das regiões 14q32 e 14q11 onde se situam os genes das Ig e o RCT, respectivamente, em diferentes tipos de LLA foi realizado.O estabelecimento de uma nova linhagem celular (cemo-1) possibilitou a clonagem de um novo oncogenese, o BCL-9.

Multidisciplinary studies of leukaemic patients may precisely identify cellular origin, clonality and genomic rearrangements, providing valuable data for diagnosis, prognosis and for the identification of genes involved in malignant transformation. These studies, herein carried out, have been most helpful in establishing a classification of lymphoblastic and lymphocytic leukaemias. Different aspects of the disease were studied in a standard group of childhooh Acute Lymphoblastic Leukaemias. Immunologic analyses, with flow cytometry, were carried out in 152 patients while molecular analyses of Immunoglobulin (Ig) and T-cell receptor (TCR) genes were used for determining the cellular origin of the acute, most-undifferentiated lymphoblastic leukaemias, biphenotypic leukaemias, and leukaemias of unknown origin previously characterized by immunophenotyping. These analyses were useful for identifying unknown cellular origin as well as for reconsidering the group of bifenotypic leukaemias in respect to myeloid or multipotent origins. The percentage of inapropriate Ig and TCR gene rearrangements in T and B acute lymphoblastic leukaemias respectively indicated that molecular analyses must be routinely used for understanding the biology of the disease. The use of PCR in conjunction with immunophenotyping for diagnosing clonality in T and B leukaemias showed consistent results in B cell leukaemias. Conversely, PCR was preferable to immunophenotyping for diagnosis of T cell leukaemias. Our protocol, once established, was also useful for monitoring minimum residual disease and relapse. Finally, general aspects of leukaemia biology, regulation of Ig and TCR genes, and involvements of l4q32 and l4qll regions, where these genes are respectively located, were studied in different types of acute leukaemias. The establishment of a new cell line (CEMO-l) lead to the identification and cloning of the new oncogene BCL-9.