Quantitative analysis of CDKN2A methylation, mRNA, and p16(INK4a) protein expression in children and adolescents with Burkitt lymphoma: biological and clinical implications.

CDKN2A is a tumor suppressor gene critical in the cell cycle regulation. Little is known regarding the role of CDKN2A methylation in the pathogenesis of Burkitt lymphoma (BL). CDKN2A methylation was investigated using pyrosequencing in 51 tumor samples. p16(INK4a) mRNA and protein levels were measured using real-time PCR and immunohistochemistry, respectively. CDKN2A methylation was detectable in 72% cases. Nuclear expression of p16(INK4a) was not detected in 41% cases. There was an association between methylation and absence of CDKN2A mRNA (P=0.003). In conclusion, CDKN2A methylation occurs at a high frequency suggesting a role in BL pathogenesis and potential therapeutic implications.

The concurrent occurrence of Leishmania chagasi infection and childhood acute leukemia in Brazil.

OBJECTIVE: This study investigated the co-existence of Leishmania chagasi infection and childhood leukemia in patients naïve to treatment; this has serious clinical and epidemiological implications. METHODS: The seroprevalence of L. chagasi antibodies prior to any treatment was investigated in children with clinical features of acute leukemia. Serological tests were performed in 470 samples drawn from under 14-year-old children from different regions of Brazil with clinical suspicion of acute leukemia. Acute leukemia subtypes were characterized by immunophenotyping using flow cytometry. Morphological analyses of bone marrow aspirates were systematically performed to visualize blast cells and/or the formation of L. chagasi amastigotes. Data analysis used a standard univariate procedure and the Pearson's chi-square test. RESULTS: The plasma of 437 children (93%) displayed antibodies against L. chagasi by indirect immunofluorescence assay and enzyme-linked immunosorbent assay tests. Of the 437 patients diagnosed from 2002 to 2006, 254 had acute lymphoblastic leukemia, 92 had acute myeloid leukemia, and 91 did not have acute leukemia. The seroprevalence of L. chagasi antibodies according to the indirect immunofluorescence assay test (22.5%) was similar in children with or without acute leukemia (p-value=0.76). The co-existence of visceral leishmanasis and acute leukemia was confirmed in 24 children. The overall survival of these children was poor with a high death rate during the first year of leukemia treatment. CONCLUSION: In the differential diagnosis of childhood leukemia, visceral leishmanasis should be considered as a potential concurrent disease in regions where L. chagasi is endemic.

The concurrent occurrence of Leishmania chagasi infection and childhood acute leukemia in Brazil

Objective: This study investigated the co-existence of Leishmania chagasi infection and childhood leukemia in patients naïve to treatment; this has serious clinical and epidemiological implications. Methods: The seroprevalence of L. chagasi antibodies prior to any treatment was investigated in children with clinical features of acute leukemia. Serological tests were performed in 470 samples drawn from under 14-year-old children from different regions of Brazil with clinical suspicion of acute leukemia. Acute leukemia subtypes were characterized by immunophenotyping using flow cytometry. Morphological analyses of bone marrow aspirates were systematically performed to visualize blast cells and/or the formation of L. chagasi amastigotes. Data analysis used a standard univariate procedure and the Pearson's chi-square test. Results: The plasma of 437 children (93%) displayed antibodies against L. chagasi by indirect immunofluorescence assay and enzyme-linked immunosorbent assay tests. Of the 437 patients diagnosed from 2002 to 2006, 254 had acute lymphoblastic leukemia, 92 had acute myeloid leukemia, and 91 did not have acute leukemia. The seroprevalence of L. chagasi antibodies according to the indirect immunofluorescence assay test (22.5%) was similar in children with or without acute leukemia (p-value = 0.76). The co-existence of visceral leishmanasis and acute leukemia was confirmed in 24 children. The overall survival of these children was poor with a high death rate during the first year of leukemia treatment. Conclusion: In the differential diagnosis of childhood leukemia, visceral leishmanasis should be considered as a potential concurrent disease in regions where L. chagasi is endemic...

O papel das infecções virais na etiologia da leucemia linfoblástica aguda comum analisado através de uma abordagem epigenômica

A leucemia linfoblástica aguda (LLA) e o principal subtipo de câncer pediátrico e, apesar do sucesso no tratamento, a sua causa continua enigmática. Acredita-se que o processo que da origem a LLA envolve dois eventos: o primeiro seria responsável por criar o clone pre-leucêmico a partir de mutações ou alterações epigenéticas e o segundo evento iria disparar a proliferação deste clone. Existem evidencias de que as infecções desempenham função importante na leucemogênese. Com o objetivo de investigar se as infecções tem um papel na etiologia das LLAs infantis foram usadas três abordagens: 1) analise do perfil de expressão, por PCR em tempo real, de genes relacionados a via do interferon a fim de avaliar a resposta imune aberrante como fator desencadeador de LLA-c; 2) analise do perfil de mitigação das LLAs, pela técnica de microarranjos, a fim de identificar possíveis alterações causadas por infecções virais e, 3) rastreamento de DNA de adenovírus em cartões do teste do pezinho, a fim de verificar se este vírus poderia ser responsável pela leucemogênese diretamente. Para os estudos de perfil de expressão gênica e mitigação foram utilizadas amostras de 121 casos de LLA infantil e para o estudo de rastreamento de AdV foram utilizadas 202 amostras de cartões Guthrie, entre casos de LLA e controles. Dentre os doze genes estudados, dois (LY6E e MX1) apresentaram expressão 4 e 2,5 vezes mais alta nos casos de LLA-c quando comparados aos outros subtipos de LLA (pré-B e pro-B), respectivamente. Existe uma associação entre os níveis de IgM anti-PVB19 e superexpressão dos genes estudados. Através da analise pelo beadarray, observou-se que os subtipos de leucemia podem ser discriminados de acordo com o perfil de mitigação de alguns genes tais como, BMP3, DAB2,NQO1, DAPK, WNT1 e IFNG. Alem disso, também foi vista uma associação entre infecção pelo PVB19 e mitilação em alguns genes, DAPK, PTGS2, MPO and NRAS. Os AdV humanos A, C e F não foram detectados por PCR no sangue de cartões do teste do pezinho em crianças que desenvolveram LLA, nem em controles saudáveis. A ausência de DNA viral nestes cartões não constitui uma evidencia negativa do papel das infecções na etiologia das leucemias. O AdV ou outro vírus, p.ex, PVB19, poderia estar influenciando o processo leucemogênico através de um mecanismo de “hit and run”, que alteraria o perfil epigenético da célula, causando hipoexpressão de genes que controlam a proliferação e diferenciação celular. Embora o mecanismo ainda não seja claro, nossos dados, corroboram a idéia de que as infecções desempenham um papel na etiologia das LLA-c e que esse efeito, muito provavelmente, e pós-natal

Acute lymphoblastic leukemia (ALL) is the main pediatric cancer subtype and, despite the success in treatment, its cause remains enigmatic. It is believed that the onset of ALL is based in a two hit model: the first hit would promote mutations or epigenetic alterations that create a preleukemic clone and the second one would precipitate ALL through a proliferative expansion of this clone. There are evidences that infections influence the leukemogenesis process. We sought to investigate whether infections have a role in the childhood ALL etiology. We used three approaches to analyze this hypothesis: 1) expression profiling of IFN related genes, by real time PCR, to evaluate the role of aberrant immune response as a trigger of c-ALL; analysis of ALL methylation profile using microarrays to identify possible alterations caused by viral infections and, 3) adenovirus (AdV) DNA screening in Guthrie cards(GC). Samples of 121 childhood ALL cases were used for expression and methylation profiling studies and 202 GC samples from children who later developed leukemia and controls were used for Adv screening. Inside a group of 12 analyzed genes, LY6E and MX1 presented higher expression levels in c-ALL when compared to the other subtypes (pro B and pre B ALL). These results suggest that somehow immune response is related to c-ALL. There is an association between anti-PVB19 IgM levels and higher expression of studied genes. Using the beadarray analysis we observed that leukemia subtypes could be distinguished based on methylation profiling of some genes such as BMP3, DAB2, NQO1, DAPK, WNT1 and IFNG. Moreover, it was observed an association between PVB19 infection and methylation of some genes for instance, DAPK, PTGS2, MPO and NRAS. AdV subtypes A, C, F were not detected by PCR in GC from ALL cases nor in controls. The absence of viral DNA in these cards does not constitute a negative evidence for the role of infections in the etiology of ALL. Adv or other viruses could influence in leukemogenesis through a hit and run mechanism which could alter cell epigenetic profile. These alterations would lead to low expression of some genes that control cell differentiation and proliferation. Although the mechanisms are not clear yet, our results sustain the idea that infections have a role in c-ALL development and this effect is probably post-natal

O papel das infecções virais na etiologia da leucemia linfoblástica aguda comum analisado através de uma abordagem epigenômica / The role of viral infections in the etiology of common acute lymphoblastic leukemia analysed through epigenomic approach

A leucemia linfoblástica aguda (LLA) é o principal subtipo de câncer pediátrico e, apesar do sucesso no tratamento, a sua causa continua enigmática. Acredita-se que o processo que dá origem a LLA envolve dois eventos: o primeiro seria responsável por criar o clone pré-leucêmico a partir de mutações ou alterações epigenéticas e o segundo evento iria disparar a proliferação deste clone. Existem evidências de que as infecções desempenham função importante na leucemogênese. Com o objetivo de investigar se as infecções têm um papel na etiologia das LLAs infantis foram usadas três abordagens: 1) análise do perfil de expressão, por PCR em tempo real, de genes relacionados à via do interferon a fim de avaliar a resposta imune aberrante como fator desencadeador de LLA-c; 2) análise do perfil de metilação das LLAs, pela técnica de microarranjos, a fim de identificar possíveis alterações causadas por infecções virais e, 3) rastreamento de DNA de adenovírus em cartões do teste do pezinho, a fim de verificar se este vírus poderia ser responsável pela leucemogênese diretamente. Para os estudos de perfil de expressão gênica e metilação foram utilizadas amostras de 121 casos de LLA infantil e para o estudo de rastreamento de AdV foram utilizadas 202 amostras de cartões Guthrie, entre casos de LLA e controles. Dentre os doze genes estudados, dois (LY6E e MX1) apresentaram expressão 4 e 2,5 vezes mais alta nos casos de LLA-c quando comparados aos outros subtipos de LLA (pre-B e pro-B), respectivamente. Existe uma associação entre os níveis de IgM anti-PVB19 e superexpressão dos genes estudados. Através da análise pelo beadarray, observou-se que os subtipos de leucemia podem ser discriminados de acordo com o perfil de metilação de alguns genes tais como, BMP3, DAB2...

Acute lymphoblastic leukemia (ALL) is the main pediatric cancer subtype and, despite the success in treatment, its cause remains enigmatic. It is believed that the onset of ALL is based in a two hit model: the first hit would promote mutations or epigenetic alterations that create a preleukemic clone and the second one would precipitate ALL through a proliferative expansion of this clone. There are evidences that infections influence the leukemogenesis process. We sought to investigate whether infections have a role in the childhood ALL etiology. We used three approaches to analyze this hypothesis: 1) expression profiling of IFN related genes, by real time PCR, to evaluate the role of aberrant immune response as a trigger of c-ALL; analysis of ALL methylation profile using microarrays to identify possible alterations caused by viral infections and, 3) adenovirus (AdV) DNA screening in Guthrie cards(GC). Samples of 121 childhood ALL cases were used for expression and methylation profiling studies and 202 GC samples from children who later developed leukemia and controls were used for Adv screening. Inside a group of 12 analyzed genes, LY6E and MX1 presented higher expression levels in c-ALL when compared to the other subtypes (pro B and pre B ALL). These results suggest that somehow immune response is related to c-ALL. There is an association between antiPVB19 IgM levels and higher expression of studied genes. Using the beadarray analysis we observed that leukemia subtypes could be distinguished based on methylation profiling of some genes such as BMP3, DAB2...