Claudete E. Klumb

Relationship of Epstein-Barr Virus and Interleukin 10 Promoter Polymorphisms with the Risk and Clinical Outcome of Childhood Burkitt Lymphoma

Epstein-Barr virus (EBV) is an important environmental factor associated to the development of Burkitt lymphoma (BL) in endemic and intermediate risk regions. However, little is known about the contribution of genetic constitution to the development and clinical response of the disease. The aim of this work was to investigate the role of EBV and Interleukin 10 (IL10) single nucleotide polymorphisms (−1082A/G, −819C/T, −592C/A) and microsatellites (IL10.R and IL10.G) in susceptibility and clinical outcome in pediatric BL patients, in a region with intermediate EBV association frequency. The frequencies of IL10 promoter Single nucleotide polymorphisms −1082A/G, −819C/T, −592C/A, and IL10.R and IL10.G microsatellites were compared in 62 pediatric patients and 216 healthy donors. IL10 −1082GG and GCC/GCC genotypes were more frequent in patients than in controls, and associated to a higher risk of BL development (GG genotype OR 2.62, 95% CI, 1.25–5.51; P = 0.008; Pc = 0.024). EBV was detected in tumor samples by EBER-ISH in 54.1% of cases. EBV+ patients exhibited a better event free survival (EFS) (P = 0.019) than EBV− patients. Carriers of IL10 R3-GCC had worse EFS (P = 0.028). Our results suggest a risk effect and an independent prognostic value of IL10 polymorphisms and EBV in childhood BL patients.

Avaliação dos métodos de detecção das alterações do gene e proteína P53 nas neoplasias linfóides

The p53 protein plays a central role in cellular responses, including cycle arrest and cell death in response to DNA damage. Dysfunction of this protein can induce abnormal cell growth, increased cell survival, and drug resistance. The p53 tumor suppressor gene is altered in many types of human cancer including hematological malignancies. Most of the p53 biologically significant mutations impair the ability of protein to act as a transcriptional regulator. Unlike functional p53, which is rapidly degraded after its synthesis, mutated forms tend to accumulate in the cells, thus becoming detectable by immunohistochemistry. Its has been reported that mutations of p53 gene occur with a frequency of approximately 12.5% in lymphoid malignancies. However, aggressive non-Hodgkins lymphomas (NHL), especially Burkitts lymphoma, show higher frequencies. Most p53 gene mutations in NHL are missense mutations, stabilising the functionally defective protein, but wild-type form of p53 protein also has a high p53 expression detected by immunohistochemistry indicating a discrepancy between gene mutation and protein detection. The aim of this work is to perform a comprehensive review of the methods used to identify gene alterations and p53 protein expression in lymphoid neoplasias in order to investigate its involvement in these neoplasias.

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 VDJCμ 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.

Resistência ao tratamento no linfoma de Burkitt: associação com mutações específicas no gene TP53?

Burkitts lymphoma (BL) originates from a germinative centre cell that loses proliferation control due to activation of the c-myc gene. Apoptosis resistance is a major cause of chemotherapy failure in most kinds of cancers, including BL. The high rate of apoptosis seen in the early steps of genesis of BL is followed by a subsequent development of inactivation of pathways leading to cell death by apoptosis. A major pathway known to be altered in BL is the one mediated by the p53 protein. This pathway is important to control cell proliferation in response to DNA damage. Data from the literature show a correlation between TP53 gene mutations and treatment resistance. However, some studies have demonstrated that distinct types of mutations have the ability to confer different cell responses to chemotherapy. We found that BL cell lines bearing distinct mutations of p53 also present different responses to drug-induced apoptosis, when using drugs that act through this pathway, such as doxorubicin. Different types of mutations might confer distinct functional phenotypes. Loss of function does not always occur which may be considered an important component of chemotherapy resistance in BL. In this article we review publications regarding the response to treatment in BL while we discuss the role of TP53 gene mutations in chemotherapy resistance of these tumors.

A step towards the cure of Burkitt's lymphoma in developing countries.

The outcome of sporadic Burkitts lymphoma (BL) in high-income countries may be considered excellent due to its overall cure rate of roughly 90%(1-4). Three major US and European childhood cancer groups [Lymphoma malignancy B-cell (LMB), BerlinFrankfurt-Munster (BFM), Childrens Cancer Group/Children Oncology Group (CCG/COG)] contributed to the most effective treatment strategies to date. Nevertheless, the treatment has pronounced toxic effects such as long periods of mucositis, hematological toxic effects as well as the potential risk of severe infection. Assuredly, the improvement of supportive care has contributed to these better outcomes(1-4). On the other hand, in low-income countries, therapeutic schemes need to be modified in accordance with local conditions in order to avoid unacceptable treatment-related mortality(5). In this issue Cunha et al.(6) present a retrospective study of 50 cases of BL in children and adolescents who were treated with chemotherapy regimens containing intermediate methotrexate doses (500 mg/m2). The probability of overall survival was 73% (median follow-up of35 months). The data show a favorable step with improvement in the results of BL treatment in Brazil even considering the high mortality rate (23.8%) observed in the study. Reinforcing the predicted tumor burden value in the prognosis of BL patients, survival was significantly lower for patients with uric acid levels > 7 mg/dL. In addition, these results may also show that the impact of methotrexate on the overall success may be of less importance in patients with lower tumor load compared with those with larger tumor masses(7). Thus, the efficacy of treatment may depend as much on the number of drugs used (at least 4-6 drugs systemically) as on their doses and so lower dose multidrug regimens may improve outcome without comparable increases in toxicity(8). Although the study by Cunha et al.(6) provides important information on the approach to BL care in Brazil, there are still several areas to be addressed. Differences among survival rates in a large country such as Brazil are worthy of comment. The mean tumor burden at diagnosis has an important impact on the outcome(9). Even though the Brazilian Government Healthcare System (SUS - Sistema Unico de Saude) covers most chemotherapy drug costs, primary healthcare should be improved for earlier diagnosis. Moreover, supportive care facilities remain inadequate in some Brazilian regions where resources are limited. Enhancement of laboratory monitoring, aggressive hydratation and the use of urate oxidase in patients with high risk of tumor lysis syndrome will result in fewer toxic events and better treatment outcomes(10). The availability of rituximab in the SUS to treat B-cell lymphomas in adult patients has ledto the possibility of obtaining excellent results in children with less toxicity by combining thisdrug with chemotherapy. However, there is a question as to whether rituximab is an effectivedrug in pediatric B-cell non-Hodgkin lymphoma (B-NHL). This drug was tested in a phaseII short-window study showing that it can be safely added to a pediatric chemotherapeuticregimen(11). Even so, only controlled clinical trials will allow an evaluation of the role of thisdrug in the treatment of pediatric B-NHL. Taking these considerations into account, results like those of high-income countries may be achieved by directing research towards designing protocol regimens in formal clinical trials with chemotherapy tailored by tumor burden. The primary goal of therapy studies in developing countries today is to define risk groups as accurately as possible, so that the patients receive therapy consistent with the best possible outcome while further reduction of acute and chronic toxicity may be achieved.

Second Epstein-Barr Virus–Associated Burkitt's Lymphoma of the CNS in a Child With Progressive Renal Failure

A 14-year-old boy was admitted to the Hematology Service in November 2002, with CNS involvement. The patient had been diagnosed with Burkitt’s lymphoma (BL) in March 1998, with pelvic mass presentation, severe ascitis, and bone marrow infiltration (St. Jude stage IV) associated with acute renal failure and tumor lysis syndrome. Magnetic resonance imaging (MRI) of the pelvis showed a solid mass, producing bone lesion by contiguity in L5 and parts of the sacrum, penetrating the right neural foramen in L5 with invasion of vertebral canal and neural root compression. Immediate Berlin-FrankfurtMuenster–based treatment resulted in complete clinical remission, although the patient continued to experience a neurogenic bladder and progressive deterioration of renal function. A month before his second admission, neurologic involvement was apparent, with seizures and temporary loss of consciousness. At his second admission, the patient showed 11,000/ L leukocyte, 343,000/ L platelets, 8.2 g/dL hemoglobin, 288 UI/L lactate dehydrogenase (normal range, 240 to 480 UI/L), 5.80 mg/dL uric acid, 8.34 mg/dL calcium, 5.60 mol/L potassium, and 3.22 mg/dL serum creatinine. Viral serology was negative for human T-cell leukemia-lymphoma virus I and II, hepatitis A virus, and hepatitis C virus. Investigation of HIV infection, performed several times along the course of his second disease and afterward, yielded negative results. Brain MRI revealed a hypodense mass in the right temporal-occipital region (Fig 1A), although abdominal computed tomography scan and a bone marrow aspirate did not show tumor involvement. A stereotactic biopsy of the cerebral mass followed by histopathologic analysis showed a CD20 , CD10 , CD3 and BCL2 –non-Hodgkin’s lymphoma, compatible with BL diagnosis, which was confirmed by interphase fluorescent in situ hybridization (I-FISH). Due to severe renal failure (creatinine clearance 20 mL/min by Cockcroft-Gault formula), whole-brain radiotherapy was administered, with 36 Gy divided in 20 fractions, and a boost of 8 Gy in four fractions. Figure 1B shows brain MRI after the end of treatment. The patient achieved complete clinical remission and is at present being observed by clinicians. He has not relapsed or shown signs of neurotoxicity related to whole-brain radiotherapy. DNA was extracted from pathology specimens of the first tumor (frozen cytospins), and from the small biopsy of the second tumor together with RNA and cell imprintings. In both tumors, Epstein-Barr virus (EBV) was detected by specific polymerase chain reaction (PCR) analyses and, in the second tumor, by EBV-encoded RNA in situ hydridization (Fig 2A), though without expressing LMP1 oncoprotein (clones CS1-4; DAKO; Glostrup, Denmark). A t(8;14)(q24;q32) translocation was detected by I-FISH with LSI IGH/MYC, CEP 8 tri-color, dual fusion translocation probes (Vysis; Downers Grove, IL). Figures 2B and 2C show I-FISH images of normal brain cells and a tumor cell, respectively. Arrows show two co-localized signals corresponding to t(8;14). Immunoglobulin (Ig) clonal gene rearrangements amplified by Framework Region 2 (FR2)–JH nested PCR showed clearly different clonality patterns. Figure 3 shows a monoallelic clonal rearrangement superimposed on a polyclonal background in the first tumor, and in the second tumor, two rearrangements of different size from the one observed in the first tumor (Fig 3). These results suggested that the tumors were clonally unrelated, and that the second tumor was a primary CNS lymphoma (PCNSL). The Variable Diversity–Joining rearrangements of the Ig variable region from the first tumor (FR2-JH fragments) and from the second tumor (cDNA amplified with VH1/7 family primers), after subcloning in a pCR2.1 vector (Invitrogen; Carlsbad, CA), were sequenced and compared with germline, V-BASE sequence data (http://www.mrc-cpe.cam.uk/) using MacVector 10.0 software (Accelrys, Inc, San Diego, CA). As expected from gel analysis, the first tumor showed a VH4-2/JH4b unmutated rearrangement coexisting with a large number of polyclonal rearrangements, mostly involving VH3 and VH4 families. Conversely, the second tumor showed two in-frame Variable Diversity–Joining rearrangements, V205/JH4b and V3-30/D3-22/JH4b, with 98.01% and 98.13% homology with germline alleles, respectively. The V3-30 allele showed intraclonal variation. In all VH genes, S (silent) and R (replacement) mutations were randomly distributed in complementarity-determining or framework regions, suggesting the absence of B-cell receptor–mediated selection in tumor cells. Activation-induced cytidin deaminase (AID) gene expression was quantified by real-time PCR in the second tumor, by a TaqMan assay (Applied Biosystems; Foster City, CA; inventoried assay), showing an approximately eight-fold lower AID expression in tumor cells with respect to quiescent, mononuclear peripheral-blood cells used for calibration. PCNSL are aggressive non-Hodgkin’s lymphomas the incidence of which has been increasing in immunocompetent patients in the last few years. More than 90% of PCNSL patients have diffuse large B-cell lymphomas (DLCBLs); the remaining 10% comprise a suite of rare and poorly-characterized lymphomas, including BL. Conversely to HIV-associated patients, PCNSLs in immunocompetent individuals A B

Chromosome 17 abnormalities and mutation of the TP53 gene: correlation between cytogenetics, flow cytometry and molecular analysis in three cases of chronic myeloid leukemia

chronic myeloid leukemia (CML) have been described. This chromosomal region contains the tumor suppressor gene TP53 that may be an important factor in the evolution of this disease. In this study, we used flow cytometry and western blotting to assess p53 protein expression and single stranded conformational polymorphism to examine TP53 gene alterations in three patients with CML who showed alterations in 17p. Only the case with del(17)(p11) had p53 expression positive by flow cytometry and an abnormal migration pattern by SSCP analysis. The importance of the correlation between the results obtained with these techniques, as well as the clinical course of the patients, are discussed.

Estudo imunomolecular dos linfomas não-Hodgkin de origem B na infância: correlação clínica e terapêutica

A proteina p53 desempenha um papel central nas respostas celulares dentre as quais se incluem o controle do ciclo e da morte celular induzida por dano ao DNA, como aquele causado por muitos agentes quimioterapicos e radiacao utilizados no tratamento do câncer. O proposito deste estudo foi descrever as funcoes biologicas da proteina p53 e investigar o seu papel nos linfomas nao Hodgkin de origem B da infância. Adicionalmente, nos tambem estudamos os tipos histologicos e a prevalencia da infeccao pelo virus Epstein-Barr e sua correlacao com os achados clinicos patologicos. Uma serie de criancas com linfoma nao Hodgkin B foi estudada com relacao aos subtipos histologicos, caracteristicas clinicas, mutacoes do gene TP53 e expressao das proteinas p53, Ki-67 e mdm2. A deteccao de mutacoes do gene TP53 foi realizada atraves da tecnica de PCR-SSCP dos exons 5-8/9 e sequenciamento direto em 49 do total de 61 pacientes da serie. As mutacoes do gene TP53 foram detectadas em 22.5por cento dos pacientes analisados, em 20por cento dos pacientes com linfoma de Burkitt. A analise das sequencias destes casos mostrou a presenca de mutacoes do tipo pontual em 10 casos e uma insercao em um caso. A expressao da proteina p53 por imunohistoquimica foi realizada em 48 do total de 61 pacientes com resultados positivos em 31por cento dos casos. A proteina mdm2 foi negativa em todos os casos testados (42casos), incluindo aqueles com mutacao do gene TP53. Observou-se uma alta concordância entre a expressao da p53 e a presenca de mutacoes (p=0.0005). Nao foi detectada uma correlacao estatisticamente significante entre mutacoes e os achados clinicos. A comparacao da sobrevida livre de eventos entre os grupos com e sem mutacao usando o teste de Long-Rank tambem nao foi significante. O virus EBV foi analisado por hibridizacao in situ e estava associado ao LB em 72por cento dos tumores(21/29 pacientes). O tipo 1 infectou a maioria dos casos(28/29). Houve uma tendencia de associacao entre idade mais baixa e os casos de linfoma de Burkitt associados ao EBV (mediana de 4 anos comparada a 6 anos, respectivamente, p=0.057). Tambem o nosso estudo sugeriu que na regiao sudeste do Brasil o LB tem uma associacao intermediaria com o EBV. Em conclusao, este estudo permitiu contribuir para uma melhor caracterizacao imunofenotipica e molecular dos linfomas nao Hodgkin B da infância no Brasil.