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Relatamos os resultados da análise cito genética de três casos de tumor de Wilms em crianças. O estudo cromossômico foi realizado a partir do material obtido de cultura de curta duração de células tumorais. Observamos alterações numéricas e estruturais, incluindo aberrações estruturais do cromossomo 1 e trissomias dos cromossomos 8 e 12.

Aberrant expression of long non-coding RNAs (lncRNAs) has been detected in several types of cancer, including acute lymphoblastic leukemia (ALL), but lncRNA mapped on transcribed ultraconserved regions (T-UCRs) are little explored. The T-UCRs uc.112, uc.122, uc.160 and uc.262 were evaluated by quantitative real-time PCR in bone marrow samples from children with T-ALL (n = 32) and common-ALL/pre-B ALL (n = 30). In pediatric ALL, higher expression levels of uc.112 were found in patients with T-ALL, compared to patients with B-ALL. T-cells did not differ significantly from B-cells regarding uc.112 expression in non-tumor precursors from public data. Additionally, among B-ALL patients, uc.112 was also found to be increased in patients with hyperdiploidy, compared to other karyotype results. The uc.122, uc.160, and uc.262 were not associated with biological or clinical features. These findings suggest a potential role of uc.112 in pediatric ALL and emphasize the need for further investigation of T-UCR in pediatric ALL.

Abstract SHOC2 scaffold protein has been mainly related to oncogenic ERK signaling through the RAS-SHOC2-PP1 phosphatase complex. In leukemic cells however, SHOC2 upregulation has been previously related to an increased 5-year event-free survival of pediatric pre-B acute lymphoid leukemia, suggesting that SHOC2 could be a potential prognostic marker. To address such paradoxical function, our study investigated how SHOC2 impact leukemic cells drug response. Our transcriptome analysis has shown that SHOC2 can modulate the DNA-damage mediated by p53. Notably, upon genetic inhibition of SHOC2 we observed a significant impairment of p53 expression, which in turn, leads to the blockage of key apoptotic molecules. To confirm the specificity of DNA-damage related modulation, several anti-leukemic drugs has been tested and we did confirm that the proposed mechanism impairs cell death upon daunorubicin-induced DNA damage of human lymphoid cells. In conclusion, our study uncovers new insights into SHOC2 function and reveals that this scaffold protein may be essential to activate a novel mechanism of p53-induced cell death in pre-B lymphoid cells.

Abstract Next-generation sequencing platforms are routinely used for molecular assignment due to their high impact for risk stratification and prognosis in medulloblastomas. Yet, low and middle-income countries still lack an accurate cost-effective platform to perform this allocation. TaqMan Low Density array (TLDA) assay was performed using a set of 20 genes in 92 medulloblastoma samples. The same methodology was assessed in silico using microarray data for 763 medulloblastoma samples from the GSE85217 study, which performed MB classification by a robust integrative method (Transcriptional, Methylation and cytogenetic profile). Furthermore, we validated in 11 MBs samples our proposed method by Methylation Array 450 K to assess methylation profile along with 390 MB samples (GSE109381) and copy number variations. TLDA with only 20 genes accurately assigned MB samples into WNT, SHH, Group 3 and Group 4 using Pearson distance with the average-linkage algorithm and showed concordance with molecular assignment provided by Methylation Array 450 k. Similarly, we tested this simplified set of gene signatures in 763 MB samples and we were able to recapitulate molecular assignment with an accuracy of 99.1% (SHH), 94.29% (WNT), 92.36% (Group 3) and 95.40% (Group 4), against 97.31, 97.14, 88.89 and 97.24% (respectively) with the Ward.D2 algorithm. t-SNE analysis revealed a high level of concordance (k = 4) with minor overlapping features between Group 3 and Group 4. Finally, we condensed the number of genes to 6 without significantly losing accuracy in classifying samples into SHH, WNT and non-SHH/non-WNT subgroups. Additionally, we found a relatively high frequency of WNT subgroup in our cohort, which requires further epidemiological studies. TLDA is a rapid, simple and cost-effective assay for classifying MB in low/middle income countries. A simplified method using six genes and restricting the final stratification into SHH, WNT and non-SHH/non-WNT appears to be a very interesting approach for rapid clinical decision-making.

Glioblastoma (GBM) is one of the deadliest tumors and has a median survival of 3 months if left untreated. Despite advances in rationally targeted pharmacological approaches, the clinical care of GBM remains palliative in intent. Since the majority of altered signaling cascades involved in cancer establishment and progression eventually affect cell cycle progression, an alternative approach for cancer therapy is to develop innovative compounds that block the activity of crucial molecules needed by tumor cells to complete cell division. In this context, we review promising ongoing and future strategies for GBM therapeutics aimed towards G2/M inhibition such as anti-microtubule agents and targeted therapy against G2/M regulators like cyclin-dependent kinases, Aurora inhibitors, PLK1, BUB, 1, and BUBR1, and survivin. Moreover, we also include investigational agents in the preclinical and early clinical settings. Although several drugs were shown to be gliotoxic, most of them have not yet entered therapeutic trials. The use of either single exposure or a combination with novel compounds may lead to treatment alternatives for GBM patients in the near future.

Câncer é um assunto frequente na mídia e no cotidiano de muitos estudantes, uma vez que ainda representa um problema de saúde pública com elevado apelo emocional e social. A falta do senso crítico no processo de ensino e aprendizagem do câncer é evidenciada pela defasagem de informações e conceitos científicos básicos durante a formação escolar. Nesse contexto, o projeto de extensão intitulado “A genética dos soldados contra o câncer”, teve como objetivo principal compartilhar conhecimentos acadêmicos acerca da gênese, genética e imunologia do câncer com alunos da rede básica de ensino e com a comunidade. O projeto foi desenvolvido na Casa da Ciência da Fundação Hemocentro de Ribeirão Preto como parte do programa educacional “Pequeno Cientista” e contou com a participação de nove alunos da rede básica de ensino, orientados por três pós-graduandos, durante 12 encontros semanais com uma hora de duração. As aulas foram expositivas argumentativas e práticas, tendo sido utilizadas como ferramentas pedagógicas jogos, debates, infográficos e vídeos. A abordagem de uma temática pertinente à realidade dos alunos e o uso de estratégias pedagógicas alternativas possibilitou a apropriação do conhecimento e a assimilação significativa dos conceitos abordados. Dessa forma, esse projeto contribuiu para o processo de formação dos alunos e difusão destes conhecimentos à comunidade, além de viabilizar o exercício didático e crítico aos pós-graduandos. Palavras-chave: Ensino de Genética; Imunologia; Câncer, Educação; Extensão Universitária Cancer genetics and immunology for students of primary school: Reporting an experience Abstract: Cancer is a common issue in the media and the daily lives of many students, once it still represents a public health problem with a high emotional and social appeal. The lack of critical sense in the process of teaching and learning about cancer is evidenced by the absence of information and basic scientific concepts during school education. In this context, the extension project entitled "The genetics of soldiers against cancer" had as main objective to share and propagate academic knowledge about genetics and cancer immunology with students of the primary education system and with the community. The project was developed at Hemocentro Foundation of Ribeirão Preto, São Paulo, Brazil, as part of the educational program "Small Scientist" and was attended by nine students from the primary education, supervised by three post-graduate students during 12 weekly meetings with one hour of duration. The classes were dialogued and argumentative lectures, with practical activities, using pedagogical tools like games, debates, infographics, and videos. The approach of themes inserted into the reality of the students and the use of alternative\didactic models led the students to the appropriation of knowledge and assimilation of important concepts on genetics, cancer, and immunology. Thus, this project contributed to the process of student educational training, and to propagate this knowledge to the community, besides allowing a didactic and critical exercise for the post-graduation students. Keywords: Teaching genetics; Immunology; Cancer; Education; University Extension Genética e inmunología del cáncer para alumnos de la escuela primaria: Relato de una experiencia Resumen: El cáncer es un tema frecuente en los medios de comunicación y en el cotidiano de muchos estudiantes, una vez que representa un problema de salud pública con alto apelo emocional y social. La falta de pensamiento crítico en el proceso de enseñanza y aprendizaje sobre cáncer es evidente por el desfase de informaciones y conceptos científicos básicos durante la formación escolar. En ese contexto, el proyecto de extensión intitulado “La genética dos soldados contra el cáncer” tuvo como objetivo principal compartir y propagar el conocimiento académico sobre la génesis, genética e inmunología del cáncer, con alumnos de la red básica de educación y con la comunidad. El proyecto fue realizado en la Casa de la Ciencia de la Fundación Hemocentro de Ribeirão Preto, São Paulo, Brasil, como parte del programa educacional “Pequeño Científico” y contó con la participación de nueve alumnos de la red básica de educación, supervisados por tres alumnos de posgrado, durante 12 encuentros semanales de una hora de duración. Las clases fueron magistrales con diálogos e argumentaciones, y prácticas, siendo usados como herramientas pedagógicas juegos, debates, gráficos y videos. El abordaje de un tema inserido en la realidad de los alumnos y el uso de modelos didácticos alternativos indujo a los alumnos a la apropiación del conocimiento y asimilación significativa de los conceptos abordados. De esa forma, este proyecto contribuyó con el proceso de formación de los alumnos y con la propagación del conocimiento para la comunidad; además de permitir un ejercicio didáctico y crítico para los alumnos de posgrado. Palabras-clave: Enseñanza de Genética; Inmunología; Cáncer; Educación; Extensión Universitaria

Propolis is a resin formed by a complex chemical composition of substances that bees collect from plants. Since ancient times, propolis has been used in folk medicine, due to its biological properties, that include antimicrobial, anti-inflammatory, antitumoral and immunomodulatory activities. Glioblastoma is the most common human brain tumor. Despite the improvements in GBM standard treatment, patients' prognosis is still very poor. The aim of this work was to evaluate in vitro the Tubi-bee propolis effects on human glioblastoma (U251 and U343) and fibroblast (MRC-5) cell lines. Proliferation, clonogenic capacity and apoptosis were analyzed after treatment with 1 mg/mL and 2 mg/mL propolis concentrations for different time periods. Additionally, glioblastoma cell lines were submitted to treatment with propolis combined with temozolomide (TMZ). Data showed an antiproliferative effect of tubi-bee propolis against glioblastoma and fibroblast cell lines. Combination of propolis with TMZ had a synergic antiproliferative effect. Moreover, propolis caused decrease in colony formation in glioblastoma cell lines. Propolis treatment had no effects on apoptosis, demonstrating a cytostatic action. Further investigations are needed to elucidate the molecular mechanism of the antitumor effect of propolis, and the study of its individual components may reveal specific molecules with antiproliferative capacity.

Chromosomal translocations are characteristic of hematopoietic neoplasias and can lead to unregulated oncogene expression or the fusion of genes to yield novel functions. In recent years, different lymphoma/leukemia-associated rearrangements have been detected in healthy individuals. In this study, we used inverse PCR to screen peripheral lymphocytes from 100 healthy individuals for the presence of MLL (Mixed Lineage Leukemia) translocations. Forty-nine percent of the probands showed MLL rearrangements. Sequence analysis showed that these rearrangements were specific for MLL translocations that corresponded to t(4;11)(q21;q23) (66%) and t(9;11) (20%). However, RT-PCR failed to detect any expression of t(4;11)(q21;q23) in our population. We suggest that 11q23 rearrangements in peripheral lymphocytes from normal individuals may result from exposure to endogenous or exogenous DNA-damaging agents. In practical terms, the high susceptibility of the MLL gene to chemically-induced damage suggests that monitoring the aberrations associated with this gene in peripheral lymphocytes may be a sensitive assay for assessing genomic instability in individuals exposed to genotoxic stress.

Fanconi anaemia (FA) is a recessive autosomal disease determined by mutations in genes of at least eleven complementation groups, with distinct distributions in different populations. As far as we know, there are no reports regarding the molecular characterisation of the disease in unselected FA patients in Brazil. OBECTIVE: This study aimed to investigate the most prevalent mutations of FANCA and FANCC genes in Brazilian patients with FA. METHODS: Genomic DNA obtained from 22 racially and ethnically diverse unrelated FA patients (mean age ± SD: 14.0 ± 7.8 years; 10 male, 12 female; 14 white, 8 black) was analysed by polymerase chain reaction and restriction site assays for identification of FANCA (delta3788-3790) and FANCC (delta322G, IVS4+4A -> T, W22X, L496R, R548X, Q13X, R185X, and L554P) gene mutations. RESULTS: Mutations in FANCA and FANCC genes were identified in 6 (27.3%) and 14 (63.6%) out of 22 patients, respectively. The disease could not be attributed to the tested mutations in the two remaining patients enrolled in the study (9.1%). The registry of the two most prevalent gene abnormalities (delta3788-3790 and IVS4 + 4 -> T) revealed that they were present in 18.2% and 15.9% of the FA alleles, respectively. Additional FANCC gene mutations were found in the study, with the following prevalence: delta322G (11.4%), W22X (9.1%), Q13X (2.3%), L554P (2.3%), and R548X (2.3%) of total FA alleles. CONCLUSION: These results suggest that mutations of FANCA and FANCC genes are the most prevalent mutations among FA patients in Brazil.

CONTEXT: Mutations of the p53 tumor suppressor gene are the most frequent alterations observed in human neoplasias affecting adults. In pediatric oncology, however, they have seldom been identified. Wilms’ tumor is a renal neoplasia commonly occurring in children and is associated with mutations of the WT1 gene. The correlation between Wilms’ tumor and alterations of the p53 gene has not been well established, with a low frequency of mutations having been reported in this type of tumor. Mutation may be associated with advanced stage disease and unfavorable histology. OBJECTIVE: To screen for mutations of the p53 gene by the PCR-SSCP method and DNA sequencing in cases of Wilms’ tumor sug-gestive of mutation. DESIGN: Case Report. CASE REPORT: Evaluations of exons 5-9 of the p53 gene in DNA samples extracted by PCR-SSCP from 10 Wilms’ tumors in children at different stages, and DNA sequencing. Changes in SSCP analy-sis were observed in exon 8 in two samples. The probable muta-tions were not confirmed by DNA sequencing. The absence of point mutations in p53 gene observed in the 10 samples of Wilms’ tumor studied agrees with literature data, with DNA sequencing being of fundamental importance for the confirmation of possible mutations.

Os rabdomiossarcomas (RMS) são considerados tumores clinicamente agressivos com origem a partir de células mesenquimais imaturas e que se caracterizam pela presença de células com diferenciação pouco definida. O emprego das técnicas citogenéticas convencionais em RMS vem contribuindo consideravelmente para a diferenciação entre os rabdomiossarcomas alveolares e os outros tumores de células pequenas e redondas, além de fornecer informações prognósticas importantes referente ao rabdomiossarcoma do tipo alveolar. Assim, este trabalho visa a realizar uma revisão das alterações citogenéticas observadas nos diferentes subtipos histológicos de RMS, enfocando não só os trabalhos de citogenética convencional, mas também novas abordagens utilizadas para o estudo de neoplasias tais como FISH, CGH, SKY e M-FISH. Tais metodologias vêm contribuindo de maneira significativa para a melhor compreensão da heterogeneidade cariotípica observada nos RMS.

CONTEXT: Despite the advances in the cure rate for acute lymphoblastic leukemia, approximately 25% of affected children suffer relapses. Expression of genes for the multiple drug resistance protein (MDR-1), multidrug resistance-related protein (MRP), and lung resistance protein (LRP) may confer the phenotype of resistance to the treatment of neoplasias. OBJECTIVE: To analyze the expression of the MDR-1, MRP and LRP genes in children with a diagnosis of acute lymphoblastic leukemia via the semiquantitative reverse transcription polymerase chain reaction (RT-PCR), and to determine the correlation between expression and event-free survival and clinical and laboratory variables. DESIGN: A retrospective clinical study. SETTING: Laboratory of Pediatric Oncology, Department of Pediatrics, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Brazil. METHODS: Bone marrow aspirates from 30 children with a diagnosis of acute lymphoblastic leukemia were assessed for the expression of messenger RNA for the MDR-1, MRP and LRP genes by semi-quantitative RT-PCR. RESULTS: In the three groups studied, only the increased expression of LRP was related to worsened event-free survival (p = 0.005). The presence of the common acute lymphoblastic leukemia antigen (CALLA) was correlated with increased LRP expression (p = 0.009) and increased risk of relapse or death (p = 0.05). The relative risk of relapse or death was six times higher among children with high LRP expression upon diagnosis (p = 0.05), as confirmed by multivariate analysis of the three genes studied (p = 0.035). DISCUSSION: Cell resistance to drugs is a determinant of the response to chemotherapy and its detection via RT-PCR may be of clinical importance. CONCLUSIONS: Evaluation of the expression of genes for resistance to antineoplastic drugs in childhood acute lymphoblastic leukemia upon diagnosis, and particularly the expression of the LRP gene, may be of clinical relevance, and should be the object of prospective studies.

CONTEXT: The p16 tumor suppressor gene encodes a cyclin-dependent kinase 4 inhibitor that blocks cell division during the G1 phase of the cell cycle. Alterations in this gene have been reported for various neoplasia types, including acute lymphoblastic leukemias (ALL), especially T-cell acute lymphoblastic leukemias (ALL). OBJECTIVE: To determine probable alterations in the p16 gene in children with acute lymphoblastic leukemias using the polymerase chain reaction (PCR) and direct DNA sequencing and also to analyze event-free survival (EFS). DESIGN: Retrospective study. SETTING: Department of Child Care and Pediatrics, Faculty of Medicine of Ribeirão Preto, Universidade Federal de São Paulo. PARTICIPANTS: Fifty-six children with ALL (mean age 4 years). Forty (71.43%) had B-cell and 12 (21.43%) had T-cell ALL; 4 (7.1%) were biphenotypic. SAMPLE: DNA samples were extracted from bone marrow upon diagnosis and/or relapse. In 2 T-cell cases, DNA from cerebrospinal fluid (CSF) was analyzed. MAIN MEASUREMENTS: Deletions or nucleotide substitutions in exons 1, 2 and 3 of the p16 gene were determined by PCR and nucleotide sequencing. Event-free survival was determined by the Kaplan-Meyer and log-rank test for patients carrying normal and altered p16. RESULTS: Deletions in exon 3 were observed in five cases. Abnormal migration in PCR was observed in seven cases for exon 1, six for exon 2, and five for exon 3. Mutations in exon 1 were confirmed by direct DNA sequencing in four cases and in exon 2 in two cases. The Kaplan-Meyer survival curves and the log-rank test showed no significant differences in 5-year EFS between children with normal or altered p16, or between patients with B-ALL carrying normal or altered p16 gene. Patients with T-ALL could not be evaluated via Kaplan-Meier due to the small number of cases. CONCLUSIONS: Our results, particularly regarding deletion frequency, agree with others suggesting that deletions in the p16 are initial events in leukemia genesis. The small number of samples did not allow stablishment of correlation between childhood ALL and the p16 point mutations found in our study. Kaplan-Meier analysis revealed no significant correlation between EFS and alterations in ALL. The p16 alterations frequency observed for B and T-ALL agreed with reports from other centers.

CONTEXT: Tumor suppressor genes act on the control of cell cycle progression. In pediatric neoplasias, some of these genes may be considered to be markers for diagnosis or relapse, thus probably representing prognostic indicators. OBJECTIVE: To study the inactivation of the p15 gene in children with acute lymphoblastic leukemia. TYPE OF STUDY: Retrospective study. SETTING: Laboratory of Molecular Biology, Department of Pediatrics, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo. PARTICIPANTS: Eighty-three children and adolescents with acute lymphoblastic leukemia were studied, with the examination of 83 bone marrow samples obtained at diagnosis, four obtained also during relapse, and two cerebrospinal fluid samples obtained from two cases of isolated relapse in the central nervous system. MAIN MEASUREMENTS: Homologous deletion of the p15 gene by multiplex polymerase chain reaction, and screening for point mutations by polymerase chain reaction/single-strand conformational polymorphism. RESULTS: Deletion of exon 2 of the p15 gene was observed in 15 children, including one case in which deletion was only verified during isolated central nervous system relapse. No case of exon 1 deletion, or that was suggestive of point mutations, was observed and no association between p15 gene inactivation and classic risk factors was established. CONCLUSION: According to the literature, inactivation of the p15 gene by deletion of exon 2 in acute lymphoblastic leukemia found in the population studied would be considered to be a molecular marker for diagnosis or relapse. However, no correlation between p15 gene deletion and clinical prognostic indicators was observed.

CONTEXT AND OBJECTIVE: Genetic investigation of central nervous system (CNS) tumors provides valuable information about the genes regulating proliferation, differentiation, angiogenesis, migration and apoptosis in the CNS. The aim of our study was to determine the prevalence of genetic polymorphisms (codon 31 and 3' untranslated region, 3'UTR) and protein expression of the cyclin-dependent kinase inhibitor 1A (CDKN1A) gene in patients with and without CNS tumors. DESIGN AND SETTING: Analytical cross-sectional study with a control group, at the Molecular Biology Laboratory, Pediatric Oncology Department, Hospital das Clínicas de Ribeirão Preto. METHODS: 41 patients with CNS tumors and a control group of 161 subjects without cancer and paires for sex, age and ethnicity were genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Protein analysis was performed on 36 patients with CNS tumors, using the Western Blotting technique. RESULTS: The frequencies of the heterozygote (Ser/Arg) and polymorphic homozygote (Arg/Arg) genotypes of codon 31 in the control subjects were 28.0% and 1.2%, respectively. However, the 3'UTR site presented frequencies of 24.2% (C/T) and 0.6% (T/T). These frequencies were not statistically different (P > 0.05) from those seen in the patients with CNS tumors (19.4% and 0.0%, codon 31; 15.8% and 2.6%, 3'UTR site). Regarding the protein expression in ependymomas, 66.67% did not express the protein CDKN1A. The results for medulloblastomas and astrocytomas were similar: neither of them expressed the protein (57.14% and 61.54%, respectively). CONCLUSION: No significant differences in protein expression patterns or polymorphisms of CDKN1A in relation to the three types of CNS tumors were observed among Brazilian subjects.

CONTEXT: Neurofibromatosis type 1 (NF-1) is the most prevalent autosomal dominant genetic disorder among humans. Moyamoya disease is a cerebral vasculopathy that is only rarely observed in association with NF-1, particularly in the pediatric age range. The present study reports an occurrence of this association in an infant. CASE REPORT: An eight-month-old female presented convulsive seizures with clonic movements. The patient suffered an ischemic stroke with hemiparesis. Magnetic resonance imaging revealed radiological findings compatible with moyamoya disease. The diagnosis of NF-1 was made at the age of 20 months. CONCLUSION: Despite the rarity of this association in childhood, children with focal neurological symptoms and a diagnosis of NF-1 deserve to be investigated for moyamoya syndrome.

CONTEXT: The CDR-3 region of heavy-chain immunoglobulin has been used as a clonal marker in the study of minimal residual disease in children with acute lymphoblastic leukemia. Southern blot and polymerase chain reaction studies have demonstrated the occurrence of bi/oligoclonality in a variable number of cases of B-lineage acute lymphoblastic leukemia, a fact that may strongly interfere with the detection of minimal residual disease. Oligoclonality has also been associated with a poorer prognosis and a higher chance of relapse. OBJECTIVES: To correlate bi/oligoclonality, detected by polymerase chain reaction in Brazilian children with B-lineage acute lymphoblastic leukemia with a chance of relapse, with immunophenotype, risk group, and disease-free survival. DESIGN: Prospective study of patients’ outcome. SETTING: Pediatric Oncology Unit of the University Hospital, Faculty of Medicine of Ribeirão Preto, University of São Paulo. PARTICIPANTS: 47 children with acute lymphoblastic leukemia DIAGNOSTIC TEST: Polymerase chain reaction using consensus primers for the CDR-3 region of heavy chain immunoglobulin (FR3A, LJH and VLJH) for the detection of clonality. RESULTS: Bi/oligoclonality was detected in 15 patients (31.9%). There was no significant difference between the groups with monoclonality and biclonality in terms of the occurrence of a relapse (28.1% versus 26.1%), presence of CALLA+ (81.2% versus 80%) or risk group (62.5% versus 60%). Disease-free survival was similar in both groups, with no significant difference (p: 0.7695). CONCLUSIONS: We conclude that bi/oligoclonality was not associated with the factors investigated in the present study and that its detection in 31.9% of the patients may be important for the study and monitoring of minimal residual disease.