Detection of Clonal Immunoglobulin and T-Cell Receptor Gene Rearrangements in Childhood Acute Lymphoblastic Leukemia Using a Low-Cost PCR Strategy

Background Imunoglobulin (Ig) and T cell receptor (TCR) gene rearrangements function as specific markers for minimal residual disease (MRD) which is one of the best predictors of outcome in childhood acute lymphoblastic leukemia (ALL) We recently reported on the prognostic value of MRD during the induction of remission through a simplified PCR method Here we report on gene rearrangement frequencies and offer guidelines for the application of the technique Procedure Two hundred thirty three children had DNA extracted from bone marrow Ig and TCR gene rearrangements were amplified using consensus primers and conventional PCR PCR products were submitted to homo/heteroduplex analysis A computer program was designed to define combinations of targets for clonal detection using a minimum set of primers and reactions Results At least one clonal marker could be detected in 98% of the patients and two markers in approximately 80% The most commonly rear ringed genes in precursor B cell ALL were IgH (75%) TCRD (59%) IgK (55%), and TCRG (54%) The most commonly rearranged genes for TALL were TCRG (100%) and TCRD (24%) The sensitivity of primers was limited to the detection of 1 leukemic cell among 100 normal cells Conclusions We propose that eight PCR reactions per ALL subtype would allow for the detection of two markers in most cases In addition these reactions ire suitable for MRD monitoring especially when aiming the selection of patients with high MRD levels (>= 10(-2)) at the end of induction therapy Such an approach would be very useful in centers with limited financial resources Pediatr Blood Cancer 2010 55 1278-1286 (C) 2010 Wiley Liss Inc

Polymorphisms of xenobiotic metabolizing enzymes and DNA repair genes and outcome in childhood acute lymphoblastic leukemia

The interindividual variation in the activity of xenobiotic metabolizing enzymes and DNA repair genes could modify an individual`s risk of recurrent malignancy and response to therapy. We investigated whether ALL outcome was related to polymorphisms in genes CYP2D6. MPO, EPHX1, NQO1, TS, XPD and XRCC1 in 95 consecutive ALL children by PCR or PCR-FRLP techniques. Polymorphisms in genes NQO1 and TS were associated with a significantly slow response to induction chemotherapy and NQO1 was also associated with a lower five-year event-free survival. This study suggests that polymorphisms of NQO1 and TS could be important for patient response to induction therapy and for treatment outcome. (C) 2009 Elsevier Ltd. All rights reserved.

New recurrent deletions in the PPAR gamma and TP53 genes are associated with childhood myelodysplastic syndrome

Myelodysplastic syndrome (MDS) is a rare hematological malignancy in children. It was performed FISH analysis in 19 pediatric MDS patients to investigate deletions involving the PPAR gamma and TP53 genes. Significant losses in the PPAR gamma gene and deletions in the tumor suppressor gene TP53 were observed in 17 and 18 cases, respectively. Using quantitative RT-PCR, it was detected PPAR gamma transcript downexpression in a subset of these cases. G-banding analysis revealed 17p deletions in a small number of these cases. One MDS therapy-related patient had neither a loss of PPAR gamma nor TP53. These data suggest that the PPAR gamma and TP53 genes may be candidates for molecular markers in pediatric MDS, and that these potentially recurrent deletions could contribute to the identification of therapeutic approaches in primary pediatric MDS. (C) 2008 Elsevier Ltd. All fights reserved.

Polymorphisms in genes encoding drugs and xenobiotic metabolizing enzymes in a Brazilian population

Polymorphic variations of several genes associated with drugs and xenobiotic metabolism have been linked to the factors that predispose to the carcinogenesis process. As considerable interindividual and interethnic variation in metabolizing enzyme activity has been associated with polymorphic alleles, we evaluated the frequency of the polymorphisms of CYP2D6, EPHX1 and NQO1 genes in 361 Brazilian individuals separated by ethnicity (European and African ancestry), using the polymerase chain reaction-restriction fragment length (PCR-RFLP) method. The allele frequencies of the variants *3 and *4 for the gene CYP2D6 were 0.04 and 0.14 for white subjects and 0.03 and 0.10 for black individuals, respectively. For the both variants of the gene EPHX1, we found higher allele frequencies among white individuals compared with mulatto subjects (0.62 vs 0.54 and 0.18 vs 0.14, respectively); however, these differences were not statistically significant (p = 0.39 and 0.56, respectively). For the NQO1 gene we observed a higher frequency of the homozygous genotype among black individuals (7.9%) compared with white subjects (6.3%) (p = 0.003). The genotype frequencies were within the Hardy-Weinberg equilibrium. We concluded that the allele frequencies of CYP2D6, EPHX1 and NQO1 gene polymorphisms in this Brazilian population showed ethnic variability when compared with those observed in other populations.

Impact of thymidylate synthase promoter and DNA repair gene polymorphisms on susceptibility to childhood acute lymphoblastic leukemia

The aim of this study was to evaluate the frequency of polymorphisms in the TYMS, XRCC1, and ERCC2 DNA repair genes in pediatric patients with acute lymphoblastic leukemia using polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) approaches. The study was conducted in 206 patients and 364 controls from a Brazilian population. No significant differences were observed among the analyzed groups regarding XRCC1 codon 399 and codon 194 and ERCC2 codon 751 and codon 312 polymorphisms. The TYMS 3R variant allele was significantly associated with a reduced risk of childhood ALL, represented by the sum of heterozygous and polymorphic homozygous genotypes (odds ratio 0.60; 95% confidence interval 0.37-0.99). The results suggest that polymorphism in TYMS may play a protective role against the development of childhood ALL.

Benefits of the Intermittent Use of 6-Mercaptopurine and Methotrexate in Maintenance Treatment for Low-Risk Acute Lymphoblastic Leukemia in Children: Randomized Trial From the Brazilian Childhood Cooperative Group Protocol ALL-99

Purpose To describe event-free survival (EFS) and toxicities in children with low-risk acute lymphoblastic leukemia (ALL) assigned to receive either continuous 6-mercaptopurine (6-MP) and weekly methotrexate (MIX) or intermittent 6-MP with intermediate-dose MTX, as maintenance treatment. Patients and Methods Between October 1, 2000, and December 31, 2007, 635 patients with low-risk ALL were enrolled onto Brazilian Childhood Cooperative Group for ALL Treatment (GBTLI) ALL-99 protocol. Eligible children (n=544) were randomly allocated to receive either continuous 6-ME/MIX (group 1, n 272) or intermittent 6-MP (100 mg/m(2)/d for 10 days, with 11 days resting) and MIX (200 mg/m(2) every 3 weeks; group 2, n = 272). Results The 5-year overall survival (OS) and EFS were 92.5% +/- 1.5% SE and 83.6% +/- 2.1% SE, respectively. According to maintenance regimen, the OS was 91.4% +/- 2.2% SE (group 1) and 93.6% +/- 2.1% SE (group 2; P=.28) and EFS 80.9% +/- 3.2% SE (group 1) and 86.5% +/- 2.8% SE (group 2; P=.089). Remarkably, the intermittent regimen led to significantly higher EFS among boys (85.7% v 74.9% SE; P=027), while no difference was seen for girls (87.0% v 88.8% SE; P=.78). Toxic episodes were recorded in 226 and 237 children, respectively. Grade 3 to 4 toxic events for groups 1 and 2 were, respectively, 273 and 166 for hepatic dysfunction (P=.002), and 772 and 636 for hematologic episodes (P=.005). Deaths on maintenance were: seven (group 1) and one (group 2). Conclusion The intermittent use of 6-MP and MIX in maintenance is a less toxic regimen, with a trend toward better long-term EFS. Boys treated with the intermittent schedule had significantly better EFS.

mRNA expression of matrix metalloproteinases (MMPs) 2 and 9 and tissue inhibitor of matrix metalloproteinases (TIMPs) 1 and 2 in childhood acute lymphoblastic leukemia: Potential role of TIMP1 as an adverse prognostic factor

This study evaluates the mRNA expression profile of genes TIMP1, TIMP2, MMP2 and MMP9 in diagnostic bone marrow samples from 134 consecutive ALL children by real-time quantitative PCR. A significant association was observed between higher expression levels of MMP9 and low risk group and absence of extramedullary infiltration and higher expression levels of TIMP2 and MMP2 with T-ALL. TIMP1 gene expression values higher than the median were associated with a significantly lower 5-year event free-survival in univariable (P = 0.04) and multivariable analysis (P = 0.01). Our data address new information in the complex interaction of the migration/adhesion genes and childhood ALL. (c) 2009 Elsevier Ltd. All rights reserved.

Resveratrol and quercetin cooperate to induce senescence-like growth arrest in C6 rat glioma cells

Glioma is the most frequent and malignant primary human brain tumor with dismal prognosis despite multimodal therapy. Resveratrol and quercetin, two structurally related and naturally occurring polyphenols, are proposed to have anticancer effects. We report here that resveratrol and quercetin decreased the cell number in four glioma cell lines but not in rat astrocytes. Low doses of resveratrol (10 mu M) or quercetin (25 mu M) separately had no effect on apoptosis induction, but had a strong effect on caspase 3/7 activation when administered together. Western blot analyses showed that resveratrol (10 mu M) and quercetin (25 mu M) caused a reduction in phosphorylation of Akt, but this reduction was not sufficient by itself to mediate the effects of these polyphenols. Most important, resveratrol and quercetin chronically administered presented a strong synergism in inducing senescence-like growth arrest. These results suggest that the combination of polyphenols can potentialize their antitumoral activity, thereby reducing the therapeutic concentration needed for glioma treatment. (Cancer Sci 2009; 100: 1655-1662).

Skin and oral lesions associated to imatinib mesylate therapy

Imatinib mesylate is a tyrosine kinase inhibitor used to treat chronic myeloid leukemia (CML) throughout all the phases of the disease. In most cases, this drug is well tolerated; however, some cases experience side effects. Skin rashes and oral lesions are uncommon and appear to be dose-dependent. The authors report two cases of CML Ph(+) in chronic phase patients who presented skin and oral lesions probably induced by imatinib therapy.

Expression of cell adhesion proteins and proteins related to angiogenesis and fatty acid metabolism in benign, atypical, and anaplastic meningiomas

Most meningiomas are benign tumours of arachnoidal origin, although a small number have high proliferative rates and invasive properties which complicate complete surgical resection and are associated with increased recurrence rates. Few prognostic indicators exist for meningiomas and further research is necessary to identify factors that influence tumour invasion, oedema and recurrence. Paraffin sections from 25 intracranial meningiomas were analysed for expression of the proteins vascular endothelial growth factor (VEGF), VEGF receptors Flt1 and Flk1, E-cadherin, metalloproteinases 2 and 9 (MMP2, MMP9), CD44, receptor for hyaluronic acid-mediated motility (RHAMM), hyaluronic acid (HA), CD45, cyclooxygenase 2 (COX2), brain fatty acid binding protein (BFABP), Ki67, and proliferating cell nuclear antigen (PCNA). Correlations among protein expression were found for several markers of proliferation (Ki67, PCNA, MI) and microvessel density (MVD). COX2 expression increased with increasing with tumour grade and correlated with Ki67, PCNA, MI, MVD, and BFABP. BFABP expression also correlated with Ki67 and PCNA expression. Relationships were also identified among angiogenic factors (VEGF, Flt1, Flk1) and proliferation markers. Oedema was found to correlate with MMP9 expression and MMP9 also correlated with proliferation markers. No correlations were found for MMP2, E-cadherin, or CD44 in meningiomas. In conclusion Ki67, PCNA, MI, MVD, BFABP, and COX2 were significantly correlated with meningioma tumour grade and with each other. These findings, by correlating both intracellular fatty acid transport and eicosanoid metabolism with tumour proliferation, as determined by Ki67 labelling and mitotic index, suggest fatty acids are involved in the progression of meningiomas.

miR-29b and miR-125a Regulate Podoplanin and Suppress Invasion in Glioblastoma

Glioblastoma is the most frequent and malignant brain tumor, characterized by an elevated capacity for cellular proliferation and invasion. Recently, it was demonstrated that podoplanin membrane sialo-glycoprotein encoded by PDPN gene is over-expressed and related to cellular invasion in astrocytic tumors; however the mechanisms of regulation are still unknown. MicroRNAs are noncoding RNAs that regulate gene expression and several biological processes and diseases, including cancer. Nevertheless, their roles in invasion, proliferation, and apoptosis of glioblastoma are not completely understood. In this study, we focused on miR-29b and miR-125a, which were predicted to regulate PDPN, and demonstrated that these microRNAs directly target the 30 untranslated region of PDPN and inhibit invasion, apoptosis, and proliferation of glioblastomas. Furthermore, we report that miR-29b and miR-125a are downregulated in glioblastomas and also in CD133-positive cells. Taken together, these results suggest that miR-29b and miR-125a represent potential therapeutic targets in glioblastoma. (C) 2010 Wiley-Liss, Inc.

Low mRNA Expression of the Apoptosis-Related Genes CASP3, CASP8, and FAS Is Associated With Low Induction Treatment Response in Childhood Acute Lymphoblastic Leukemia (ALL)

Background. Defects in apoptosis signaling have been considered to be responsible for treatment failure in many types of cancer, although with controversial results. The objective of the present study was to assess the expression profile of key apoptosis-related genes in terms of clinical and biological variables and of the survival of children with acute lymphoblastic leukemia (ALL). Procedure. The levels of mRNA expression of the apoptosis-related genes CASP3, CASP8, CASP9, FAS, and BCL2 were analyzed by quantitative real-time PCR in consecutive samples from 139 consecutive children with ALL at diagnosis treated by the Brazilian protocol (GBTLI-ALL 99). Gene expression levels and clinical and biological features were compared by the Mann-Whitney test. Event-free survival (EFS) was calculated by Kaplan-Meier plots and log-rank test. Results. A significant correlation was detected between CASP3, CASP8, CASP9, and FAS expression levels (P<0.01) in ALL samples. Higher levels of BCL2 were significantly associated with white blood cell (WBC) count <50,000/mm(3) at diagnosis (P=0.01) and low risk group classification (P=0.008). Lower expression levels of CASP3, CASP8 and FAS gene were associated with a poor response at day 7 according the GBTLI-ALL 99 protocol (P=0.03, P=0.02 and P=0.008, respectively). There was a relationship between FAS gene expression lower than the 75th percentile and lower 5-year EFS (P=0.02). Conclusion. These findings suggest an association between lower expression levels of the pro-apoptotic genes and a poor response to induction therapy at day 7 and prognosis in childhood ALL. Pediatr Blood Cancer 2010;55:100-107. (C) 2010 Wiley-Liss, Inc.

Cryptic SYT/SXX1 fusion gene in high-grade biphasic synovial sarcoma with unique complex rearrangement and extensive BCL2 overexpression

Synovial sarcomas are high-grade malignant mesenchymal tumors that account for 10% of all soft-tissue sarcomas. Almost 95% of these tumors are characterized by a nonrandom chromosomal abnormality, t(X;18)(p11.2;q11.2), that is observed in both biphasic and monophasic variants. In this article, we present the case of a 57-year-old woman diagnosed with high-grade biphasic synovial sarcoma in which conventional cytogenetic analysis revealed the constant presence of a unique t(18;22)(q12;q13), in addition to trisomy 8. The rearrangement was confirmed by fluorescence in situ hybridization. The use of the whole chromosome painting probes WCPX did not detect any rearrangements involving chromosome X, although reverse-transcriptase polymerase chain reaction (PCR) analysis demonstrated the conspicuous presence of a SYT/SXX1 fusion gene. Spectral karyotyping (SKY) was also performed and revealed an insertion of material from chromosome 18 into one of the X chromosomes at position Xp11.2. Thus, the karyotype was subsequently interpreted as 47,X,der(X)ins(X;18) (p11.2;q11.2q11.2),der(18)del(18)(q11.2q11.2)t(18;22)(q12;q13),der(22)t(18;22). Real-time PCR analysis of BCL2 expression in the tumor sample showed a 433-fold increase. This rare finding exemplifies that thorough molecular-cytogenetic analyses are required to elucidate complex and/or cryptic tumor-specific translocations. (C) 2010 Elsevier Inc. All rights reserved.

Syndromic and non-syndromic aneurysms of the human ascending aorta share activation of the Smad2 pathway

Common features such as elastic fibre destruction, mucoid accumulation, and smooth muscle cell apoptosis are co-localized in aneurysms of the ascending aorta of various aetiologies. Recent experimental studies reported an activation of TGF-beta in aneurysms related to Marfan (and Loeys-Dietz) syndrome. Here we investigate TGF-beta signalling in normal and pathological human ascending aortic wall in syndromic and non-syndromic aneurysmal disease. Aneurysmal ascending aortic specimens, classified according to aetiology: syndromic MFS (n = 15, including two mutations in TGFBR2), associated with BAV (n = 15) or degenerative forms (n = 19), were examined. We show that the amounts of TGF-beta 1 protein retained within and released by aneurysmal tissue were greater than for control aortic tissue, whatever the aetiology, contrasting with an unchanged TGF-beta 1 mRNA level. The increase in stored TGF-beta 1 was associated with enhanced LTBP-I protein and mRNA levels. These dysiregulations of the extracellular ligand are associated with higher phosphorylated Smad2 and Smad2 mRNA levels in the ascending aortic wall from all types of aneurysm. This activation correlated with the degree of elastic fibre fragmentation. Surprisingly, there was no consistent association between the nuclear location of pSmad2 and extracellular TGF-beta 1 and LTBP-I staining and between their respective mRNA expressions. In parallel, decorin. was focally increased in aneurysmal media, whereas biglycan was globally decreased in aneurysmal aortas. In conclusion, this study highlights independent dysregulations of TGF-beta retention and Smad2 signalling in syndromic and non-syndromic aneurysms of the ascending aorta. Copyright (C) 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Maternal and Developmental Toxicity of Ayahuasca in Wistar Rats

INTRODUCTION: Ayahuasca is a psychotropic plant beverage initially used by shamans throughout the Amazon region during traditional religious cult. In recent years, ayahuasca has also been used in ceremonies of a number of modern syncretic religious groups, including pregnant women. However, no documented study has been performed to evaluate the risk of developmental toxicity of ayahuasca. METHODS: In the present work, maternal and developmental toxicity was evaluated in Wistar rats. Ayahuasca was administered to pregnant rats in three different doses [the equivalent typical dose (TD) administered to humans, five-fold TD and 10-fold TD] during the gestational period (6-20 days). RESULTS: Dams treated with the highest ayahuasca dose showed maternal toxicity with decrease of weight gain and food intake. Visceral fetal findings were observed in all treatment groups. Skeletal findings were observed in the intermediate- and high-dose groups. The fetuses deriving from the highest dose group also presented a decrease in body weight. CONCLUSIONS: From these results, it is possible to conclude that there is a risk of maternal and developmental toxicity following ayahuasca exposure and that the level of toxicity appears to be dose-dependent. Birth Defects Res (Part B) 89:207-212, 2010. (C) 2010 Wiley-Liss, Inc.