Krepischi A.C.V.,National Institute of Science and Technology in Oncogenomics INCITO |
Rossi B.M.,Barretos Cancer Hospital Pio XII Foundation |
Carraro D.M.,National Institute of Science and Technology in Oncogenomics INCITO
BMC Medical Genetics | Year: 2012
Background: Familial adenomatous polyposis (FAP) is a hereditary colorectal cancer syndrome caused by a loss of function of the APC gene. Large deletions in APC are a common cause of FAP; despite the existence of a variety of gene dosage detection methodologies, most are labor intensive and time and resource consuming.Methods: We describe a new duplex qPCR method for gene dosage analysis based on the coamplification of a target and a reference gene in a SYBR Green reaction, followed by a comparison of the ratio between the target and the reference peaks of the melting curve for the test (patient) and control samples. The reliability of the described duplex qPCR was validated for several genes (APC, HPRT1, ATM, PTEN and BRCA1).Results: Using this novel gene dosage method, we have identified an APC gene deletion in a FAP patient undergoing genetic testing. Comparative genomic hybridization based on microarrays (aCGH) was used to confirm and map the extent of the deletion, revealing a 5.2 MB rearrangement (5q21.3-q22.3) encompassing the entire APC and 19 additional genes.Conclusion: The novel assay accurately detected losses and gains of one copy of the target sequences, representing a reliable and flexible alternative to other gene dosage techniques. In addition, we described a FAP patient harboring a gross deletion at 5q21.3-q22.3 with an unusual phenotype of the absence of mental impairment and dysmorphic features. © 2012 Torrezan et al.; licensee BioMed Central Ltd.
Torrezan G.T.,A C Camargo Hospital |
Da Silva F.C.C.,A C Camargo Hospital |
Santos E.M.M.,National Institute of Science and Technology in Oncogenomics INCITO |
Santos E.M.M.,A C Camargo Hospital |
And 8 more authors.
Orphanet Journal of Rare Diseases | Year: 2013
Background: Patients with multiple colorectal adenomas are currently screened for germline mutations in two genes, APC and MUTYH. APC-mutated patients present classic or attenuated familial adenomatous polyposis (FAP/AFAP), while patients carrying biallelic MUTYH mutations exhibit MUTYH-associated polyposis (MAP). The spectrum of mutations as well as the genotype-phenotype correlations in polyposis syndromes present clinical impact and can be population specific, making important to obtain genetic and clinical data from different populations. Methods. DNA sequencing of the complete coding region of the APC and MUTYH genes was performed in 23 unrelated Brazilian polyposis patients. In addition, mutation-negative patients were screened for large genomic rearrangements by multiplex ligation-dependent probe amplification, array-comparative genomic hybridization, and duplex quantitative PCR. Biallelic MUTYH mutations were confirmed by allele-specific PCR. Clinical data of the index cases and their affected relatives were used to assess genotype-phenotype correlations. Results: Pathogenic mutations were identified in 20 of the 23 probands (87%): 14 in the APC gene and six in the MUTYH gene; six of them (30%) were described for the first time in this series. Genotype-phenotype correlations revealed divergent results compared with those described in other studies, particularly regarding the extent of polyposis and the occurrence of desmoid tumors in families with mutations before codon 1444 (6/8 families with desmoid). Conclusions: This first comprehensive investigation of the APC and MUTYH mutation spectrum in Brazilian polyposis patients showed a high detection rate and identified novel pathogenic mutations. Notably, a significant number of APC-positive families were not consistent with the predicted genotype-phenotype correlations from other populations. © 2013 Torrezan et al.; licensee BioMed Central Ltd.
Carraro D.M.,National Institute of Science and Technology in Oncogenomics INCITO |
Andrade V.P.,Ac Camargo Cancer Center
Bioscience Reports | Year: 2014
The spread of mammographic screening programmes around the world, including in developing countries, has substantially contributed to the diagnosis of small non-palpable lesions, which has increased the detection rate of DCIS (ductal carcinoma in situ). DCIS is heterogeneous in several ways, such as its clinical presentation, morphology and genomic profile. Excellent outcomes have been reported; however, many questions remain unanswered. For example, which patients groups are overtreated and could instead benefit from minimal intervention and which patient groups require a more traditional multidisciplinary approach. The development of a comprehensive integrated analysis that includes the radiological, morphological and genetic aspects of DCIS is necessary to answer these questions. This review focuses on discussing the significant findings about the morphological and molecular features of DCIS and its progression that have helped to uncover the biological and genetic heterogeneity of this disease. The knowledge gained in recent years might allow the development of tailored clinical management for women with DCIS in the future. © 2014 The Author(s).
PubMed | São Paulo State University and National Institute of Science and Technology in Oncogenomics INCITO
Type: Clinical Trial | Journal: Molecular oncology | Year: 2015
A significant association between DNA losses on 22q13.31 and head and neck squamous cell carcinomas (HNSCC) was previously reported by our group. Our data indicated that PHF21B gene, mapped on 22q13.31 and encoding a protein with function of chromatin-mediated transcriptional regulation, might be a putative tumor suppressor gene. To test this hypothesis, gene copy number was assessed in 75 HNSCC and 49 matched peripheral blood samples. PHF21B losses were detected in 43 tumors and were significantly associated with patients with familial history of cancer (P < 0.0001); i.e., 36/43 cases showed a positive family history of cancer and 22/36 had first-degree relatives with cancer (P = 0.049). In attempt to investigate other mechanisms for PHF21B loss of function, DNA sequencing was performed and no mutations were detected. We next evaluated the gene expression levels after inhibition of DNA methylation in nine HNSCC and breast carcinoma cell lines. Additionally, PHF21B expression levels were evaluated in colon cancer HCT116 cells as well as in its counterpart DKO (double knockout of DNMT1 and DNMT3B). The higher expression levels of PHF21B gene detected in DKO cells were inversely correlated with the DNA methylation. Further, DNA methylation in the specific promoter-associated CpG Island was investigated. Interestingly, gene hypermethylation was detected in 13/37 tumors: 5/13 HNSCC cases had family history of cancer in first-degree relatives and 8/13 showed both, DNA methylation and PHF21B losses in the tumor sample. One patient had PHF21B loss in the peripheral blood cells and PHF21B methylation in the tumor sample. Additionally, overexpression of PHF21B in cell lines drastically reduces clonogenic and migratory abilities. These data suggest that PHF21B is a novel tumor suppressor gene that can be inactivated by genetic and epigenetic mechanisms in the human cancer.
PubMed | Camargo Cancer Center, margo Cancer Center, International Research Center Cipe margo Cancer Center and National Institute of Science and Technology in Oncogenomics INCITO
Type: Journal Article | Journal: PloS one | Year: 2015
Lynch syndrome (LS) accounts for 3-5% of all colorectal cancers (CRC) and is inherited in an autosomal dominant fashion. This syndrome is characterized by early CRC onset, high incidence of tumors in the ascending colon, excess of synchronous/metachronous tumors and extra-colonic tumors. Nowadays, LS is regarded of patients who carry deleterious germline mutations in one of the five mismatch repair genes (MMR), mostly in MLH1 and MSH2, but also in MSH6, PMS1 and PMS2. To comprehensively characterize 116 Brazilian patients suspected for LS, we assessed the frequency of germline mutations in the three minor genes MSH6, PMS1 and PMS2 in 82 patients negative for point mutations in MLH1 and MSH2. We also assessed large genomic rearrangements by MLPA for detecting copy number variations (CNVs) in MLH1, MSH2 and MSH6 generating a broad characterization of MMR genes. The complete analysis of the five MMR genes revealed 45 carriers of pathogenic mutations, including 25 in MSH2, 15 in MLH1, four in MSH6 and one in PMS2. Eleven novel pathogenic mutations (6 in MSH2, 4 in MSH6 and one in PMS2), and 11 variants of unknown significance (VUS) were found. Mutations in the MLH1 and MSH2 genes represented 89% of all mutations (40/45), whereas the three MMR genes (MSH6, PMS1 and PMS2) accounted for 11% (5/45). We also investigated the MLH1 p.Leu676Pro VUS located in the PMS2 interaction domain and our results revealed that this variant displayed no defective function in terms of cellular location and heterodimer interaction. Additionally, we assessed the tumor phenotype of a subset of patients and also the frequency of CRC and extra-colonic tumors in 2,365 individuals of the 116 families, generating the first comprehensive portrait of the genetic and clinical aspects of patients suspected of LS in a Brazilian cohort.
Francisco A.L.N.,University of Campinas |
Correr W.R.,University of Sao Paulo |
Pinto C.A.L.,Ac Camargo Cancer Center |
Filho J.G.,Ac Camargo Cancer Center |
And 6 more authors.
Oral Oncology | Year: 2014
Oral cancer is a public health problem with high prevalence in the population. Local tumor control is best achieved by complete surgical resection with adequate margins. A disease-free surgical margin correlates with a lower rate of local recurrence and a higher rate of disease-free survival. Fluorescence spectroscopy is a noninvasive diagnostic tool that can aid in real-time cancer detection. The technique, which evaluates the biochemical composition and structure of tissue fluorescence, is relatively simple, fast and, accurate. Objectives This study aimed to compare oral squamous cell carcinoma lesions to surgical margins and the mucosa of healthy volunteers by fluorescence spectroscopy. Materials and methods The sample consisted of 56 individuals, 28 with oral squamous cell carcinoma and 28 healthy volunteers with normal oral mucosa. Thirty six cases (64.3%) were male and the mean age was 60.9 years old. The spectra were classified and compared to histopathology to determine fluorescence efficiency for diagnostic discrimination of tumors. Results In the analysis of the other cases we observed discrimination between normal mucosa, injury and margins. At two-year follow up, three individuals had local recurrence, and in two cases investigation fluorescence in the corresponding area showed qualitative differences in spectra between the recurrence area and the area without recurrence at the same anatomical site in the same patient. Conclusion In situ analysis of oral mucosa showed the potential of fluorescence spectroscopy as a diagnostic tool that can aid in discrimination of altered mucosa and normal mucosa. © 2014 Elsevier Ltd. All rights reserved.
Jardim J.F.,University of Campinas |
Francisco A.L.N.,National Institute of Science and Technology in Oncogenomics INCITO |
Gondak R.,Federal University of Santa Catarina |
Damascena A.,International Center for Cancer Research |
And 2 more authors.
International Journal of Oral and Maxillofacial Surgery | Year: 2015
Perineural invasion (PNI) and lymphovascular invasion (LVI) have been associated with the risk of local recurrences and lymph node metastasis. The aim of this study was to evaluate the prognostic impact of PNI and LVI in patients with advanced stage squamous cell carcinoma of the tongue and floor of the mouth. One hundred and forty-two patients without previous treatment were selected. These patients underwent radical surgery with neck dissection and adjuvant treatment. Clinicopathological data were retrieved from the medical charts, including histopathology and surgery reports. Univariate analysis was performed to assess the impact of studied variables on survival. Overall survival was negatively influenced by six tumour-related factors: increasing T stage (P = 0.003), more than two clinically positive nodes (P = 0.002), extracapsular spread of lymph node metastasis (P < 0.001), tumour thickness (P = 0.04), PNI (P < 0.001), and LVI (P = 0.012). Disease-free survival was influenced by PNI (P = 0.04), extracapsular spread of lymph node metastasis (P = 0.008), and N stage (P = 0.006). Multivariate analysis showed PNI to be an independent predictor for overall survival (P = 0.01) and disease-free survival (P = 0.03). Thus the presence of PNI in oral carcinoma surgical specimens has a significant impact on survival outcomes in patients with advanced stage tumours submitted to radical surgery and adjuvant radiotherapy/radiochemotherapy. © 2014 International Association of Oral and Maxillofacial Surgeons.
Roffe M.,International Research Center |
Roffe M.,National Institute of Science and Technology in Oncogenomics INCITO |
Lupinacci F.C.,International Research Center |
Lupinacci F.C.,National Institute of Science and Technology in Oncogenomics INCITO |
And 6 more authors.
Cellular Signalling | Year: 2015
The 90. kDa ribosomal S6 kinases (RSK) are effectors of the Ras-ERK1/2 signaling pathway. RSK signaling controls proliferation and protein synthesis, and is altered in several types of tumors. BI-D1870 and SL0101 are two widely used inhibitors of RSK. After revision of the literature, discrepancies in the effects of the inhibitors were identified. Herein we report that while SL0101 inhibited mTORC1-p70S6K signaling, BI-D1870 increased p70S6K activation. Both effects were independent of ERK1/2 and RSK, and thus nonspecific. We also demonstrated how these opposite nonspecific effects mislead the identification of the RSK-dependent phosphorylation of rpS6 (S235/236), a known RSK and p70S6K substrate. Phosphorylation of tuberin at S1798 by RSK was proposed to mediate ERK1/2-dependent activation of mTORC1-p70S6K signaling. In glioblastoma-derived cells, phosphorylation of tuberin was abolished after RSK depletion or ERK1/2 inhibition, suggesting that RSK is its main kinase. However, RSK depletion did not reduce PMA-dependent p70S6K phosphorylation, which suggests that tuberin phosphorylation at S1798 is not the main mediator of ERK1/2-dependent activation of mTORC1. Remarkably, tuberin phosphorylation (S1798) followed the activation status of RSK in different cells and experimental conditions, suggesting that phosphorylation of that residue could be used as readout for RSK activation in cells. We confirmed the difference in the effects of SL0101 and BI-D1870 in cellular proliferation assays. Rapamycin potentiated the inhibition of proliferation induced by BI-D1870, but not by SL0101. We thus conclude that SL0101 and BI-D1870 induce distinct off-target effects in mTORC1-p70S6K signaling, and thus, the functions previously ascribed to RSK based on these inhibitors should be reassessed. © 2015 Elsevier Inc.
PubMed | Ac Camargo Cancer Center, Molecular Morphology Group, National Institute of Science and Technology in Oncogenomics INCITO and International Research Center
Type: Journal Article | Journal: International journal of molecular sciences | Year: 2015
Xeroderma pigmentosum (XP) is a rare autosomal recessive disorder characterized by DNA repair defects that cause photophobia, sunlight-induced cancers, and neurodegeneration. Prevalence of germline mutations in the nucleotide excision repair gene XPA vary significantly in different populations. No Brazilian patients have been reported to carry a germline mutation in this gene. In this study, the germline mutational status of XPA was determined in Brazilian patients exhibiting major clinical features of XP syndrome. The study was conducted on 27 unrelated patients from select Brazilian families. A biallelic inactivating transition mutation c.619C>T (p.Arg207Ter) was identified in only one patient with a history of neurological impairment and mild skin abnormalities. These findings suggest that XP syndrome is rarely associated with inherited disease-causing XPA mutations in the Brazilian population. Additionally, this report demonstrates the effectiveness of genotype-phenotype correlation as a valuable tool to guide direct genetic screening.
Olivieri E.H.R.,Camargo Cancer Center |
De Andrade Franco L.,International Cancer Center |
Pereira R.G.,International Cancer Center |
Carvalho Mota L.D.,Camargo Cancer Center |
And 4 more authors.
Biopreservation and Biobanking | Year: 2014
A critical issue in defining protocols for biobanking practices is the preservation of total RNA for assessing the whole transcriptome and ensuring that it can be utilized in clinically oriented studies. Storage conditions, such as temperature and the length of time that tissues and purified RNA stay frozen, may directly impact RNA preservation. In this study, we evaluated a) the quality of RNA (as measured by RNA Integrity Number) purified from head and neck tumor tissues stored at -140 C for distinct time intervals of up to 7 years, and b) the quality of their respective RNAs stored for 4 years at -80 C when diluted at either 250 ng/μL or 25 ng/μL, with repeated freezing and thawing. Additionally, we generated a profile of the RNA collection of human tumors from different body sites stored at the AC Camargo Biobank. Our results showed no significant change in RIN values according to length of storage at -140 C. With respect to RNA aliquots stored at -80 C, RNA integrity at 250 ng/μL was preserved, while statistically significant degradation was observed at 25 ng/μL after only 8 months of storage. The RNA collection from most of the human tumors stored at the AC Camargo Biobank exhibited high quality, with average RIN around seven. However, ovary and stomach samples had the greatest RNA degradation. Taken together, the results show that both the temperature of preservation and the concentration of RNA should be strictly controlled by the biobank staff involved in macromolecule purification. Moreover, the RNAs from our biobank can be useful for the most demanding methods of gene expression analysis by virtue of adherence to optimal standard operating procedures for both tissue and macromolecule laboratories. © Mary Ann Liebert, Inc.