Expresión y amplificación del gen HER2 en el cáncer gástrico avanzado / HER2 gene amplification and overexpression in advanced gastric cancer

Background:Overexpression/amplification of the HER2 gene in advanced gastric cancer is a predictor of response to adjuvant therapy with monoclonal antibodies.Aim: To determine the frequency of HER2 gene overexpression and amplificationin advanced gastric cancer. Material and Methods: One hundred nine advancedgastric cancer biopsy specimens, from 76 men and 33 women aged 67 ± 14 and 62± 12 years respectively, were selected. Three histological patterns (diffuse, intestinaland mixed) were recognized. Automated immunohistochemistry was performedwith monoclonal c-erbB-2 (NCL-356) Novocastra. Fluorescent in situ hybridization (FISH) for HER2 was performed in positive cases. Results: In 39% of cases,immunohistochemical staining was negative. It was 1+, 2+ and 3+ positive in 15,36 and 11% of cases, respectively. It was positive in 16% and 3% of intestinal typeand mixed carcinomas, respectively. It was negative in all diffuse carcinomas. FISHwas performed in 39 (2 +) cases and in 11 (3 +) cases. The gene amplification waspositive in two (2 +) and 11 (3 +) cases (11.9%). The overall concordance betweenimmunohistochemical staining and in situ hybridization was 85%. Conclusions: Inadvanced gastric cancer, HER2 gene overexpression or amplification was observed in11% and 12% of cases, respectively.

Novel polymerase chain reaction-restriction fragment length polymorphism assay to determine internal transcribed spacer-2 group in the Chagas disease vector, Triatoma dimidiata (Latreille, 1811)

Triatoma dimidiata is the most important Chagas disease insect vector in Central America as this species is primarily responsible for Trypanosoma cruzi transmission to humans, the protozoan parasite that causes Chagas disease. T. dimidiata sensu lato is a genetically diverse assemblage of taxa and effective vector control requires a clear understanding of the geographic distribution and epidemiological importance of its taxa. The nuclear ribosomal internal transcribed spacer 2 (ITS-2) is frequently used to infer the systematics of triatomines. However, oftentimes amplification and sequencing of ITS-2 fails, likely due to both the large polymerase chain reaction (PCR) product and polymerase slippage near the 5' end. To overcome these challenges we have designed new primers that amplify only the 3'-most 200 base pairs of ITS-2. This region distinguishes the ITS-2 group for 100% of known T. dimidiata haplotypes. Furthermore, we have developed a PCR-restriction fragment length polymorphism (RFLP) approach to determine the ITS-2 group, greatly reducing, but not eliminating, the number of amplified products that need to be sequenced. Although there are limitations with this new PCR-RFLP approach, its use will help with understanding the geographic distribution of T. dimidiata taxa and can facilitate other studies characterising the taxa, e.g. their ecology, evolution and epidemiological importance, thus improving vector control.

Mutation and genomic amplification of the PIK3CA proto-oncogene in pituitary adenomas

The tumorigenesis of pituitary adenomas is poorly understood. Mutations of the PIK3CA proto-oncogene, which encodes the p110-α catalytic subunit of PI3K, have been reported in various types of human cancers regarding the role of the gene in cell proliferation and survival through activation of the PI3K/Akt signaling pathway. Only one Chinese study described somatic mutations and amplification of the PIK3CA gene in a large series of pituitary adenomas. The aim of the present study was to determine genetic alterations of PIK3CA in a second series that consisted of 33 pituitary adenomas of different subtypes diagnosed by immunohistochemistry: 6 adrenocorticotropic hormone-secreting microadenomas, 5 growth hormone-secreting macroadenomas, 7 prolactin-secreting macroadenomas, and 15 nonfunctioning macroadenomas. Direct sequencing of exons 9 and 20 assessed by qPCR was employed to investigate the presence of mutations and genomic amplification defined as a copy number ≥4. Previously identified PIK3CA mutations (exon 20) were detected in four cases (12.1%). Interestingly, the Chinese study reported mutations only in invasive tumors, while we found a PIK3CA mutation in one noninvasive corticotroph microadenoma. PIK3CA amplification was observed in 21.2% (7/33) of the cases. This study demonstrates the presence of somatic mutations and amplifications of the PIK3CA gene in a second series of pituitary adenomas, corroborating the previously described involvement of the PI3K/Akt signaling pathway in the tumorigenic process of this gland.

Genetic diversity of breeding popcorn lines determined by SSR markers / Diversidad genética de líneas de mejoramiento del Zea mays determinada por marcadores SSR

Information about genetic dissimilarity is very important to corroborate genealogical relationships and to predict the most heterozygotic hybrid combinations. Eight popcorn S6 lines of diverse germplasm types were evaluated using simple sequence repeats (SSR) markers. Of a total of 51 evaluated polymorphic primers, 15 were used for polymerase chain reaction (PCR) amplification. The genetic distance was estimated by Rogers’ modified distance. The different popcorn breeding programs in Brazil are possibly using highly similar base-populations. The genetic similarity of lines P1-3 and P8-1 was lowest, while P3-3 and P8-2 were genetically more similar. The cophenetic correlation showed that the Unweighted Pair-Group Method Using Arithmetic Averages (UPGMA) was reliable to discriminate the genotypes in five groups. The clusters were consistent with the estimates of genetic identity. There was a moderate coincidence degree between the groups and genealogy of lines. Higher levels of heterozygosity are expected from crosses between the group containing lines P3-3 and P7-3 with that of P1-3 and P7-4. Crosses between lines P1-3 and P8-1 are also promising.

Prevalence of chimerism after non-myeloablative hematopoietic stem cell transplantation / Prevalência do quimerismo após transplante de células hematopoiéticas não-mieloablativo

Context and objective: Non-myeloablative hematopoietic stem cell transplantation (NMA-HSCT) is performed in onco-hematological patients who cannot tolerate ablative conditioning because of older age or comorbidities. This approach does not completely eliminate host cells and initially results in mixed chimerism. Long-term persistence of mixed chimerism results in graft rejection and relapse. Involvement of graft-versus-host disease is concomitant with complete chimerism and graft-versus-tumor effect. The aim of this study was to evaluate the prevalence of chimerism in onco-hematological patients who underwent NMA-HSCT. Desingn and setting: Observational clinical study on chimerism status after human leukocyte antigen-identical NMA-HSCT at the Discipline of Hematology and Hemotherapy of Universidade Federal de São Paulo. Methods: We sequentially analyzed the amplification of APO-B, D1S80, DxS52, FVW, 33.6, YNZ-2 and H-ras primers using variable number of tandem repeats (VNTR) on 17 pairs and fluorescent in situ hybridization (FISH) with the XY probe and SRY primer on 13 sex-unmatched pairs. RESULTS: The informativeness of the primers using VNTR was 60 percent for APO-B, 75 percent D1S80, 36 percent DxS52, 14 percent FVW, 40 percent YNZ-22 and 16 percent H-ras. The SRY primer was informative in female receptors with male donors. The XY-FISH method was informative in 100 percent of the sex-unmatched pairs. Conclusion: These methods were sensitive and informative. In VNTR, the association of APO-B with D1S80 showed 88 percent informativeness. The quantitative FISH method was more sensitive, but had the disadvantage of only being used for sex-unmatched pairs.

Contexto e objetivo: O transplante de células hematopoiéticas não-mieloablativo é realizado em pacientes com doenças onco-hematológicas que não suportam condicionamentos ablativos devido à elevada idade ou ao acometimento por comorbidades. Esta abordagem não elimina completamente as células do hospedeiro, resultando, inicialmente, em quimerismo misto. A persistência do quimerismo misto na evolução de longo prazo resulta na rejeição ao enxerto e recaída. O acometimento pela doença do enxerto contra hospedeiro é concomitante ao quimerismo completo e ao efeito enxerto versus tumor. O objetivo deste estudo foi avaliar a prevalência do quimerismo em doenças onco-hematológicas tratadas com o transplante não-mieloablativo de células hematopoiéticas. Tipo de estudo e local: Estudo clínico observacional do estado de quimerismo após transplante antígenos leucocitários humanos-idêntico não-mieloabaltivo realizado na Disciplina de Hematologia e Hemoterapia da Universidade Federal de São Paulo. Métodos: Analisamos sequencialmente a amplificação dos primers APO-B, D1S80, DxS52, FVW, 33,6, YNZ-22, H-ras pelo VNTR (variable number of tandem repeats) em 17 pares e FISH (fluorescent in situ hybridization) pela sonda XY e do primer SRY em 13 pares de não relacionados a sexo. Resultado: A informatividade dos primers pelo VNTR foi de 60 por cento para APO-B; 75 por cento D1S80; 36 por cento DxS52; 14 por cento FVW; 40 por cento YNZ-22 e 16 por cento H-ras. O primer SRY foi informativo em receptores femininos com doadores masculinos. O método XY-FISH foi informativo em 100 por cento dos pares de não relacionado a sexo. Conclusão: Estes métodos foram sensíveis e informativos. No VNTR, a associação do APO-B com D1S80 mostrou 88 por cento de informatividade. O FISH, método quantitativo, foi mais sensível, porém com desvantagem de ser usado somente nos pares não relacionados a sexo.

Gene amplification in carcinogenesis

Gene amplification increases the number of genes in a genome and can give rise to karyotype abnormalities called double minutes (DM) and homogeneously staining regions (HSR), both of which have been widely observed in human tumors but are also known to play a major role during embryonic development due to the fact that they are responsible for the programmed increase of gene expression. The etiology of gene amplification during carcinogenesis is not yet completely understood but can be considered a result of genetic instability. Gene amplification leads to an increase in protein expression and provides a selective advantage during cell growth. Oncogenes such as CCND1, c-MET, c-MYC, ERBB2, EGFR and MDM2 are amplified in human tumors and can be associated with increased expression of their respective proteins or not. In general, gene amplification is associated with more aggressive tumors, metastases, resistance to chemotherapy and a decrease in the period during which the patient stays free of the disease. This review discusses the major role of gene amplification in the progression of carcinomas, formation of genetic markers and as possible therapeutic targets for the development of drugs for the treatment of some types of tumors.

Cloning and expression of Vibrio cholerae virulence gene, accessory cholera enterotoxin (ace).

The cholera enterotoxin (CT) has been considered a major virulence factor of Vibrio cholerae. The accessory cholera enterotoxin (ace) gene is the third gene of V. cholerae virulence cassette. The gene coding for the Ace toxin was amplified from V. cholerae isolates producing a single band of 314 bp. The presence of ace gene was confirmed by hybridization as well as by sequencing. The gene was successfully expressed in Escherichia coli (LMG194) using expression, pBAD/Thio-TOPO vector. Optimal conditions for expression included choice of host strain, temperature used for culturing, and concentration of antibiotic and arabinose inducer. The Ace protein was obtained from the cell supernatant as a fusion protein with a molecular mass 34 kDa which was detected using an anti V5-HRP epitope tagged antibody.

Patrón de metilación génico en el cáncer de mama / Promoter methylation profile in breast cancer

BACKGROUND: Genomic DNA methylation, mutations and allelic deletions explain the inactivation of genes involved in cell proliferation and cell cycle control mechanisms. AIM: To analyze the methylation pattern of important genes related to different carcinogenic mechanisms in patients with breast cancer and the relationship with its biological behavior. MATERIAL AND METHODS: Seventy fresh-frozen breast cancer samples were selected. The methylation specific PCR (MSP) test was used to analyze promoter methylation status for genes CDKN2A (p16), hMLH1, APC, CDH1 (Cadherin E) and FHIT. RESULTS: We found methylation in at least one of the genes studied in 88% of cases and in 3 or more genes in 40.5% of cases. The frequencies of promoter hypermethylation of CDKN2A, hMLH1, APC, CDH1 and FHT were 41.4%, 11.4%, 52.9%, 70% and 42.9%, respectively. We found a relationship between CDKN2A methylatlon and better survival (p=0.002). CDH1 methylation and poor histological differentiation (p=0.007), hMLH1 methylation and non-Mapuche ethnicity (p=-0.03), APC methylation and larger tumor size (p<0.05), FHIT methylatton and lack of estrogen rectptor IHC expression (p<0.05). CONCLUSIONS: The high frequency of promoter methylation in patients with breast cancer confirms its role in breast carcinogenesis. The finding of alterations in the methylation pattern of genes studied and its association with prognostic factors is a helpful tool in the search of new criteria for clinical and therapeutic decision making.

Polimorfismo do exon 20 do gene da elastina em indivíduos portadores de doença diverticular dos cólons / Polymorphism of 20 of elastin gene in individuals with colonic diverticular disease

A doença diverticular dos cólons (DDC) é relacionada à dieta, pressão intraluminal elevada, bem como alterações estruturais da parede intestinal. Pacientes com alterações genéticas do gene da elastina (ELN), como a estenose aórtica supravalvar e a cútis laxa, podem manifestar hérnia, diverticulose e disfunção urinária. Recentemente, uma mutação pontual no exon 20 do ELN foi demonstrada em pacientes com hérnia inguinal. No presente estudo é demonstrada uma mutação pontual (AGTGGT) no códon 422 do exon 20 do ELN em 5/14 pacientes com DDC e em 0/26 controles. Foi observada uma associação significativa desta mutação com o desenvolvimento da DDC. /Colonic diverticular disease (CDD) is related to diet, increased intraluminal pressure and structural changes within intestinal wall. Patients carrying genetic disorders of elastin (ELN) gene, such as supravalvular aortic stenosis and cutis laxa, may present hernias, diverticulosis and bladder dysfunction. Recently, a punctual mutation in exon 20 of ELN gene was detected in patients with inguinal hernia. Present study demonstrates a punctual mutation (AGTGGT) within codon 422 of ELN gene in 5/14 patients carrying CDD and in 0/26 among controls. This investigation demonstrated a significant association between the mutation found and CDD development...

Alterações moleculares do gene da elastina nos pacientes portadores de hérnia inguinal / Molecular alterations of elastin gene in inguinal hernia patients

O objetivo do presente estudo foi verificar a ocorrência de mutações do gene da elastina nos pacientes portadores de hérnia inguinal. Estudou-se o DNA genômico de 19 pacientes com hérnia inguinal e 16 controles. Os 34 exons do gene foram amplificados e foi realizada a análise conformacional de fita simples. Observou-se bandas anormais no exon 20. O seqüenciamento do exon 20 revelou uma dupla substituição de bases no códon 404 em 78 por cento e 50 por cento dos pacientes idosos e adultos com hérnia inguinal respectivamente e a substituição de uma base no códon 422 em todos os pacientes com hérnia inguinal, em todos os indivíduos controles idosos e em 37,5 por cento dos indivíduos controles adultos. É possível que estas mutações possam estar relacionadas à produção de moléculas de elastina alteradas e desempenhem papel na gênese da hérnia inguinal.The present study was designed to verify the presence of elastin gene mutations from genomic DNA in patients with inguinal hernia. Nineteen adult patients with diagnosed inguinal hernia and 16 controls were analyzed. All 34 exons were amplified and mutations were sought by single strand conformation polymorphism. DNA amplification revealed an abnormal band in exon 20. Sequencing showed a double base substitution in codon 404 in 78 per cent of the elderly hernia patients and in 50 per cent of the younger hernia group. It also revealed a single base substitution in codon 422 in all inguinal hernia patients, in all the elderly controls as well as in 37,5 per cent of the younger controls. It is possible that these elastin mutations may be responsible for the production of abnormal elastic fibers, thus playing a role in inguinal hernia genesis...

Whole CagA gene amplification of Helicobacter pylori and its fingerprinting by restriction fragment length polymorphism / 华中科技大学学报（医学）（英德文版）

To set up a method of amplification for the whole CagA gene of Helicobacter pylori and its fingerprinting by restriction fragment length polymorphism (RFLP), nested PCR was employed in combination with TD-PCR to amplify the gene and EcoRI and Hind III were used to generate the RFLP fingerprinting. Target DNA fragments from 13 of 20 samples were successfully amplified and the relevant RFLP fingerprintings were obtained. It is concluded that the method can be used to amplify the whole CagA gene and CagA gene has apparent diversity of RFLP profile.

Tumorogénesis y proteína mdm2 / Tumorogenesis and mdm2 protein

Tumorogenesis is associated with several events by which a normal cell transforms itself into a tumour cell with an increased proliferation rate. One of the most important research initiatives in this area is the characterization of the molecular mechanisms involved in tumorogenesis and cancer. Oncogenes and tumour suppressor genes are directly involved in the cell cycle, differentiation, and apoptosis. The cellular oncogene MDM2 seems to be abnormally elevated in several human tumours, specially in sarcomas. The MDM2 gene product, mdm2 protein, pS3 and retinoblastoma (Rb) proteins, play crucial roles in the control of the cell cycle. The molecular interactions between mdm2, pS3 and Rb in cancer, are associated with a loss of control in the G1 phase of the cell cycle leading to uncontrolled cell proliferation. Studies by gene amplification appear to show an incomplete picture of mdm2 protein levels in tumour cells. The simultaneous determination of mdm2 protein and mRNA levels seems to give a more accurate interpretation of the abnormal function of the mdm2 protein. Thus, in addition to gene amplification, different mechanisms by which mdm2 is overexpressed in cancer cells also play an important role in tumorogenesis

Genetic alterations in head and neck squamous cell carcinomas

The genetic alterations observed in head and neck cancer are mainly due to oncogene activation (gain of function mutations) and tumor suppressor gene inactivation (loss of function mutations), leading to deregulation of cell proliferation and death. These genetic alterations include gene amplification and overexpression of oncogenes such as myc, erbB-2, EGFR and cyclinD1 and mutations, deletions and hypermethylation leading to p16 and TP53 tumor suppressor gene inactivation. In addition, loss of heterozygosity in several chromosomal regions is frequently observed, suggesting that other tumor suppressor genes not yet identified could be involved in the tumorigenic process of head and neck cancers. The exact temporal sequence of the genetic alterations during head and neck squamous cell carcinoma (HNSCC) development and progression has not yet been defined and their diagnostic or prognostic significance is controversial. Advances in the understanding of the molecular basis of head and neck cancer should help in the identification of new markers that could be used for the diagnosis, prognosis and treatment of the disease

Nova variante do gene do prionio em uma familia psicotica / New variant of the prion protein gene in a psychotic family

As doencas de prionio sao caracterizadas pelo acumulo no cerebro da PrP-sc, uma proteina prionica infectante e protease resistente. A Prp-sc difere da PrP-c, de funcao desconhecida, apenas em termos conformacionais. As doencas humanas de prionio conhecidas sao Kuru, Gertsman Straussler (GSS), Insonia Familiar Fatal(IFF) e doencas de Creutzfeldt-Jakob (CJD)...

The significance of HER-2/neu/c-erbB-2 gene amplification in benign and malignant breast disease.

This study was carried out to investigate the amplification of HER-2/neu oncogene in 66 patients with primary breast cancer and 90 samples from benign breast disease (BBD). The amplification of HER-2/neu oncogene in the DNA of paraffin-embedded specimens was determined by differential PCR. Nineteen out of 66 (28.8%) breast cancer patients showed amplification of the gene. No gene amplification was found in benign breast disease. There was no significant correlation of HER-2/neu amplification with, age, menopausal status, the number of positive nodes, tumor size, estrogen receptor, however, amplification of HER-2/neu gene was strongly correlated with nodal status (p = 0.0049). In node positive patients, the incidence of HER-2/neu amplification was high (43%). These findings indicate that the amplification of HER-2/neu gene may be of pathogenetic significance in breast cancer and may have a poor prognosis in node positive breast cancer patients while no gene amplification in benign breast disease suggests that HER-2/neu amplification is a late molecular alteration event in the pathogenesis of breast cancer.

A 28-fold increase in secretory protein synthesis is associated with DNA puff activity in the salivary gland of Bradysia Hygida (Diptera, Sciaridae)

When the first group of DNA puffs is active in the salivary gland regions S1 and S3 of Bradysia hygida larvae, there is a large increase in the production and secretion of new salivary proteins demonstrable by [3H]-Leu incorporation. The present study shows that protein separation by SDS-PAGE and detection by fluorography demonstrated that polypeptides range in molecular mass from about 23 to 100 kDa. Furthermore, these proteins were synthesized mainly in the S1 and S3 salivary gland regions where the DNA puffs C7,C5,C4 and B10 are conspicuous, while in the S2 region protein synthesis was very low. Others have shown that the extent of amplification for DNA sequences that code for mRNA in the DNA puffs C4 and B10 was about 22 and 10 times, respectively. The present data for this group of DNA puffs are consistent with the proposition that gene amplification is necessary to provide some cells with additional gene copies for the production of massive amounts of proteins within a short period of time (Spradling AC and Mahowald AP (1980) Proccedings of the National Academy of Sciences, USA, 77: 1096-1100).

Molecular cytogenetics of human cancer.

Cancer may arise from the genetic transformation of a single precursor cell, which proliferates to form a clone. Chromosomal abnormalities are associated with many types of tumours. Some of the chromosomal rearrangements such as translocation, deletion and insertion involve breakage of chromosomes close to known oncogenes. The close linkage between the chromosomal changes, the gene modifications and consequently altered protein function seen in malignant cells suggest that cancer is a genetic disease. Analysis of chromosomal abnormalities and oncogene amplifications in malignant cells have been found to be related to their malignant potential and hence may be utilized in the clinical management of patients with cancer.

DNA sequence amplification in sciarid flies: results and perspectives

The discovery of Dna sequence amplification in sciarid flies and investigations into its control and biological significance are reviewed. Results thus far show that amplification of specific salivary gland polytene chromosome bands is a general phenomenon in sciarids. It brought about as part of a final endoreplication cycle by the rising titer of ecdysterone that occurs as the Larvae approach the prepupal period. Amplification and transcription of these bands is a late, multistep effect of this hormone.The Dna puffs which form in amplified region produce mRNAs which are translated into polypeptides that appear to be involved in coccon formation. Application of molecular cloning techniques to the study of Dna amplification has allowed precise quantitation of amplification for several Dna puffs and is yielding maps of their transcription units.These techniques will ultimately help to define the origins of Dna puff replication and contribute to an understanding of the mechanism and control of the amplification phenomenon in sciaridae. Projections for future experimental approaches are presented

Caracterización de la amplificación génica de un segmento de 294 bases del genoma de mycobacterium tuberculosis / Characterization of genic amplification of a segment of 294 bases of mycobacterium tuberculosis genoma

Se presentan los resultados de una prueba diagnóstica novedosa, basada en la amplificación de una secuencia nucleótida conocida de un ADN blanco. La reacción de amplificación génica (PCR), descrita en este trabajo, permite una amplificación * 10 elevado a 6 -veces un segmento de 294 bp de un gene que codifica a un antígeno de 65 KDa- correspondiente a una proteína de respuesta al estrés térmico, presente en Mycobacterium tuberculosis y Mycobacterium bovis BCG. Esta reacción requiere grandes cantidades de ADN blanco, pero con la técnica utilizada fue posible detectar amplificación comenzando con 10 elevado a -12 g de ADN; cantidad que corresponde a 10-100 micobacterias/equivalentes ADN. Este nivel detectado representa un mejoramiento respecto a la tinción Ziehl-Neelsen, el cual necesita sobre 10.000 micobacterias para ser leído como positivo. La concentración de Mg elevado a +2 es crítica en la reacción, la cual corresponde a 3-5 mM. El análisis de la secuencia muestra que la amplificación de ésta es comparable a la secuencia localizada entre las posiciones 781-1075 del gene 65 KDa. Se realizaron pruebas específicas que demostraron que la señal de amplificación es posible de encontrar con ADN obtenido de M. tuberculosis y M. bovis BCG. Con otras micobacterias la señal de amplificación es muy baja y equivalente a aquéllas encontradas con ADN micobacterial no relacionado. La reacción fue probada con cuatro muestras clínicas, las cuales fueron positivas para la presentación de micobacterias; en tres de ellas se encontró el segmento amplificado 294 bp. Continúan desarrollándose ensayos de sensibilidad y especificidad en muestras clínicas

Gene amplification in Rhynchosciara (1955-1987)

A review is made of the evidence indicating the existence of gene amplification in Rhynchosciara, from the early cytological work to the more recent studies using cloned sequences from the DNA puffs. Mention is made of work still in progress which indicates that the transcription unit of a DNA puff is surprisingly complex.