Angélica Melo

Description of Echinococcus granulosus genotypes in human hydatidosis in a region of southern Chile.

INTRODUCTION Echinococcus granulosus species has a wide variety in both geography and hosts; indeed, 10 genotypes have been reported in studies on material of animal origin. The aim of this study was to genotype E. granulosus obtained from human hydatid cysts. MATERIALS AND METHODS The hydatid fluid and sand was collected from patients who underwent surgery for hepatic and pulmonary hydatidosis at Hospital Regional in Temuco, Chile, between 2004 and 2005. Two PCR systems were used: PCR Eg 9 and PCR Eg 16. The RsaI enzyme was used for RFLP. The genotype was confirmed using the sequence of one fragment of 366 bp from a mitochondrial gene (cox1). RESULTS The DNA of protoscolices from 24 samples was analyzed, 4 of them from pulmonary cysts and 20 from hepatic cysts. The 366 bp fragment was amplified in 20 out of 24 samples (83.3%). Enzymatic digestion revealed the presence of 3 possible genotypes: in 20 out of 21 samples (95,2%), a restriction was observed corresponding to the G1 or G7 genotypes; in the remaining sample genotype G4 or G7 was observed. Sequencing confirmed the presence of G1 genotype for 19 samples and G6 genotype for the remaining sample (G4 or G7 according to PCR-RFLP). CONCLUSION The PCR-RFLP technique enabled three possible genotypes present (G1 or G7, G4 or G7) to be established. Sequencing allowed us to decisively identify the G1 and G6 genotypes in our study group. Previous studies agree with the identification of the G1 genotype in our country. We consider it significant that the G6 genotype is present in Chile for its epidemiological implications.

p53 tumour suppressor gene protein expression in early and advanced gallbladder carcinoma.

Gallbladder carcinoma is one of the most frequent malignant tumours occurring in Chile and the mortality rate in both sexes ranks among one of the highest in the world. Mutation of p53 tumour suppressor gene has been demonstrated in many tumours. Our aim was to determine protein expression of p53 gene in early and advanced gallbladder carcinoma.

Promoter methylation profile in gallbladder cancer.

BackgroundMethylation in the promoter region of genes is an important mechanism of inactivation of tumor suppressor genes. Our objective was to analyze the methylation pattern of some of the genes involved in carcinogenesis of the gallbladder, examining the immunohistochemical expression of proteins, clinical features, and patient survival time.MethodsTwenty cases of gallbladder cancer were selected from the frozen tumor bank. The DNA extracted was analyzed by means of a methylation-specific polymerase chain reaction test for the CDKN2A (p16), MLH1, APC, FHIT, and CDH1 (E-cadherin) genes. Morphological and clinical data and follow-up information were obtained.ResultsAll cases were in an advanced stage: histologically moderate or poorly differentiated tumors (95%). Methylation of the promoter area of genes was observed in 5%, 20%, 30%, 40%, and 65% of cases, and an altered immunohistochemical pattern (AIP) in 5%, 35%, 21%, 25%, and 66% for the MLH1, CDKN2A, FHIT, APC, and CDH1 genes, respectively. The Kappa concordance index between methylation of the promoter area and AIP for the MLH1 and CDH1 genes was very high (K > 0.75) and substantial for APC (K > 0.45). No correlation was found between survival time and the methylation of the genes studied.ConclusionsThe high frequency of gene methylation (with the exception of MLH1) and the high agreement between AIP and methylation of the gene promoter area for the MLH1, APC, and CDH1 genes suggest that the inactivation of tumor suppressor genes and of the genes related to the control of cellular proliferation through this mechanism is involved in gallbladder carcinogenesis.

Viability and Fertility of Human Hepatic Hydatid Cysts

IntroductionThe adequate treatment for hydatidosis requires a knowledge of certain aspects related to the survival of infectious agents, especially protoscoleces. The aim of this study is to evaluate the viability of protoscoleces in human hepatic hydatid cysts in order to determine the prevalence of their fertility and to study the association with variables typical of the host and of the parasite.MaterialsA cross-sectional study was done in biological material (the fluid from human hepatic hydatid cysts). The viability criteria used were: ovoid form, invaginated scolices and intact calcareous corpuscles, the presence of vibrating movements, and the absence of “vital” staining. The cysts were grouped as univesicular cysts (UVC), multivesicular cysts (MVC) and abscessed cysts (LAHO). Fertility was defined as living protoscoleces in relation to the total number of protoscoleces. Descriptive statistics were for the calculation of the prevalence of fertility, analytical statistics for the comparison of groups, and a multivariate analysis for the examination of the association between cyst fertility and clinical variables.ResultsA total of 163 cysts with a median diameter of 15 cm were studied in this way. Of these lesions, 79 (48.5%) were UVC, 54 (33.1%) MVC, and 30 (18.4%) LAHO. On 99 occasions (60.7%), macroscopic communication was evident in the bile duct, and a prevalence of general fertility of 57.1% (94.4% for MVC, 53.2% for UVC, and 0% for LAHO, with a value of P < 0.001). Association with location, type, and diameter of the cyst, and presence of biliary communications was verified by applying a bivariate analysis, and association between fertility and the variables of the type of the cyst and the existence of biliary communications was verified employing a multivariate analysis (P values of 0.004 and < 0.001, respectively).ConclusionsThe prevalence of fertility found was low. The main prevalence of fertility was observed in MVCs. Fertility is associated with the type of cyst and the presence of biliary communications.

Mutación del gen p53 en el cáncer de la vesícula biliar

33 formalin fixed paraffin embedded samples of gallbladdercarcinoma (30 women, age range 32-86 years) were selected. Pancreatic cancer tissue with K-ras mutations was used as control. DNA was extracted from the histological section by mean ofmicrodissection and K-ras mutations in codon 12 were detected by polymerase chain reactionand restriction fragment length polymorphism (RFLP), using previously reported technique.

Estudio del patrón de metilación génico en el cáncer gástrico en Chile

Background: Promoter genomic DNA methylation is an important inactivation mechanism of tumor suppressor genes. This genetic-molecular pathway for cancer may separate a subset of patients with different prognoses and eventually different responses to specific therapies. Aim: To analyze the methylation pattern of important genes related to different carcinogenic mechanisms in patients with gastric cancer (GC) and the relationship with its morphological features and biological behavior. Material and methods: Forty-seven fresh-frozen GC samples were selected. The methylation-specific PCR (MSP) test was used to analyze promoter methylation status for genes MLH1, CDKN2A (p16), APC, CDH1 (Cadherin E) and FHIT. Follow-up and complete morphological features were obtained for all cases. Results: We found methylation in at least one of the genes studied in 83% of the cases. The frequencies of promoter hypermethylation of MLH1, CDKN2A, APC, CDH1 and FHIT were 31%, 43%, 46%, 80% y 62%, respectively. We found a relationship between APC methylation and good histological differentiation (p=0.03); CDH1 methylation with diffuse type by Lauren and 3 or more metastasic lymph nodes (p <0.05); FHIT, CDKN2A and CDH1 methylation and female condition (p <0.04). We also found a non-significant relationship between CDKN2A methylation and better survival (p=0.07). Conclusions: The high frequency promoter methylation found confirms its importance in gastric 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 for new criteria in clinical and therapeutic decision making (Rev Med Chile 2005; 133: 874-80). (Key Words: DNA methylation; Promoter regions (Genetics); Stomach neoplasms)

Efecto de la fijación en la calidad del ADN: estudio controlado con cinco fijadores

Resumen Antecedentes Los tejidos fijados y embebidos en parafina son una fuente importante de material para diagnostico e investigacion. La amplificacion de ADN desde este tipo de tejidos, mediante reaccion en cadena de la polimerasa (PCR), es afectada por el tipo de fijador y los tiempos de fijacion empleados. Para determinar el parametro que mejor se adecue a las condiciones de trabajo de nuestro laboratorio de Patologia Molecular, evaluamos el efecto de cinco fijadores sobre la calidad del ADN bajo condiciones controladas. Material y Metodo Muestras de mucosa gastrica fueron fijadas, embebidas en parafina y luego procesadas para extraccion de ADN empleando un protocolo basado en digestion con proteinasa K. La calidad del ADN se evaluo mediante amplificacion de tres fragmentos del gen β-globina (268, 536 y 989 pb). Resultados No observamos mayores diferencias entre fijadores ni tiempos de fijacion en la amplificacion de ADN de 268 y 536 pb. No obstante, la amplificacion del fragmento mayor (981 pb) se vio alterada al aumentar el tiempo de fijacion, a excepcion de aquellas muestras fijadas en etanol 70% que presentaron una banda de similar intensidad a la obtenida para muestras control (tejido fresco congelado). Conclusiones Estos resultados nos proporcionan una pauta para el diseno de experimentos de acuerdo a la calidad del material archivado, optimizando recursos humanos e insumos.

TRANSCRIPTOS DE FUSIÓN DEL GEN BCR/ABL EN PACIENTES CON LEUCEMIA MIELOIDE CRÓNICA

La anormalidad citogenetica mas comun en la leucemia mieloide cronica (LMC) es el cromosoma Philadelphia, producida por la t(9;22), cuya expresion molecular es el gen de fusion BCR-ABL, que codifica proteinas con actividad tirosinquinasa. Segun el punto de ruptura de los genes BCR o ABL se produce una proteina de fusion de 210-kD(p210) o 190-kD(p190). La presencia de este gen de fusion en pacientes con LMC tiene implicancia diagnostica. Con el proposito de detectar transcriptos de fusion del gen BCR/ABL en pacientes con leucemia mieloide cronica, procedentes de la IX Region de Chile, se estudiaron 14 muestras de sangre de 11 pacientes con LMC. A 2 de ellos, se les realizo seguimiento durante su tratamiento con Gleevec. Se aplico la tecnica de reaccion en cadena de la polimerasa con transcriptasa reversa (RT-PCR), usando una PCR en nido. Para la deteccion de los transcriptos de fusion p210 y p190 del gen BCR/ABL, se utilizaron 4 pares de iniciadores. En 9/14 muestras se detecto el transcripto de fusion p210 y en 5/14 los transcriptos de fusion p210 y p190. En los 2 pacientes en seguimiento, hubo desaparicion del transcripto p190, permaneciendo el transcripto p210. Estos resultados reafirman la importancia de detectar transcriptos de fusion BCR/ABL para el diagnostico y seguimiento durante el tratamiento de la LMC

Mutación del gen p53 en el cáncer de colon y recto

Background: Genetic events associated to colorectal carcinoma are well characterized, but there is scanty information about this issue in Chilean subjects. Aim: To determine the frequency and distribution of exons 5, 6, 7, 8 and 9 mutations and the immunohistochemical expression of p53 gene in biopsy samples of colorectal carcinoma. Material and methods: p53 gene exons 5, 6, 7, 8 and 9 were directly sequenced in 42 biopsy samples of colorectal carcinoma. Immunohistochemical expression of p53 was determined in 35 samples. Results: Thirty one discrete mutations (12 transitions, 11 transversions and 8 insertions) were observed in 21 samples (60%). Nine samples had mutations in exon 5, twelve samples had mutations in exon 6, seven samples had mutations in exon 7 and three samples had mutations in exons 8 and 9. Immunohistochemical expression of p53 protein was observed in 18 of 35 cases. There was a high correlation between the genetic alteration and immunohistochemistry, when p53 was expressed in more the 20% of cells. The positive and negative predictive values of p53 expression were 87 and 80% respectively. There was a non significant lower mortality among patients with mutations in their biopsies. Conclusions: These results confirm the involvement of p53 gene mutations in colonic carcinogenesis. Immunohistochemical methods for the detection of p53 protein have a high predictive value (Rev Med Chile 2000; 128: 996-1004)

Virus papiloma humano y Chlamydia trachomatis según número de parejas sexuales y tiempo de actividad sexual en estudiantes universitarias en la Región de La Araucanía, Chile

BACKGROUND Human papilloma virus (HPV) and Chlamydia trachomatis are the most prevalent sexually transmitted infections (STIs), among teenagers and young people, with risk factors: active sex life and multiple partners. Chlamydia trachomatis infection may favor HPV infection and this, the development of cervical cancer. Both infections can lead to consequences on sexual and reproductive health. OBJECTIVE To determine frequency of HPV and C. trachomatis in asymptomatic university women less than 25 years, associating them with number of sexual partners (n°SxP) and time of sexual activity (TSxA). Material andMethods: 151 cervical samples for HPV and C. trachomatis, were processed by conventional and in real time reaction polymerase chain. RESULTS HPV 21, 8%, C. trachomatis 11, 2% and co-infection (HPV/C.trachomatis), 4.6%. Aimong HPV +, 80, 6% showed high risk HPV. The n°SxP was strongly associated with HPV. Aimong young coinfected HPV/C. trachomatis, 71.4% had 3 or more PSx. Chlamydia trachomatis was more frequent (64,7%) that HPV within range of 3-5 years according to the TSxA, Discussion: A high prevalence of HPV and C. trachomatis was observed. Young women with coinfection HPV/C. trachomatis could be a high-risk group need to monitor their infections. It suggests the implementation of university programs in education, counseling and prevention in sexual health.