Gutemberg Almeida

The R337H mutation in TP53 and breast cancer in Brazil

BackgroundGermline mutations in p53 are associated with the Li-Fraumeni Syndrome which is characterized by childhood cancers, including pediatric adrenal cortical carcinomas and early onset breast cancer. The high incidence of adrenal cortical carcinomas in southern Brazil is mostly attributed to the R337H mutation in TP53. The relatively high population frequency of this mutation in southern Brazil, along with the clustering of early onset breast cancer in Li-Frameni families, suggests this mutation may also be a low-penetrance breast cancer susceptibility polymorphism.MethodsWe undertook this study to evaluate the frequency of the R337H mutation in breast cancer patients from Rio de Janeiro, Brazil. R337H mutation status was determined in 390 unselected breast cases and 324 controls identified from clinics in Rio de Janeiro, Brazil using a PCR-based assay.ResultsTwo of the breast cancer cases (0.5%) and none of the controls carried the mutation. Both cases had an early age at diagnosis (< 40 years old) and a family history of breast and other cancers.ConclusionsThese data suggest genetic screening of young onset breast cancer patients should include testing for the R337H mutation.

HIV/HPV co-infection during pregnancy in southeastern Brazil: Prevalence, HPV types, cytological abnormalities and risk factors

OBJECTIVE HIV(+) pregnant women are at a higher risk of HPV infection and development of cervical cancer. Our objectives were to assess the prevalence and HPV types in HIV(+) pregnant women and to identify risk factors for HPV infection and cytological abnormalities. METHODS Cervicovaginal smears were collected during pregnancy from 140 women. Partial HPV L1 gene and the exon 4 of the human TP53 gene (containing codon 72) were PCR-amplified and sequenced. Amplified products indicating multiple HPV infection were further cloned and sequenced. The association of demographic, obstetric and HIV-related clinical variables with HPV infection and cervical lesions was tested by univariate analyses, and significant factors were subsequently tested by logistic regression multivariate analysis. RESULTS HPV DNA tested positive for 118 patients and HPV types were identified in 104 samples. Twenty-eight different types were found, HPV-16 and HPV-58 being the most prevalent. High-risk types were present in 79.8% of samples and multiple infections in 16.3%. Abnormal cervical smears were found in 44 patients (31.4%). Absolute CD4(+) T-cell counts below 350 were associated with HPV infection. Younger age was associated with cervical abnormalities and higher CD4(+) T-cell count was an apparent protective factor. CONCLUSIONS We found a high prevalence of HPV infection and high-risk types in this cohort. Our results highlighted the relevance of immune system integrity rather than TP53 variants for protecting this highly vulnerable population to HPV infection and carcinogenesis.

The presence of methylation of the p16INK4A gene and human papillomavirus in high-grade cervical squamous intraepithelial lesions.

Methylation is a chemical modification in which a methyl group (CH3) is added to the cytosine in the promoter region of the gene. It involves a very frequent epigenetic event that is found in many human cancers. Currently, there is no consensus on whether methylation of the p16 gene could be used as a biomarker in cervical intraepithelial neoplasia. The authors studied the presence of methylation of the p16INK4a gene and human papillomavirus (HPV) DNA, and a possible relationship between them in high-grade squamous intraepithelial lesions of the cervix. This case-control study analyzed 27 high-grade squamous intraepithelial lesion samples and 20 normal cytology samples. To detect p16INK4a methylation, methylation-specific polymerase chain reaction was used, and for HPV DNA detection the polymerase chain reaction was performed by using MY09/MY11 and GP5+/GP6+ consensus primers. The presence of methylation of the promoter region of the p16INK4a gene was detected in 55.6% of the samples from the case group, whereas it was detected only in 20% of the samples from the control group (P=0.005). HPV DNA was found in 66.7% of the samples from the case group, whereas only 15% from the control group (P=0.0001). The relationship between the presence of methylation of the p16INK4a gene and HPV DNA did not prove statistically significant in the case group (P=0.67) or the control group (P=0.51). In conclusion, the presence of methylation of the p16INK4a gene constituted an occurrence that was early but independent of the presence of HPV DNA.

Análise do emprego de retalhos fasciocutâneos para reconstrução vulvar imediata

OBJECTIVE To analyze the use of immediate reconstruction techniques of the vulva after surgical resection, with fasciocutaneous flaps of the medial and/or posterior thigh. METHODS We conducted a transversal, retrospective study to analyse the outcome of immediate surgical reconstruction with fasciocutaneous flaps in nine patients who underwent vulvectomy from May 2009 to August 2010. RESULTS Mean age was 61 years (range 36-82). In 56% of cases, diagnosis was vulvar intraepithelial neoplasia (VIN), usual type. Radical vulvectomy was performed in 45% of patients, simple vulvectomy in 33% and wide resections in 22%. Eleven fasciocutaneous flaps were made, of which 36.3% were flap transpositions from the posterior thigh, 18.2% from the medial thigh, 18.2% were in advancement flaps, 18.2% simple advancement flaps and 9.1% flap rotation from the posterior thigh. There were no major losses of the flaps made. CONCLUSION Thigh fasciocutaneous flaps are currently the best options for immediate reconstruction after resection of vulvar cancer due to the preservation of sensibility and tissue availability in the donor areas. The association of the Plastic Surgeon with the Gynecologist offers tranquility for patients and provides good postoperative results.

Promoter hypermethylation patterns of death-associated protein kinase and p16 genes in vulvar lichen sclerosus.

Objective: This article aimed to investigate the hypermethylation of promoter regions of tumor suppressor genes, such as death-associated protein kinase (DAPK) and p16, in vulvar lichen sclerosus (LS). Materials and Methods: The promoter hypermethylation of DAPK and p16 was investigated from 15 vulvar biopsies of patients with LS who had had no previous treatment. DNA was treated with sodium bisulfate and underwent methylation-specific polymerase chain reaction of these genes. The amplified polymerase chain reaction products were analyzed by 10% polyacrylamide gel. Results: The mean age of the patients was 57 years (most were postmenopausal). Methylation of the promoter region of DAPK was found in 2 (13%) of 15 patients analyzed, and p16 promoter region methylation was found in 7 patients (47%). The samples that showed DAPK methylation also showed p16 methylation. Conclusions: Methylation of DAPK and p16 represent alterations that might occur in cell cycle control in LS. The hypothesis is that patients who had methylated genes in this study, mainly the 2 cases in which there has been methylation in both studied genes, may be more susceptible to the development of differentiated vulvar intraepithelial neoplasia or vulvar cancer. Methylation may play a role in progress of vulvar carcinogenesis.

Evaluation of DAPK gene methylation and HPV and EBV infection in cervical cells from patients with normal cytology and colposcopy

ObjectiveEvaluation of promoter methylation of the death-associated protein kinase (DAPK) gene and HPV and EBV infections in cervical cells from patients with normal cytology and colposcopy.Study designTwenty women, who had been patients at the Institute of Gynecology of the Federal University of Rio de Janeiro (UFRJ) for routine examinations and who showed normal cytology and colposcopy, were selected for this work. Cervical brushings were used for DNA extraction, and the analysis of methylation patterns of the DAPK gene was done through chemical modification with sodium bisulfite. Analysis of viral infection was done using polymerase chain reaction (PCR).ResultsOf the 20 patients studied, six (30%) presented methylation of the DAPK gene, five (25%) presented infection with EBV and three (15%) presented coinfection with HPV/EBV. Associating methylation with viral infection, we found methylated DAPK in one patient (16%) with EBV, in two patients (33%) with co-infection and in three patients (50%) with no viral infection.ConclusionsIn the present study, we verified, for the first time, the methylation pattern of the DAPK gene in cervical smears from patients with normal cytology and colposcopy. The results also showed the presence of viral infections in these patients. EBV infection, irrespective of whether associated with HPV or not, may contribute to cervical carcinogenesis as a cofactor. Methylation of the DAPK gene is associated with cell transformation, suggesting that DAPK methylation might be an important marker for the development of cervical epithelial neoplasias.

Bonafide, type-specific human papillomavirus persistence among HIV-positive pregnant women: predictive value for cytological abnormalities, a longitudinal cohort study

This study investigated the rate of human papillomavirus (HPV) persistence, associated risk factors, and predictors of cytological alteration outcomes in a cohort of human immunodeficiency virus-infected pregnant women over an 18-month period. HPV was typed through L1 gene sequencing in cervical smears collected during gestation and at 12 months after delivery. Outcomes were defined as nonpersistence (clearance of the HPV in the 2nd sample), re-infection (detection of different types of HPV in the 2 samples), and type-specific HPV persistence (the same HPV type found in both samples). An unfavourable cytological outcome was considered when the second exam showed progression to squamous intraepithelial lesion or high squamous intraepithelial lesion. Ninety patients were studied. HPV DNA persistence occurred in 50% of the cases composed of type-specific persistence (30%) or re-infection (20%). A low CD4+T-cell count at entry was a risk factor for type-specific, re-infection, or HPV DNA persistence. The odds ratio (OR) was almost three times higher in the type-specific group when compared with the re-infection group (OR = 2.8; 95% confidence interval: 0.43-22.79). Our findings show that bonafide (type-specific) HPV persistence is a stronger predictor for the development of cytological abnormalities, highlighting the need for HPV typing as opposed to HPV DNA testing in the clinical setting.

TIMP-2 gene methylation in cervical precursor and invasive lesions

OBJECTIVE To analyze the presence of HPV-DNA and TIMP-2 gene methylation in cervical precursor and invasive lesions, as well as to study the associations among the latter, the presence of HPV-DNA, and the clinical evolution of such lesions. METHODS Cross-sectional study that includes 49 biopsy or brush smear samples from women with a normal cervix, LSIL, HSIL, microinvasive carcinoma and invasive carcinoma. The presence of HPV-DNA and specific methylation was analyzed using PCR. Thirty-eight biopsy samples for HSIL, microinvasive carcinoma and frank invasive carcinoma as well as 11 brush smear samples for LSIL and normal cervices were analyzed. RESULTS TIMP-2 gene methylation was detected in 86.8% (33/38) of the samples from the group with lesions and 50% (4/8) of the normal samples (p=0.03). HPV-DNA was detected in 81.6% (31/38) of the samples from the group with lesions and 25% (2/8) of the normal samples (p=0.003). HPV-DNA was more frequent in the methylated samples (50%), and the group with methylation had a higher risk of unfavorable evolution than the group without methylation; however, such observations were not statistically significant (p=0.19). CONCLUSION TIMP-2 gene methylation and the presence of HPV-DNA were characteristic of the group with cervical lesions. Methylation was not associated with the presence of HPV-DNA or an unfavorable clinical evolution.

HPV DNA genotyping and methylation of gene p16INK4A in cervical LSIL

BACKGROUND DNA methylation is the most important epigenetic change involved in the control of gene expression in human cells. Methylation of the p16(INK4a) gene occurs early in the development of cervical cancer. Low-grade squamous intraepithelial lesions (LSILs) are prevalent, and their behavior is variable. OBJECTIVE To identify the HPV DNA type, detect the methylation status of the p16(INK4A) gene, and analyze their association with the cytological evolution of LSIL over a period of two years. METHODS We conducted a cohort study with 40 participants. Cervical scrapings were collected for cytological and molecular analysis. HPV DNA detection and typing were performed by means of polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). Methylation-specific PCR was performed to detect methylation. RESULTS HPV DNA was detected in 87% of the cases, and type 16 was the most frequent type. Methylation was detected in 11% of the cases and did not exhibit a significant correlation with the HPV type. Unfavorable cytological evolution exhibited a significant association with the presence of methylation. CONCLUSION HPV 16 was the most frequently detected type of HPV in LSIL. Methylation of the p16(INK4A) gene was infrequent and occurred independent of the presence of HPV DNA. Methylation of the p16(INK4a) gene exhibited a significant correlation with persistence/progression of LSIL.

The association of HPV genotype with the regression, persistence or progression of low-grade squamous intraepithelial lesions ☆

BACKGROUND Human papillomavirus (HPV) is a highly prevalent sexually transmitted virus causing cytological alterations that precede cervical cancer. Approximately 130 genotypes have been sequenced. Low-grade squamous intraepithelial lesions (LSIL) are the most frequent cytological alteration and have an uncertain behavior. OBJECTIVES To analyze the frequency of HPV types in LSIL and their association with the regression, persistence or progression of these lesions. METHODS A cohort study of forty patients with LSIL cytology was conducted from December 2007 to March 2011. The follow-up lasted two years and included cytology and colposcopy. HPV detection was performed using PCR, and genotyping was performed using PCR-specific and RFLP techniques. RESULTS DNA-HPV was detected in 87% (35/40) of the cases, with oncogenic HPV accounting for 76%; type 16 in 32% (11/35) and type 18 in 20%. LSIL regression, persistence and progression rates at the end of the study were 60%, 23% and 17%, respectively. There was 50% regression in lesions in the high oncogenic risk group (types 16 and 18). CONCLUSION HPV 16 was the most frequent genotype found in LSIL. The persistence and progression of the LSIL were related to the persistence of oncogenic HPV. The longer the follow-up time, the lower the LSIL persistence rate and the higher its regression rate; the progression rate remained stable. In addition to the presence of oncogenic HPV, other factors are necessary for the progression of LSIL.