Expression of human papillomavirus E6 and E7 oncoprotein mRNA in women with low-grade squamous intraepithelial lesions or less.

We verified the prevalence of human papillomavirus (HPV) E6/E7 protein mRNA expression in patients with low-grade squamous intraepithelial lesions (LSILs) and negative cervicovaginal cytology. To investigate the relationship between mRNA expression and viral infection type, we assessed genotyping in single infections. Samples from 825 women were submitted to the E6/E7 survey. We noticed a larger percentage of E6/E7 mRNA expression in the atypical squamous cells of undetermined significance (ASC-US) and LSIL cytologies. Negative results of mRNA expression were in accordance with negative cytologies. In positive cases, the infection by a single HPV type was most common, with type 16 being most prevalent. The expression of mRNA was most prevalent in ASC-US and LSIL cytologies, compared with the negative cytology. The infection by a HPV type was more frequent in cases of positive expression, with HPV type 16 being found most frequently. Patients with LSIL cytologies had a higher percentage of multiple infections.

HPV infection-associated anogenital cyto-colpo-histological findings and molecular typing in HIV-positive women.

HIV and human papillomavirus (HPV) coinfection is increasing, especially in the anal canal (AC) and cervico-vaginal regions. We identified anal epithelium abnormalities related to high-risk HPV (HR-HPV) lesions in the lower genital tracts (LGTs) of HIV-positive women, described the HPV genotypes identified, and assessed the expression of E6/E7 oncogenes in coinfected patients. Ninety-eight women were enrolled in groups combining HIV status and presence or absence of HPV in the LGT. Anal and cervical smears were collected for cytology and HR-HPV assays using Cobas(®) and/or PapilloCheck(®). Samples with highly oncogenic HPV genotypes were confirmed by NucliSENS EasyQ(®). Forty-two HIV-positive (25-52; mean age 39.5) and 56 HIV-negative (18-58; mean age 35.7) patients were included. E2 and C1 groups presented AC alterations (P = 0.002); altered images for high-resolution anoscopy were higher in E1 and C2 (P < 0.001). Of the 29 women with alterations, 41.38% were HIV-negative and 58.62% were HIV-positive (P < 0.001). HIV-positive patients accounted for 29% of the anal high-grade squamous intraepithelial lesions (P = 0.015). The Cobas(®) positive result frequency was higher in three AC groups than in the other groups. There was variation in the number of HPV types in the cervico-vaginal samples among the study groups (P < 0.001). Anal cytology and anoscopy showed more altered findings in HIV-positive patients with HPV in the LGT. HR-HPV anal infections by various genotypes are common and are associated with cervical infections in HIV-positive patients. E6/E7 expression is apparently more common in the AC of HIV-positive women.

Accuracy of the cytopathology, bacterioscopy, and vaginal flora culture.

INTRODUCTION: An over-population of vaginal microorganisms causing inflammatory processes renders it difficult to properly assess the cytopathological exam that aims to screen precedent cervical lesions. On the contrary, the occurrence of the microbial flora saprophyte does not influence correct cythodiagnosis. OBJECTIVE: To assess the composition of vaginal tract aerobic microorganisms of asymptomatic women in menacme and post-menopause, and to analyze the accuracy of cytopathologic, bacterioscopic exams, and culturing of the flora. METHODS: The women were first submitted to a focused anamnestic interrogatory and then submitted to gynecological exam. A sample of the vaginal fluid was collected with a culture swab and a smear was made on two glass slides for stained bacterioscopic exam (GRAM). The collection of material was then compiled in a cytopathologic smear analysis. All women signed the free and informed consent letter and the project was approved by the Ethics Research Board of Hospital São Paulo - UNIFESP. RESULTS: Bacterioscopy and culture proved to be better than the cytopathologic exam in featuring the bacilli and cocci. The bacterioscopy provided a better detection of the presence of bacilli (p < 0.001); no statistical difference was seen between both exams with respect to the detected cocci. The beta-hemolytic Streptococcus group was of significance in post-menopausal women (p < 0.05). CONCLUSION: In this study, the bacterioscopic and culture exams of the vaginal fluid were more effective in assessing the vaginal flora and in the detection of bacilli, compared to the cytopathological exam.

Depth of glandular crypts and its involvement in squamous intraepithelial cervical neoplasia submitted to large loop excision of transformation zone (LLETZ).

BACKGROUND: The authors aimed to confirm the depth of six mm in order to achieve an optimal eradication of the lesion. MATERIALS AND METHODS: This is a retrospective observational study of 94 cervical surgical pieces from women aged 17 to 22 years with a cyto-colpo-histopathological diagnosis of high-grade squamous cervical intraepithelial neoplasia (CIN II and/or CIN III) submitted to large loop excision of transformation zone (LLETZ). The glandular crypts and margins, both exposed or not to CIN, were assessed. The compromise and the maximum depth of the glandular crypts were noticed. RESULTS: After LLETZ, 23 (24.47%) cases presented a neoplasic impairment of endocervical margin and ten (10.64%) of the ectocervical margin. The largest noticed crypt measured 4.500 mm and the shortest 0.100 mm, with an average of 2.148 mm. CONCLUSIONS: Squamous CIN more frequently show the exposure of surgical margins to LLETZ. The deeper location of glandular crypts in the cases studied was 4.500 mm, while the largest neoplastic extension was 3.000 mm.The therapeutic method depends on this knowledge.

Efficacy of vaginal use of topical estriol in postmenopausal women with urogenital atrophy.

OBJECTIVE: This study evaluates the effect of intravaginal estriol on urogenital atrophy, Pap smear parameters, colposcopy parameters and discomfort during gynecological examination. METHODS: 31 postmenopausal women who had not used hormone therapy in the previous six months were studied. All women used intravaginal estriol, 1 mg/day for 21 days. The following variables were analyzed before and after treatment: complaints of urogenital atrophy; cytological parameters, colposcopic parameters, and subjective evaluation of discomfort during gynecologic examination. RESULTS: All urogenital atrophy complaints improved after treatment. At the first visit, 45.2% of women presented a predominance of profound cells, 51.6% with predominance of intermediate cells, and 3.2% with predominance of superficial cells. At the second visit, these rates were 35.5%, 64.5%, and 0%, respectively. Evaluation of the maturation index showed that 83.9% of women had atrophic Pap smears, and 16.1% showed good estrogenic level before treatment. At the second visit, atrophic smears occurred in 12.9%, and 87.1% of women exhibited good estrogenic level (chi2 = 20.045; p = 0.000). Colposcopy showed that 71% of women had atrophic colpitis and/or petequiae before treatment, while atrophy after therapy was present in only 6.4%. The evaluation of other colposcopic parameters also improved after treatment. Great discomfort was reported by 19.4% before and by 0% after treatment. CONCLUSION: Intravaginal estriol 1 mg/day for a period of 21 days was efficient in improving urogenital atrophy, Pap smear parameters and colposcopic evaluation in postmenopausal women.

Immunohistochemical expression of granzyme B and vascular endothelial growth factor (VEGF) in normal uterine cervices and low and high grade squamous intraepithelial lesions.

PURPOSE: This study aimed to evaluate the immunoexpression of granzyme B and vascular endothelial growth factor (VEGF) in the variants of cervical squamous intraepithelial neoplasia. METHODS: Granzyme B immunohistochemical expression was studied in the epithelium, stroma and in both the epithelium + stroma of 142 fragments of uterine cervix; there were 34 grade 1 cervical intraepithelial neoplasias (CIN 1), 36 grade 2 cervical intraepithelial neoplasias (CIN 2), 33 grade 3 cervical intraepithelial neoplasias (CIN 3) and 39 uterine cervix fragments without abnormalities - control group. Immunoexpression of VEGF was studied in 160 uterine cervix fragments, with 43 grade 1 cervical intraepithelial neoplasias (CIN 1), 33 grade 2 cervical intraepithelial neoplasias (CIN 2), 31 grade 3 cervical intraepithelial neoplasias (CIN 3) and 53 uterine cervix fragments without abnormalities--control group. RESULTS: In the stroma, immunoexpression of granzyme B in grade 1 cervical intraepithelial neoplasias was smaller than in grade 3 cervical intraepithelial neoplasias. High VEGF immunoexpression was found in grade 3 cervical intraepithelial neoplasias while it was low in grade 1 cervical intraepithelial neoplasias and in the control group. CONCLUSION: The higher the severity of the cervical intraepithelial lesion, the higher the immunoexpression of granzyme B. A progressive increase in VEGF immunoexpression was found in the intense grade, according to the severity of the cervical intraepithelial neoplasia.

Cytopathological screening in indigenous women from Parque Indigena do Xingu.

Over the years, there have been many deaths due to cervical cancer among indigenous women of the Parque Indigena do Xingu as a consequence of low screening coverage. Since 2005, however, the coverage index of cervical lesion screening has been high: 97.6% among at-risk women in 2005 and 92.6% in 2007. Cytological alterations occurred in 12.6% and 6% of the cases in the respective years. After complete diagnosis and treatment of uterine lesions, by staff trained in lower tract pathology, negative results were seen in all cases of high-grade lesions and invasive neoplasia and no case of invasive carcinoma was detected in 2007. We conclude, therefore, that health actions have been effective in decreasing the incidence of cytological alterations and invasive carcinoma.

Uterine cervical neoplasia prevention in Parque Indigena do Xingu.

Results of preventive health measures, diagnosis and treatment applied to Parque Indigena do Xingu native women were studied. Thirty-seven cases of uterine cervical intraepithelial lesions and invasive neoplasias were treated in the local villages without referral to an advanced medical center. LEEPs were carried out in 32 women, three cold knife conizations, one vaginal hysterectomy and one Wertheim Meigs procedure. Results of 53.1% of LEEP surgical procedures did not have margin involvement by the lesions. Bleeding complications were seen in 15.6%. Regular follow-up with two or three cytologic and colposcopic tests in 32 women was carried out. All cases were negative for lesions. Five women were not followed-up due mainly to logistical reasons. Health endeavors adopted in the period 2005-2007 brought about a significant reduction of precursor lesions in this native aboriginal population without screening resources.

Fas/CD95 promoter polymorphism gene and its relationship with cervical carcinoma.

OBJECTIVE: Apoptosis is an important fail-safe control in human papillomavirus (HPV)-associated carcinogenesis. We tested the hypothesis that the A/G polymorphism at -670 of Fas promoter is associated with an increased risk for cervical cancer, using a matched case-control setting. METHODS: The material in this case-control study consisted of 91 patients with cervical carcinoma and 176 population-based control subjects, recruited between 2002 and 2004; all the ethnic Brazilian women had histologically confirmed cervical carcinoma. Control subjects were age-matched; healthy women who were selected following a negative cervical cytology and normal colposcopy. Fas genotyping was performed using a PCR-RFLP technique. RESULTS: No significant difference existed in the distribution of the Fas polymorphisms (wild, heterozygous, mutant) between the cases and controls. The heterozygous (OR: 4.85, 95% CI: 1.1-22.6) genotypes among the younger (< 48 yrs) cancer patients were almost 5-fold increased, as compared with the wild type. No such increase was observed among the patients older than 48 years. CONCLUSIONS: Our data suggest that 670A/G polymorphism in the promoter region of the death receptor Fas is associated with an increased risk of cervical cancer among Brazilian women under 48 years. The mechanisms would be the inhibition of apoptosis by Fas -670G allele-mediated down-regulation of Fas transcription.

Prognostic markers of low-grade squamous intraepithelial lesions: the role of topoisomerase IIalpha and active caspase-3.

PURPOSE: To study the relationship between topoisomerase IIalpha, active caspase-3 expressions and HPV DNA in uterine cervices with low-grade squamous intraepithelial lesions (LSIL). METHODS: Forty women with LSIL and 32 without cervical neoplasia diagnosed through cytologic and histopathologic examination were evaluated regarding topoisomerase IIalpha and active caspase-3 expressions and HPV DNA detection using PCR (GP5/GP6) in cervicovaginal smears. RESULTS: The mean percentage of cells immunomarked by topoisomerase in the group with LSIL was 11.62% while in the control it was 4.13% (p < 0.0001). In the presence of HPV DNA, topoisomerase expression was higher in the group with productive viral infection than in nonneoplastic tissue (p = 0.004). Caspase-3 expression was observed in 17 patients with LSIL (42.5%) and in five without cervical neoplasia (15.63%). CONCLUSION: The use of topoisomerase IIalpha and active caspase-3 in cervical biopsies may help to define the prognosis of HPV cervical infection.

Polymorphisms of p53, GSTM1 and GSTT1, and HPV in uterine cervix adenocarcinoma.

OBJECTIVE: To analyze the participation of glutathione-S-transferase (GST) M1 and T1 polymorphisms associated or not with protein p53 polymorphism at codon 72 and in the presence of HPV in the carcinogenesis of uterine cervix adenocarcinoma. METHODS: Forty-three samples of uterine cervix adenocarcinoma were studied and 86 samples of endocervical cells of women without tumors formed the control group. The presence of HPV was determined in order to genotype the isoforms of p53 at codon 72, GSTM1, GSTM1*0, GSTT1 and GSTT1*0 which were evaluated by the PCR method. RESULTS: HPV was present in 97.67% of the adenocarcinoma cases and in 31.40% of the control group. Statistical analysis showed differences (p = 0.001) and an OR of 113.3 (CI 95%: 13.67-947.14). GSTT1 and GSTT1*0 analysis showed a significant difference between the groups (p = 0.001) with an OR of 4.58 (CI 95%: 2.041-10.28) (p < 0.001) for the presence of GSTT1*0. When it was associated with HPV OR was 6.6 (CI 95%: 0.04-0.50). Analyses of p53 and GSTM1 and GSTM1*0 either alone or associated with HPV were not significant. CONCLUSION: The presence of GSTT1*0 increased the risk for uterine cervix adenocarcinoma development while the allele GSTT1 had a protective action. The other isoforms did not appear to participate in the carcinogenesis of uterine cervix adenocarcinoma.

Carbon dioxide laser treatment of Bartholin's gland cyst.

The purpose of this study is to present the results of laser surgery for Bartholin's cyst which shows to be a method that is safe, efficient, of low morbidity, and can be performed in an outpatient clinic under local anesthesia. Of the 22 cases operated on, only two had relapses which were resolved using a second procedure. The average surgery time was short, without bleeding during the operation. Complete epithelization was achieved within a maximum 4-week span with minimal or no postoperative discomfort.

Grade 3 vulvar and anal intraepithelial neoplasia in a HIV seropositive child--therapeutic result: case report.

A case report of a HIV seropositive 8-year-old child with vulvar and anal border neoplasia, both grade 3, and the adopted therapeutic management are presented. The mother reported the history of a progressively growing verrucous lesion in the vulva since the age of three and a half years. On physical examination a pigmented and elevated lesion was observed in the whole vulvar region extending to the anal region and intergluteal sulcus. After biopsies and anatomic pathological examination, antiretroviral therapy, adequate for age, and topical application of podophyllotoxin associated with Thuya officinalis extract was started. Three months afterwards vaporization and CO2 laser excision were performed in five sequential sessions, thereafter associated with topical imiquimod application. After the first two sessions of laser therapy early relapses occurred. After four weeks of imiquimod use, already a significant improvement of the lesions was observed, making the following laser therapy sessions easier. We conclude that antiretroviral therapy associated with podophyllotoxin and Thuya was not effective regarding regression of the lesions. Laser therapy alone led to early relapses. The local use of imiquimod associated with laser was effective in decreasing and controling the lesions.

Granzyme B as a prognostic marker of cervical intraepithelial neoplasia.

Granzyme B (GrB) is a serine protease synthesized in T lympocytes (CTL), released after T-cell activation resulting from exogenous stimulation. With perforin, GrB discharges apoptotic signals to a target cell and therefore constitutes a marker to identify activated CTL. We aimed to quantify GrB expression by immunohistochemistry staining in 12 tissue fragments of cervical carcinoma, 33 cervical intraepithelial neoplasias treated by LLTEZ and nine cervical pieces without disease. Activated cytotoxic lymphocyte mean values (20 HPF-400x) in both epithelial and stromal pars were 7.11 cells in tissue without neoplasia, 33.45 cells in cervical intraepithelial neoplasia and 139.75 cells in carcinoma samples, with a statistical difference between them. Comparative analysis in the CIN group showed an expressive difference between cases with disease recurrence (19.28 cells) and without recurrence (37.26 cells). Thus, the relation between number of activated CTLs found at the moment of treatment and clinical evolution determined in this study, suggest GrB use as a prognostic marker.

Relationship between angiogenesis and grade of histologic differentiation in endometrial adenocarcinoma.

The objective of the study was to quantify vessels and to relate them to the degree of histologic differentiation in endometrial adenocarcinoma. We studied 35 cases of which ten were G1, 13 G2 and 12 G3 adenocarcinomas. The control group consisted of 11 atrophic and 10 proliferative endometria. From each case two histologic sections were obtained: one for hematoxylin-eosin staining and the other for immunohistochemical study with anti-CD34. Vessel count was performed by morphometric study. Mean vessel count was 15.3 for G1; 19 for G2 and 22.7 for G3 adenocarcinomas; in the control group it was 11.6 for atrophic and 13.2 for proliferative endometria. Slightly differentiated adenocarcinoma presented greater angiogenesis than normal and well-differentiated carcinoma. In contrast, moderately differentiated carcinoma showed greater angiogenicity as related to normal endometrium, but did not differ from other tumoral endometria.

Angiogenesis in squamous intraepithelial neoplasia of the uterine cervix in HIV-seropositive women.

OBJECTIVES: This study aimed to quantify angiogenesis in squamous intraepithelial lesions of the uterine cervix in seropositive HIV patients as well as to establish a relationship between vascular density and variations in the CD4+ lymphocyte titer and the viral load of human immunodeficiency virus (HIV). METHODS: 125 patients, 55 HIV seropositive and 70 seronegative, were allocated with respect to grade of squamous intraepithelial lesion (SIL). The obtained samples were stained with an immunohistochemical marker for CD34 antigen and vessel counts were performed in ten consecutive fields at 400x magnification. The seropositive HIV patients were distributed into groups according to the CD4+ index and HIV viral load. RESULTS: Seropositive HIV patients presented a higher mean vascular density (MVD) than the control group, even in the absence of cervical intraepithelial lesions. High- and low-grade lesions in the presence of HIV seropositivity presented higher MVD than that found in seronegative HIV patients. There was no significant variation in the MVD and CD4+ count ratio or viral RNA-HIV load, except for high-grade (H)SIL. CONCLUSIONS: Infection with HIV influenced angiogenesis of uterine cervix in the presence of squamous intraepithelial lesions and more significantly in HSIL.

Low-dose 5-fluorouracil adjuvant in laser therapy for HPV lesions in immunosuppressed patients and cases of difficult control.

The authors established a protocol for the use of 5-fluorouracil (5FU) adjuvant in lasertherapy for clinical and subclinical HPV infection in immunosuppressed patients, persistent lesions and as reinforcement treatment in cases of poor progress. Sixty-four patients were evaluated, of whom 26 were immunosuppressed, 34 presented persistent lesions and four received intravaginal reinforcement treatment with 2.5 g 5% 5FU every two weeks, or biweekly vulvar reinforcement after lasertherapy. On average, five 5FU courses were used, but in the immunossuppressed patients its use was maintained indefinitely. The rate of complete response was 66%, but the immunossuppressed patients showed less response (46.2%) when compared with the persistent lesion/reinforcement treatment group (78.9%). The responses were positive in the two groups when compared to that with no response. We deem the use of low-dose 5FU an excellent alternative in cases of difficult HPV progress, presenting a low cost and minimal side-effects.

Evaluation of Langerhans' cells in human papillomavirus-associated squamous intraepithelial lesions of the uterine cervix.

Current research has been evaluating morphological modifications and density of Langerhans' cells in women with histopathological HPV lesions of the uterine cervix. Fourteen women with subclinical HPV infections underwent clinical, colpocytological colposcopy examinations, and paired biopsies of the uterine cervix. Histopathological, HPV hybrid capture and S-100 immunohistochemical examinations were performed in biopsy specimens. Groupings of viral lesions and normal tissue were analyzed by the Wilcoxon rank-sum test. Langerhans' cells in the specimens were frequently located in the intermediate and basal layers of the epithelium. A significant reduction of cytoplasmic profiles occurred in viral lesions (144.08 profiles/mm2) when compared to normal tissue (256.27 profiles/mm2) of the epithelium. An inverse modulation occurred in the cytoplasmatic profiles/nuclei ratio with 2.80 in viral lesions and 4.89 in normal tissue of the stroma. A local immunodeficiency based on cytoplasmic changes of Langerhans' cells has been postulated as a mechanism by which HPV could be involved in the genesis of neoplasia.

P27 expression in normal epithelium and condylomas of the vulva in HIV positive and negative women.

PURPOSE: The objective of the present study was to evaluate the expression of the p27 protein in the normal epithelium and vulvar condylomas in human immunodeficiency (HIV) positive and negative patients. METHODS: Eight samples of normal vulvar epithelium were evaluated (Group A), ten of the HIV negative vulvar condyloma patients (Group B) and another eight of the vulvar condyloma HIV positive patients (Group C). The DNA of human papillomavirus (HPV) was identified by means of polymerase chain reaction (PCR). Immunohistochemistry was the method used to evaluate the expression of p27 using monoclonal mouse antibody (Monoclonal Mouse, anti-human p27, Clone Sx 53 G8). The immunoexpression was evaluated at a magnification of 400x, counting a minimum of 1,000 cells per slide. RESULTS: The results obtained were the following: a) comparing groups A and B and groups A and C there was a significant difference in relation to the expression of the p27 protein which was 63.32% in group A and only 13.35% and 18.89% in groups B and C, respectively; b) comparing groups B and C among them, there was no significant difference. CONCLUSION: We concluded that in normal vulvar tissue the p27 protein is present in a large number of cells and that in vulval condylomas its expression is very much lowered both in HIV positive and negative cases.