Analysis of human kallikrein 7 expression as a potential biomarker in cervical neoplasia.

Overexpression of kallikrein 7, a proteolytic enzyme important for epithelial cell shedding, may be causally involved in carcinogenesis, particularly in tumor metastasis and invasion. In this study, we have evaluated hK7 (human kallikrein 7) protein levels by immunohistochemistry in 367 cervical histological samples including 35 cases of cervicitis, 31 low-grade cervical intraepithelial neoplasia, 51 high-grade cervical intraepithelial neoplasia (H-SIL), 197 squamous cervical carcinomas (SCC) and 53 cervical adenocarcinomas. We have observed that hK7 staining increased with the severity of cervical disease. Intense hK7 staining was found in 15.2% of cervicitis samples, in contrast to 55% of H-SIL and 68% of SCC. Moreover, 92.5% of adenocarcinomas also exhibited intense hK7 staining. Differences in the expression of hK7 could potentially be used as a biomarker for the characterization of different stages of cervical disease.

Monocarboxylate transporters 1 and 4 are associated with CD147 in cervical carcinoma.

Due to the highly glycolytic metabolism of solid tumours, there is an increased acid production, however, cells are able to maintain physiological pH through plasma membrane efflux of the accumulating protons. Acid efflux through MCTs (monocarboxylate transporters) constitutes one of the most important mechanisms involved in tumour intracellular pH maintenance. Still, the molecular mechanisms underlying the regulation of these proteins are not fully understood. We aimed to evaluate the association between CD147 (MCT1 and MCT4 chaperone) and MCT expression in cervical cancer lesions and the clinico-pathological significance of CD147 expression, alone and in combination with MCTs. The series included 83 biopsy samples of precursor lesions and surgical specimens of 126 invasive carcinomas. Analysis of CD147 expression was performed by immunohistochemistry. CD147 expression was higher in squamous and adenocarcinoma tissues than in the non-neoplastic counterparts and, importantly, both MCT1 and MCT4 were more frequently expressed in CD147 positive cases. Additionally, co-expression of CD147 with MCT1 was associated with lymph-node and/or distant metastases in adenocarcinomas. Our results show a close association between CD147 and MCT1 and MCT4 expressions in human cervical cancer and provided evidence for a prognostic value of CD147 and MCT1 co-expression.

Increasing expression of monocarboxylate transporters 1 and 4 along progression to invasive cervical carcinoma.

Solid tumor cells are known to be highly glycolytic and, to prevent apoptosis by cellular acidosis, cells increase proton efflux through pH regulators, such as monocarboxylate transporters (MCTs). However, the role of these membrane proteins in solid tumor development and survival is not fully understood. We aimed to evaluate the expression of the MCT isoforms 1, 2, and 4 in a large series of cervical lesions (neoplastic and non-neoplastic) and assess its clinical-pathologic significance. The series analyzed included 29 chronic cervicitis, 30 low-grade squamous intraepithelial lesions, 32 high-grade squamous intraepithelial lesions, 49 squamous cell carcinomas, 51 adenocarcinomas, and 30 adenosquamous carcinomas of the uterine cervix. Analysis of the expression of MCT isoforms 1, 2, and 4 was performed by immunohistochemistry with specific antibodies. Immunoreactions were evaluated both qualitatively and semiquantitatively. We found a significant increase in MCT expression from preinvasive to invasive squamous lesions and from normal glandular epithelium to adenocarcinomas. This is the first study evaluating the significance of MCT expression in lesions of the uterine cervix, including invasive carcinomas, and the results found herein led us to believe that these membrane proteins are involved in the progression to invasiveness in uterine cervix carcinoma.

Pero Vaz de Caminha: an-interchange program for quality control between Brazil and Portugal.

OBJECTIVE: To start an interexchange program for quality control in cervical cytology and discuss conceptual criteria for diagnosis. STUDY DESIGN: Slides were selected in the archives of the 2 institutes and included cases with unsatisfactory, negative and positive results. Sets of slides were changed between the partners every 3 months. At the end of each year a senior cytopathologist was invited to discuss the major discrepancies found in the study. RESULTS: A total of 1,041 cases were analyzed. Full concordance was obtained in 74.4% (774) of cases and discrepancies in 25.6% (267 cases). Full agreement was achieved in 276 (39%) of 707 cases categorized as negative. In 421 negative cases from laboratory A, this concordance represents 65.5% and 96.5% for laboratory B, which submitted 286 negative cases. The main discordance was the high number of atypical squamous cells of undetermined significance cases: 3.1% for A and 128 (33.2%) for B. Samples with discrepancies related to the quality of the material was another controversial issue: of 16 cases from laboratory A, 6 (37.5%) unsatisfactory cases were the same and 10 (62.5%) different. Laboratory B presented 20 unsatisfactory cases, and 14 (70.0%) had other diagnoses. Low grade squamous intraepithelial lesion and high grade squamous intraepithelial lesion concordance ranged from 75% to 80%, and invasive carcinoma has 4 discordances (28.5%), 3 previously screened as high grade squamous intraepithelial lesion and 1 as atypical squamous cells of undetermined significance. The kappa value obtained was 0.65, indicating substantial agreement. CONCLUSION: Our results indicated that atypical squamous cells of undetermined significance diagnoses are the crucial point of controversies and concern the quality of routine diagnosis in cytopathology.

100% rapid rescreening for quality assurance in a quality control program in a public health cytologic laboratory.

OBJECTIVE: To verij5 the efficacy of the quality control (QC) program in a cytologic laboratwy with a rapid rescreening (RR) protocol. STUDY DESIGN: RR, according to the Turret RR method, of all samples initially screened as negative at the Laboratory of Cytology, Adolfo Lutz Institute, was performed. The slides were reviewed for 60 seconds. Suspect smears were fully checked by 2 reviewers to determine the final diagnoses. A total of 2954 sequential cytologic results were considered in this study. Of the 2954, 2568 (86.9%) were considered initially negative according to our internal QC, and these cases underwent RR. Also, 10% were randomly selected from these negative cases for full reviewing. The internal QC in our laboratory includes review of cases selected according to clinical and cytomorphologic criteria. RESULTS: Among the 2954 total cases, QC detected 386 (13%) atypias with final diagnoses reported according to The Bethesda System 2001 as follows: 82 (2.18%) low grade squamous intraepithelial lesions (LSILs), 35 (1.18%) high grade squamous intraepithelial lesions (HSILs), 2 (0.06%) squamous cell carcinomas, 105 (3.5%) atypical cells of undetermined significance (ASC-US), 4 (0.12%) atypical endocervical cells (AECs) and 158 (5.3%) unsatisfactory samples. RR of 2568 smears initially considered negative selected 194 (7.5%) slides. Of the 194, 146 (75.3%) were negative, 28 (14.4%) ASC-US, 5 (2.6%) AEC, 1 (0.5%) LSIL and 14 (7.2%) unsatisfactory. Full review of a 10% random fraction of the 2568 cases interpreted as negative did not detect lesions but did detect 5 (1.95%) unsatisfactory samples. CONCLUSION: Internal QC used in our laboratory based on clinical and cytomorphologic criteria to select cases for review proved to be an efficient method of detecting HSIL and cervical cancer. The consensus basis of this program strongly limits the false positive and false negative rates and also provides subjects with continuing education. One hundred percent RR is more efficient than 10% full reviewing in detecting cervical abnormalities.

Pap test in a high-risk population comparison of conventional and liquid-base cytology.

Liquid-based cytology (LBC) is believed to have better sensitivity than conventional smears (CSs) and offers the possibility to perform molecular assay. The goal of this work was to study the performance of CS and LBC in a high-risk population and to compare the results with the hybrid capture (HC) II for high-risk human papillomavirus (HPV). Samples were collected from selected women with clinical suspicions of low genital tract lesion at Pérola Biygnton Hospital (São Paulo, Brazil). After CS preparation, the brush was introduced in the endocervix and a new sample was collected and rinsed in the preservative medium of the system. The residual material was used to HC2. From 925 cases, LBC was unsatisfactory in 4 (1.51%) cases and CS was unsatisfactory in 100 cases (10.81%); among theses cases HC2(+) reactions was observed in 54 (54%) CSs and 3 (21.4%) LBCs. Considering cases with atypia of undetermined significance (squamous and glandular), 85 (77.27%) cases from LBC and 44 (86.4%) from CS were positive for HC2 assay for high-risk DNA-HPV. The difference among the methods was not significant (P < 0.38). The diagnosis improvement of LBC in comparison with CS was 86% in satisfactory samples, 92.76% in undetermined atypical lesions (including glandular), 83% in positive low-grade squamous intraepithelial lesions (LSIL(+)), and 86.84% in high-grade SIL(+) (HSIL(+)). HC2(+) reactions were observed in 144 CS cases and 266 LBC cases with abnormalities. Our results have showed that LBC was superior to CS in a high-risk population to detect lesions with high concordance with HC2(+) reactions; CSs also exhibit a high concordance with HC2 assay but with inferior performance to detect lesions.

Hybrid capture II and polymerase chain reaction for identifying HPV infections in samples collected in a new collection medium: a comparison.

OBJECTIVE: To compare the performance of human papillomavirus (HPV) DNA detection by polymerase chain reaction (PCR) and Hybrid Capture II (HCII) test (Digene, Gaithersburg, Maryland, U.S.A.) in residual cells left in the collection vials of the DNACitoliq system (Digene Brasil, São Paulo, Brazil). STUDY DESIGN: A series of 263 cervical samples collected for liquid-based cytology with the DNACitoliq system was tested for oncogenic HPV types first with HCII and subsequently with PCR. After DNA purification with GFX Genomic Blood DNA Purification Kit (Amersham, Piscataway, New Jersey, U.S.A.), PCR was performed using AmpliTaq Gold DNA polymerase (Applied Biosystems). PGMY09/11 L1 consensus primers and GH20/PCO4 primers for human beta-globin target were coamplified. RESULTS: Altogether, 260 samples were positive for beta-globin, and 3 negative ones were excluded from the analysis. PCR and HCII yielded concordant results in 199 cases (76.5%) (102 positive and 97 negative), with Cohen's kappa of .577 (95% CI .477-.677) and weighted kappa of .733 (95% CI .659-.791). HPV prevalence in different categories of cytologic abnormalities was practically identical with HCII and PCR assays (P=.989). Among the 61 (23.5%) discrepant cases, 28 samples were HCII+/PCR- cases. Of these, 27 of 28 samples showed a low viral load, and 1 had an intermediate viral load. CONCLUSION: The data suggest that residual material from the DNACitoliq system adequately preserves HPV DNA for detection by HCII and PCR, with performance similar to that of specimen transport medium.

[Critical evaluation of the diagnostic nomenclatures of cervical cytopathological exams used in the Brazilian Unified Health System (SUS)]. / Avaliação crítica das nomenclaturas diagnósticas dos exames citopatológicos cervicais utilizadas no Sistema Único de Saúde (SUS).

PURPOSE: to identify the nomenclature for reporting cervical cytological diagnoses used by laboratories which render services to the Brazilian Unified Health System (SUS) and which participate in External Quality Monitoring (MEQ). To evaluate the information acquired from gynecologists of the SUS regarding the various diagnostic classifications that they receive in the cervical cytology diagnostic reports. METHODS: we evaluated 94 cytology reports issued by laboratories which participate in the MEQ in the State of São Paulo, Brazil, and 126 questionnaires applied to gynecologists who work for the SUS. RESULTS: out of the 94 laboratories, 81 (86.2%) use one diagnostic classification: 79 (97.6%) use the Brazilian Nomenclature for Cytological Reports (NBLC), 1 (1.2%) uses the Papanicolaou classification and 1 (1.2%) uses the Richart diagnostic classification. Of the 13 (13.8%) laboratories that use more than one classification, 5 use 2 types and 8 use 3 to 4 types, with 9 including the Papanicolau diagnostic classification. The study showed that 52 (55.3%) laboratories presented more than one descriptive diagnosis in the same report. Out of the 126 gynecologists who filled out a questionnaire evaluating the cytopathology reports, 78 (61.9 %) stated that they received laboratory reports with only one diagnostic classification, 48 (38.1%) received reports with more than one classification and 2 received reports with all 4 classifications. Among the 93 (73.8%) gynecologists who prefer only one classification, 56 (60.2%) claimed that the NBLC contributes to clinical practice, 13 (14.0%) opted for the Richart classification, 8 (8.6%) for the Reagan classification and 16 (17.2%) for the Papanicolaou classification. Out of 33 (26.2%) gynecologists who prefer more than one classification, 5 opted for the 4 classifications. CONCLUSIONS: these data suggest that there is still resistance on the part of pathologists about using the official nomenclature in cytology reports for SUS. There is discrepancy between the information that gynecologists would like to see in the reports and the information provided by the pathologists. Greater efforts should be made to stimulate the use of the official nomenclature.

Simultaneous Chlamydia trachomatis and HPV infection in pregnant women.

Pregnancy is associated with HPV infection and with Chlamydia trachomatis (CT) infection mostly due to the natural immunosuppression. In addition, pregnancy associated to CT infection can lead to adverse conditions to the woman and fetus, and CT is also believed to be a co-factor in human immunodeficiency virus infection and HPV-induced cervical cancer. The aim of this study was to establish the odds ratios (OR) of CT infection in to HPV-infected pregnant women and vice versa of women stratified by age (<25 years) and marital status. This work is part of a national multicentric transversal study carried out in six Brazilian cities supported by the Ministry of Health of Federal Government of Brazil in 2003. Cervical scrapes of 371 pregnant women were sampled. We performed a hybrid capture-2 technique to diagnose these samples on HPV and CT infection, and the women responded a questionnaire. Significant association was observed between nonstable marital status and hr-HPV infection [OR = 2.61 (1.38-4.97) P = 0.003)], and age <25 years old [OR = 2.26 (1.09-4.71) P = 0.029]. Nonstable marital status was also associated with lr-HPV infection [OR = 2.67 (1.59-4.50) P < 0.001), and age <25 years old [OR = 2.55 (1.51-4.32) P < 0.001). Fifty of the 371 pregnant women were infected with hr-HPV (13.5%) and 111 (30.0%) were infected with lr-HPV. The coinfections of HPV and CT were found in 31 women, that is, 8.36% of the pregnant women (P < 0.001). The high rate of simultaneous CT and HPV infection in pregnant women favors the recommendation to screen pregnant women for both CT and HPV.

Avaliação do desempenho da mistura verniz/xilol na diafanização de lâminas de citopatologia coradas com a técnica de Papanicolaou / Valuation of performance of solution varnish/xylene in the diaphanization of citopatology slides stained with Papanicolaou's technique

Introdução: O xilol é necessário para a diafanização de amostras citopatológicas. Pode trazer problemas de ordem ocupacional, proteção ao meio ambiente e de custos. Objetivo:Avaliar o desempenho de várias concentrações da mistura verniz/xilol em relação à diafanização e conservação em amostras de citologia coradas com a técnica dePapanicolaou (CP). Material e métodos: Foram avaliadas 75 lâminas de raspado bucal (RB) e 8.773 esfregaços cérvico-vaginais (CV). As lâminas foram coradas pela CP, com a retirada das etapas de álcool/xilol e xilol e foram secas à temperatura ambiente porvinte minutos antes da montagem com lamínula. A montagem foi realizada com soluções de verniz/xilol de concentrações 75/25%, 70/30%, 60/40%, 50/50% e 40/60%. As lâminas foram distribuídas de forma aleatória aos profissionais para avaliação da técnica, sendo conceituadas como boas, regulares ou ruins e reavaliadas após nove meses dearquivamento. A concentração de verniz/xilol que apresentou melhor qualidade final nas lâminas de RB foi aplicada também nos CV. Resultados: A concentração de 75/25% deverniz/xilol apresentou melhor desempenho para as duas situações, em RB e CV. Após nove meses, a qualidade foi mantida. Conclusão: A concentração de 75/25% apresentou melhor resultado.

Lymphatic vessel density and epithelial D2-40 immunoreactivity in pre-invasive and invasive lesions of the uterine cervix.

OBJECTIVE: We sought to determine the significance of lymphatic vessel density (LVD) in pre-malignant lesions and carcinomas of the uterine cervix and to evaluate the prognostic value of lymphatic invasion and D2-40 positivity in tumor cells in the three histological types of invasive lesions. The correlation of LVD, lymphatic invasion and D2-40 positivity in tumor cells with EGFR and COX-2 expressions was also evaluated. METHODS: We studied 50 cervicitis, 50 low-grade squamous intraepithelial lesions (LSIL) (CIN1), 51 high-grade squamous intraepithelial lesions (HSIL) (CIN2/CIN3), 49 invasive squamous cells carcinomas (SCC), 43 adenocarcinomas (AC) and 30 adenosquamous cells carcinomas (ASC). The immunoreaction assay was performed using the monoclonal antibody D2-40. RESULTS: Significant differences in LVD were found among all categories of pre-invasive and invasive lesions (p=0.001 and p<0.001, respectively). LVD in invasive lesions was significantly greater than in pre-invasive lesions (p<0.001) and no significant association was found between LVD in invasive lesions and both lymph node invasion and/or metastasis. D2-40 positivity in tumor cells was associated with a better prognosis in ASC cases. EGFR and COX-2 expressions in invasive lesions were not associated with LVD; however, they correlated with both lymphatic invasion and D2-40 positivity in tumor cells. CONCLUSIONS: Lymphatic neovascularization begins early in intraepithelial lesions and continues to increase towards malignancy. Both lymphatic invasion and decrease in D2-40 expression in tumor cells appear to have a prognostic value.

Análise das discordâncias diagnósticas dos exames citopatológicos do Programa de Monitoramento Externo de Qualidade no Estado de São Paulo, Brasil, 2000-2010 / Analysis of the diagnostic disagreements in cytopathologic exams from the External Quality Assurance Program in the State of São Paulo, Brazil, 2000-2010

Introdução: O Programa de Monitoramento Externo de Qualidade (MEQ) dos exames citopatológicos cervicovaginais realizados pelo Sistema Único de Saúde (SUS ) foi proposto pelo Ministério da Saúde (MS) para melhorar o desempenho dos laboratórios de citologia da Rede Pública de Saúde. Objetivo: Avaliar as discordâncias diagnósticas dos exames citopatológicos cervicovaginais submetidos à revisão pelo Programa de MEQ do Estado de São Paulo, no período de 2000 a 2010. Método: Avaliação comparativa retrospectiva dos diagnósticos emitidos pelos lab-SUS e dos diagnósticos de revisão dos exames citopatológicos cervicovaginais. Resultados: Das 123.002 amostras revisadas,16.581 (13,48%) apresentaram discordância diagnóstica, sendo que, em 14.313 (11,64%) casos, houve divergência entre a conduta adotada e a preconizada pelo Instituto Nacional de Câncer José Alencar Gomes da Silva (INCA). Foram considerados exames falso-positivos 2.530 (2,06%) e falso-negativos 2.154 (1,75%). A sensibilidade foi de 92,02% e especificidade de 96,49%. Houve concordância boa entre os diagnósticos originais e de revisão (kappa=0,77).Conclusão: Além do conjunto de atividades básicas propostas pelo MS, o MEQ do Estado de São Paulo destaca asações educativas implementadas, adotadas para promover a educação continuada no sentido da uniformização de critérios citomorfológicos e a consequente redução dos resultados falso-negativos e falso-positivos

The association of p16(INK4A) and fragile histidine triad gene expression and cervical lesions.

OBJECTIVE: This cross-sectional study was intended to assess the association between immunohistochemical analysis of p16 and fragile histidine triad (FHIT) and the presence of precancerous cervical lesions. MATERIALS AND METHODS: Women seen at Pérola Byington Hospital, São Paulo, Brazil, with histologically confirmed cervicitis (n = 31), cervical intraepithelial neoplasia (CIN) 1 (n = 30), CIN 2,3 (n = 30), and cervical cancer (n = 7) had also cervical material collected for liquid-based cytology, human papillomavirus Hybrid Capture 2 (HC2) test, and p16 and FHIT immunohistochemical reactions. RESULTS: p16 and FHIT reactions were scored as the following: <1%, 1% to 5%, >5% to 25%, and >25%. Receiver operating curve analysis was used to select p16 and FHIT score cutoffs for further categorical analyses. All but one of the 37 CIN 2,3/cancer cases had a p16 score of greater than 1% to 5%. Among the 61 cervicitis/CIN 1 cases, 46 (75%) had a p16 score lower than 1% to 5%. In contrast, no association of FHIT expression and severity of cervical lesions could be demonstrated in this data set. Receiver operating curve analyses suggested the score of 1% to 5% for p16 as the cutoff that best discriminates CIN 2,3/cancer from cervicitis/CIN 1. No cutoff for FHIT scores could be suggested with data set. CONCLUSIONS: p16, but not FHIT expression, has the potential to be used as complementary diagnostic tool to investigate human papillomavirus-induced cervical lesions, if these results are confirmed in larger studies.

Associação multivariada entre alterações citológicas do colo uterino e comportamento de risco em mulheres profissionais do sexo / Multivariate association between the cervix cytological abnormalities and the risk behavior female sex workers

Neste trabalho foram identificadas as associações multivariadas entre as alterações citológicas (AC) do colo uterino e o comportamento de risco das profissionais do sexo (PS) atendidas no Centro de Referência em Saúde Sexual e Reprodutiva (CRESSER) – Sumaré, SP. Das 90 PS participantes, foram coletados dados sociocomportamentais por meio de questionário e amostras cérvico-vaginais para realização do exame de Papanicolaou. Para o teste estatístico foi utilizada a Análise Hierárquica de Agrupamentos (AHA). AC foram detectadas em 33 PS (36,7 por cento), sendo 18 (20,0 por cento) atipias de significado indeterminado possivelmente não neoplásica (ASC-US), quatro (4,4 por cento) atipias de significado indeterminado sem excluir lesão de alto grau (ASC-H), sete (7,8 por cento) lesão intraepitelial de baixo grau (LSIL) e quatro (4,4 por cento) lesão intraepitelial de alto grau (HSIL). Comparando os dados das PS com citologia normal e alterada, houve diferenças estatisticamente significativas quanto ao tabagismo, início da atividade sexual, relações com pessoas do mesmo sexo e detecção de Gardnerella vaginalis. AHA classificou as PS em quatro grupos distintos, em que as PS com idade média superior apresentaram maior proporção de DST, HIV, AC e pouco uso do preservativo. As PS com menor idade média mostraram menor frequência de AC, DST, atividade sexual precoce e HIV negativo.

Avaliação crítica das nomenclaturas diagnósticas dos exames citopatológicos cervicais utilizadas no Sistema Único de Saúde (SUS) / Critical evaluation of the diagnostic nomenclatures of cervical cytopathological exams used in the Brazilian Unified Health System (SUS)

OBJETIVO: identificar as nomenclaturas diagnósticas dos exames citopatológicos cervicais utilizadas pelos laboratórios que atendem o Sistema Único de Saúde (SUS) e participantes do Monitoramento Externo de Qualidade (MEQ). Avaliar as informações adquiridas de profissionais ginecologistas que atuam no SUS sobre os tipos de classificação diagnóstica que recebem nos laudos citopatológicos cervicais. MÉTODOS: foram avaliados 94 laudos citopatológicos liberados pelos laboratórios participantes do MEQ no Estado de São Paulo e 126 questionários aplicados aos ginecologistas que atenderam o SUS. RESULTADOS: dos 94 laboratórios, 81 (86,2 por cento) utilizam uma única nomenclatura diagnóstica: 79 (97,6 por cento) utilizam a Nomenclatura Brasileira para Laudos Citopatológicos (NBLC), 1 (1,2 por cento) utiliza a classificação de Papanicolaou e 1 (1,2 por cento) utiliza a de Richart. Dos 13 (13,8 por cento) laboratórios que utilizam mais de uma nomenclatura, 5 apresentam 2 tipos, e 8, de 3 a 4, 9 dos quais incluem a classificação de Papanicolaou. O estudo demonstrou que 52 (55,3 por cento) laboratórios apresentaram mais de um diagnóstico descritivo num mesmo laudo. Dos 126 ginecologistas que responderam ao questionário de avaliação dos laudos citopatológicos, 78 (61,9 por cento) disseram receber laudos dos laboratórios com apenas uma classificação diagnóstica, 48 (38,1 por cento), laudos com mais de uma classificação, e 2 receberam as 4 classificações. Entre os 93 (73,8 por cento) ginecologistas que preferem uma classificação, 56 (60,2 por cento) alegaram que a NBLC contribui para a conduta clínica, 13 (14,0 por cento) optaram pela nomenclatura de Richart, 8 (8,6 por cento), de Reagan e 16 (17,2 por cento), a de Papanicolaou. De 33 (26,2 por cento) ginecologistas que preferem mais de uma nomenclatura, 5 optaram pelas 4 classificações...

PURPOSE: to identify the nomenclature for reporting cervical cytological diagnoses used by laboratories which render services to the Brazilian Unified Health System (SUS) and which participate in External Quality Monitoring (MEQ). To evaluate the information acquired from gynecologists of the SUS regarding the various diagnostic classifications that they receive in the cervical cytology diagnostic reports. METHODS: we evaluated 94 cytology reports issued by laboratories which participate in the MEQ in the State of São Paulo, Brazil, and 126 questionnaires applied to gynecologists who work for the SUS. RESULTS: out of the 94 laboratories, 81 (86.2 percent) use one diagnostic classification: 79 (97.6 percent) use the Brazilian Nomenclature for Cytological Reports (NBLC), 1 (1.2 percent) uses the Papanicolaou classification and 1 (1.2 percent) uses the Richart diagnostic classification. Of the 13 (13.8 percent) laboratories that use more than one classification, 5 use 2 types and 8 use 3 to 4 types, with 9 including the Papanicolau diagnostic classification. The study showed that 52 (55.3 percent) laboratories presented more than one descriptive diagnosis in the same report. Out of the 126 gynecologists who filled out a questionnaire evaluating the cytopathology reports, 78 (61.9 percent) stated that they received laboratory reports with only one diagnostic classification, 48 (38.1 percent) received reports with more than one classification and 2 received reports with all 4 classifications. Among the 93 (73.8 percent) gynecologists who prefer only one classification, 56 (60.2 percent) claimed that the NBLC contributes to clinical practice, 13 (14.0 percent) opted for the Richart classification, 8 (8.6 percent) for the Reagan classification and 16 (17.2 percent) for the Papanicolaou classification. Out of 33 (26.2 percent) gynecologists who prefer more than one classification, 5 opted for the 4 classifications...

Molecular strategies for identifying human papillomavirus infection in routinely processed samples: focus on paraffin sections.

OBJECTIVE: To optimize the protocols of human papillomavirus (HPV) detection in clinical samples, we used polymerase chain reaction (PCR)-based techniques in paraffin-embedded tissue sections and compared the results with those obtained with PCR and Hybrid Capture II (HC2) performed in liquid-based cytology (LBC) preservation medium. MATERIALS AND METHODS: Forty-five consecutive cervical biopsy specimens were taken from women participating in the ongoing Latin American Screening Study at Leonor Mendes de Barros Hospital, São Paulo, Brazil, during 2003 and 2004. The biopsy specimens were analyzed for HPV by a modified GP5+/GP6+ PCR protocol, and the results were compared with those obtained by PGMY PCR and HC2 in samples collected in LBC preservation medium. RESULTS: beta-Globin was detected in 100% of the multiplex PCR system from LBC samples and 66.7% with PCO4+/PCO3+ PCR in biopsy specimens. Of the three methods, PGMY PCR system and HC2 were equally effective in detecting HPV; both detected 13 cases in 45 samples (28.9%). The GP5+/GP6+ PCR applied in biopsy specimens showed a 20% HPV detection rate (9/45). CONCLUSIONS: Our PCR protocols worked reasonably well and allowed us to compare the three molecular methods with histological and cytological findings. The reproducibility of the results makes the technique applicable in archival materials.

DCS liquid-based system is more effective than conventional smears to diagnosis of cervical lesions: study in high-risk population with biopsy-based confirmation.

OBJECTIVE: To compare the performances of Papanicolaou test (PapTest) and of a new liquid-based cytology method, DNA-Citoliq System (DCS), in a high-risk population, with histology confirmation. METHODS: Paired specimens of exfoliated cervical cells were collected under split-sample protocol. All patients were submitted to colposcopy and a biopsy taken when any atypical transformation zone was seen. Sensitivity, specificity, positive and negative predictive values, and overall accuracy of both conventional and DCS methods were computed in relation to histology. RESULTS: A total of 1095 patients were analyzed by two cytology methods and, in 425 (38.8%), histologically. There were significantly more adequate samples with DCS (98.63%) than with conventional (89.6%) smears (P < 0.001). ASCUS was diagnosed significantly more with DCS than with conventional Pap (P < 0.001). Conventional Pap misclassified as normal 55.4% (158/285) of cases with either LSIL or HSIL or cancer at histology, whereas DCS misclassified 31.2% (89/285) of cases (P < 0.001). DCS had a significantly higher sensitivity (70% and 91.3%) than the conventional Pap (49.8% and 72.8%) to detect both LSIL+ and HSIL+ at histology, respectively. On the other hand, specificity of conventional smear (88.2% and 85.2%) was significantly higher than DCS (75.4% and 70.9%) considering both LSIL+ and HSIL+ at histology, respectively. CONCLUSIONS: This study confirms the superiority of the liquid-based cytology system DCS to detect cervical lesions. The rate of adequate DSC slides was significantly higher than with conventional cytology.

Immunocytochemical expression of p16INK4A and Ki-67 in cytologically negative and equivocal pap smears positive for oncogenic human papillomavirus.

This study was designed to analyze the cross-sectional comparison of the p16 and Ki-67 immunocytochemical expression in negative and equivocal (atypical squamous cells of undetermined significance (ASC-US)) liquid-based cytology (LBC) samples testing positive for high-risk human papillomavirus (HPV) types with HC2 assay or polymerase-chain reaction (PCR). A series of 199 consecutive LBC specimens derived from the same number of women participating in the ongoing Latin American Screening Study at Leonor Mendes de Barros Hospital, Sao Paulo, were analyzed using immunocytochemistry for expression of p16 and Ki-67 in negative and equivocal LBC samples testing positive for high-risk HPV types with hybrid capture II test (HC2) or PCR. All patients with at least one test positive (cytology, PCR, and/or HC2) were followed each 6 months for 3 years. The follow-up procedure consisted of visual examination, colposcopic inspection, cytology, and HC2 assay. Among the negative cytologic samples, 101 were HPV-positive and 55 HPV-negative. Of the HPV-positive group, 59 of 101 cases (58.4%) were positive for both p16 and Ki67 immunostaining, and 17 of 101 (16.8%) were negative for both. The proportion of Ki-67-positivity increased almost in parallel with the increasing grade of p16-positivity (p = 0.0001 for linear trend). In the HPV-negative group, both markers were negative in 41 of 55 cases (74.5%), and no statistical relationship was observed between the two markers (Pearson, p = 0.595). HPV-positive ASC-US samples demonstrated a simultaneous positive immunoreaction for p16 and Ki67 in 11 of 16 cases (68.7%), whereas 3 (18.7%) were concurrently negative. The relationship between the two markers was of borderline significance (Pearson, p = 0.053), but no linear relationship was found between the graded p16 and Ki-67 expression (p = 0.065 for linear trend). In the HPV-negative ASC-US group, there was no statistical association between the graded p16 and Ki-67 positivity (Pearson, p = 0.281). After 36 months of follow-up of the ASC-US patients, 6 women still displayed ASC-US smear, of which 4 of 6 were HPV-positive and expressed both p16 and Ki-67 markers. Two of 43 ASC-US smears had high-grade squamous intraepithelial lesions diagnosed (4.6%), and 1 had low-grade squamous intraepithelial lesion (2.3%). All of those were positive for HPV, p16 and Ki-67. Patients with ASC-US diagnosis and positive high-risk HPV status and positive for p16 Ki67 should be carefully observed to exclude occurrence of a squamous intraepithelial lesion. The combination of these two markers can be a useful implement for management of women with equivocal cytology.

Estudo retrospectivo dos diagnósticos cito-histopatológicos nas atipias de significado indeterminado / Retrospective study on cyto-histological diagnoses in the atypical squamous cells of undertermined signifance

No presente estudo foi avaliada a história de realização dos exames citológicos (EC) prévios e posteriores com diagnóstico citológico de células escamosas atípicas (ASC) e sua correlação com os resultados de exames de biópsias. Para tanto, foi realizado o estudo retrospectivo, de 60 casos de EC cérvico-vaginais com diagnóstico de ASC e biópsias correlatas, os exames prévios e posteriores de ASC e a periodicidade de exames realizados. Das 60 análises selecionadas, 57 apresentaram diagnóstico citológico anterior de ASC-US (possivelmente não neoplásico), 30 (52,6%) foram negativos no exame de biópsia, 16 (28,1%) de NIC 1, 11 (19,3%) com diagnósticos discrepantes acima de 2 graus; 3 (100%) casos de ASC-H (não se pode afastar lesão de alto grau) foram NIC 1 na biópsia. Dentre os 60 casos, 44 (73,3%) que tiveram exames anteriores, 14 (23,3%) eram positivos; 53 (88,3%) que fizeram EC posteriores à biópsia, 10 (16,7%) eram positivos. Quanto à periodicidade dos exames, 26 (43,3%) realizaram o exame com intervalo de até 1 ano, 29 (48,3%) de 1 a 3 anos, 1 (1,7%) em intervalo maior que 3 anos. De acordo com os achados observados neste estudo, os diagnósticos citológicos de ASC, quando comparado com os exames da biópsia, estavam mais frequentemente associados às alterações morfológicas reacionais. Estes dados mostram o valioso papel da correlação cito- histológica, uma vez que a execução apenas do seguimento citológico pode ser insuficiente para obter o diagnóstico morfológico. É salientada, ainda, a necessidade de efetuar o exame de acompanhamento de seis em seis meses, para realizar o controle evolutivo dessas atipias, bem como para auxiliar na conduta terapêutica das pacientes.

Frequência de diagnóstico de lesões do colo uterino por faixa etária em mulheres atendidas no Programa de Rastreamento Viva Mulher no período de 2004 a 2008 / Review on the occurrence of uterine cervix lesions according to age group among women enrolled in the Viva Mulher – Prevention Program during the period from 2004 to 2008

No presente estudo foi realizado o levantamento retrospectivo de diagnóstico citopatológico das lesões pré-neoplásicas e neoplásicas pela método de Papanicolaou e sua distribuição por faixa etária das mulheres atendidas no período de 2003 a 2008. Das 222.024 amostras analisadas, 3.674 (1,65%) foram classificadas como insatisfatórias, 206.439 (92,98%) foram negativas e 11.911 (5,36%) apresentaram algum tipo de atipias nucleares. Dentre as alterações epiteliais atípicas, 6.437 (54,04%) foram de ASC-US e 210 (1,76%) de ASC-H; 3.264 (27,40%) de LSIL; 1.279 (10,74%) de HSIL; 131 (1,10%) de SCC; 552 (4,63%) por AGC-US e 23 (0,19%) de AGC-H; 7 (0,06%) de AIS e 8 (0,07%) de ADENOCA. Quanto à ocorrência e distribuição dos diagnósticos de lesões intraepiteliais escamosas e glandulares, de acordo com a faixa etária em intervalos de 5 anos, foi observada maior frequência de diagnóstico de LSIL entre as mulheres mais jovens (15-25 anos) e os diagnósticos de ASC-US, ASC-H, AGC-US, HSIL, SCC e ADENOCA nas mulheres >50 anos de idade. As alterações glandulares, como AGC-H e AIS, ocorreram em mulheres na faixa etária entre 30-34 e 45-49 anos. Foram detectados dois (0,16%) casos de HSIL em pacientes <14 anos e em 51 (3,99%) pacientes na faixa entre 15-19 anos. Os dados da presente avaliação enfatiza a importância de campanhas orientativas, rastreamento e seguimento das pacientes para garantir a melhoria e a efetividade dos programas de prevenção.