Molecular Signatures of High-Grade Cervical Lesions.

Cervical cancer is the fourth most common neoplasia in women and the infection with human papilloma virus (HPV) is its necessary cause. Screening methods, currently based on cytology and HPV DNA tests, display low specificity/sensitivity, reducing the efficacy of cervical cancer screening programs. Herein, molecular signatures of cervical cytologic specimens revealed by liquid chromatography-mass spectrometry (LC-MS), were tested in their ability to provide a metabolomic screening for cervical cancer. These molecules were tested whether they could clinically differentiate insignificant HPV infections from precancerous lesions. For that, high-grade squamous intraepithelial lesions (HSIL)-related metabolites were compared to those of no cervical lesions in women with and without HPV infection. Samples were collected from women diagnosed with normal cervix (N = 40) and from those detected with HSIL from cytology and colposcopy (N = 40). Liquid-based cytology diagnosis, DNA HPV-detection test, and LC-MS analysis were carried out for all the samples. The same sample, in a customized collection medium, could be used for all the diagnostic techniques employed here. The metabolomic profile of cervical cancer provided by LC-MS was found to indicate unique molecular signatures for HSIL, being two ceramides and a sphingosine metabolite. These molecules occurred independently of women's HPV status and could be related to the pre-neoplastic phenotype. Statistical models based on such findings could correctly discriminate and classify HSIL and no cervical lesion women. The results showcase the potential of LC-MS as an emerging technology for clinical use in cervical cancer screening, although further validation with a larger sample set is still necessary.

Comparing the Copenhagen Index (CPH-I) and Risk of Ovarian Malignancy Algorithm (ROMA): Two equivalent ways to differentiate malignant from benign ovarian tumors before surgery?

AIM: To evaluate the prediction of malignancy in women with pelvic masses using the Copenhagen Index (CPH-I) and Risk of Ovarian Malignancy Algorithm (ROMA). PATIENTS AND METHODS: Three hundred eighty four women operated due to an ovarian mass were enrolled between January 2010 and June 2015. All patients had histopathological diagnosis, HE4 and CA125 measurement. CPH-I and ROMA were calculated and their performances compared in two distinct scenarios: 1) for the discrimination of benign ovarian disease from epithelial ovarian cancer (EOC), non-epithelial ovarian cancer, borderline ovarian tumors (BOT) and ovarian metastases, and 2) for the discrimination of benign disease from EOC. Receiver Operator Characteristics' Areas Under the Curves (AUC) were calculated for CPH-I and ROMA and compared. RESULTS: Of the 384 women, 224 presented a benign ovarian tumor, 32 BOT, 87 EOC, 26 non-epithelial ovarian cancer, and 15 had ovarian metastases. The best AUCs were obtained for the discrimination of EOC from benign tumors. CPH-I performed slightly better than ROMA, and both approached 89% sensitivity and 85% specificity. When all malignant tumors (EOC, BOT, ovarian metastases and non-epithelial ovarian cancer - entire cohort) were included, the performance of CPH-I and ROMA declined to nearly 72%, although the specificity remained close to 85%. CONCLUSION: CPH-I and ROMA performed similarly well for the discrimination of EOC from benign ovarian tumors. However, caution is necessary since, in practical situations, where all the histological possibilities for malignant ovarian tumors must be considered, the sensitivity of CPH-I and ROMA may not surpass 70%.

Retrieval of HPV oncogenes E6 and E7 mRNA from cervical specimens using a manual open technology protocol.

BACKGROUND: HPV oncogenes mRNA detection gains momentum as an adjuvant for HPV-related cervical abnormalities diagnosis, but is based on costly detection assays not allowing viral type targeting. OBJECTIVE: To assess detection rate of HPV oncogenes E6/E7 mRNA from cervical specimens using a manual, open technology, fully customizable protocol and determine whether HPV-related epidemiological features influence mRNA retrieval. We reviewed literature and compared our retrieval rate with automated technologies. METHODS: We used 60 samples positive for HPV DNA types 16, 18, 31 and/or 45. We extracted mRNA with a TRizol-based protocol, and tested mRNA purity and concentration using light absorbance. We reverse-transcribed mRNA into cDNA for E6/7 detection. RESULTS: HPV oncogenes E6/E7 mRNA was retrieved from 36 (60%) out of 60 specimens. No HPV load-related clinical or epidemiological feature was significantly associated with mRNA retrieval. Presence of HPV-DNA 16/18 was associated with mRNA retrieval (OR = 9.08; 95% CI 1.26 to 65.32 for HPV 16; and 18.2; IC95% 1.86 to 391.44 for HPV 18). CONCLUSIONS: The open-technology protocol yielded an mRNA detection rate similar to that of automated technologies. Advantages are lower costs and target HPV type customization.

[Prevalence of HPV 16, 18, 45 and 31 in women with cervical lesions]. / Prevalência dos HPV 16, 18, 45 e 31 em mulheres com lesão cervical.

PURPOSE: To determine the prevalence of HPV 16, 18, 31 and 45 in cervical screening samples of women with cellular changes and/or colposcopy suggestive of persistent high grade or low grade lesion who were submitted to conization. METHODS: A total of 120 women were included in the study. Histological analysis of the cervical cones revealed 7 cases of cervicitis, 22 of CIN1, 31 of CIN2, 54 of CIN3, and 6 invasive carcinomas. The cervical screening samples were analyzed before conization for the presence of HPV-DNA by PCR using the consensus primers PGMY09/11. HPV-DNA-positive samples were tested for the presence of HPV16, 18, 31 and 45 using type-specific primers for these HPV. RESULTS: HPV-DNA was detected in 67.5% of the studied women. HPV 16 (40%) was the most prevalent type in most ilesions, followed by HPV 31 (13.3%), 45 (13.3%), and 18 (4.1%). Multiple infections occurred in 15% of the cases and infections with other HPV types were detected in 14% of the sample. CONCLUSIONS: HPV 16 and 18 infections do not always occur as a single infection, and may be associated with other HPV types on different occasions.

Prevalência dos HPV 16, 18, 45 e 31 em mulheres com lesão cervical / Prevalence of HPV 16, 18, 45 and 31 in women with cervical lesions

OBJETIVO: avaliar a prevalência dos HPV 16, 18, 31 e 45 em amostras de raspado cervical de mulheres com alterações celulares e/ou colposcopia sugestiva de lesão de alto grau ou lesão de baixo grau persistente submetidas à conização. MÉTODOS: Foram incluídas 120 mulheres. A análise histológica dos cones cervicais revelou 7 casos de cervicite, 22 de NIC1, 31 de NIC2, 54 de NIC3 e 6 carcinomas invasores. Foram analisadas as amostras de raspado cervical coletadas antes da conização para a presença do DNA-HPV por PCR com os primers de consenso, PGMY09/11. As amostras positivas para DNA de HPV foram testadas para presença do HPV16, 18, 31 e 45 utilizando-se primers tipo específico para esses HPV. RESULTADOS: O DNA-HPV foi detectado em 67,5 por cento das mulheres. O HPV 16 (40 por cento) foi o tipo mais prevalente na maioria das lesões, seguido dos HPV 31 (13,3 por cento), 45 (13,3 por cento) e 18 (4,1 por cento). Infecções múltiplas ocorreram em 15 por cento dos casos e as infecções por outros tipos de HPV foram detectadas em 14 por cento da amostra. CONCLUSÕES: as infecções pelos HPV 16 e 18 nem sempre ocorrem de maneira solitária (infecção única), estando associadas a outros tipos de HPV em diversas ocasiões.

PURPOSE: to determine the prevalence of HPV 16, 18, 31 and 45 in cervical screening samples of women with cellular changes and/or colposcopy suggestive of persistent high grade or low grade lesion who were submitted to conization. METHODS: a total of 120 women were included in the study. Histological analysis of the cervical cones revealed 7 cases of cervicitis, 22 of CIN1, 31 of CIN2, 54 of CIN3, and 6 invasive carcinomas. The cervical screening samples were analyzed before conization for the presence of HPV-DNA by PCR using the consensus primers PGMY09/11. HPV-DNA-positive samples were tested for the presence of HPV16, 18, 31 and 45 using type-specific primers for these HPV. RESULTS: HPV-DNA was detected in 67.5 percent of the studied women. HPV 16 (40 percent) was the most prevalent type in most ilesions, followed by HPV 31 (13.3 percent), 45 (13.3 percent), and 18 (4.1 percent). Multiple infections occurred in 15 percent of the cases and infections with other HPV types were detected in 14 percent of the sample. CONCLUSIONS: HPV 16 and 18 infections do not always occur as a single infection, and may be associated with other HPV types on different occasions.

Phylogenetic classification of human papillomavirus genotypes in high-grade cervical intraepithelial neoplasia in women from a densely populated Brazilian urban region.

CONTEXT AND OBJECTIVE: Differences in human papillomavirus (HPV) types may correlate with the biological potential and invasion risk of high-grade cervical intraepithelial neoplasia (CIN 2 and CIN 3). The objective of this study was to determine the relationship between different combinations of HPV types and CIN severity. DESIGN AND SETTING: Cross-sectional study, at Universidade Estadual de Campinas (Unicamp). METHODS: Cervical samples from 106 women treated due to CIN 2 (18) or CIN 3 (88) were examined for specific HPV genotypes using Roche Linear Array (LA-HPV). The proportions of CIN 2 and CIN 3 in groups of women infected with the HPV phylogenetic groups A7 and A9 were compared. Three groups were formed: women with single infections; multiple infections; and the whole sample. RESULTS: Multiple infections were detected in 68 samples (64.7%). The most frequent high-risk genotypes detected (single/multiple) were HPV 16 (57.1%), HPV 58 (24.7%), HPV 33 (15.2%), HPV 52 (13.3%), HPV 31 (10.4%), HPV 51 (7.6%) and HPV 18 (6.6%). Women without infection with HPV species Alpha 9 were less likely to have CIN 3 than were their Alpha 9 HPV-infected counterparts. HPV 16 and/or HPV 18, with or without associations with other viral types, were more frequently found in women with CIN 3 than in those with CIN 2. CONCLUSIONS: The severity of high-grade CIN may be aggravated by the presence of HPV types included in the Alpha 9 phylogenetic classification and by infections including HPV 16 and 18, singly or in combination with other HPV genotypes.

Phylogenetic classification of human papillomavirus genotypes in high-grade cervical intraepithelial neoplasia in women from a densely populated Brazilian urban region / Classificação filogenética dos genótipos de papilomavírus humano em neoplasia intraepitelial cervical de alto grau em mulheres de uma região urbana brasileira densamente povoada

CONTEXT AND OBJECTIVE: Differences in human papillomavirus (HPV) types may correlate with the biological potential and invasion risk of high-grade cervical intraepithelial neoplasia (CIN 2 and CIN 3). The objective of this study was to determine the relationship between different combinations of HPV types and CIN severity. DESIGN AND SETTING: Cross-sectional study, at Universidade Estadual de Campinas (Unicamp). METHODS: Cervical samples from 106 women treated due to CIN 2 (18) or CIN 3 (88) were examined for specific HPV genotypes using Roche Linear Array® (LA-HPV). The proportions of CIN 2 and CIN 3 in groups of women infected with the HPV phylogenetic groups A7 and A9 were compared. Three groups were formed: women with single infections; multiple infections; and the whole sample. RESULTS: Multiple infections were detected in 68 samples (64.7 percent). The most frequent high-risk genotypes detected (single/multiple) were HPV 16 (57.1 percent), HPV 58 (24.7 percent), HPV 33 (15.2 percent), HPV 52 (13.3 percent), HPV 31 (10.4 percent), HPV 51 (7.6 percent) and HPV 18 (6.6 percent). Women without infection with HPV species Alpha 9 were less likely to have CIN 3 than were their Alpha 9 HPV-infected counterparts. HPV 16 and/or HPV 18, with or without associations with other viral types, were more frequently found in women with CIN 3 than in those with CIN 2. CONCLUSIONS: The severity of high-grade CIN may be aggravated by the presence of HPV types included in the Alpha 9 phylogenetic classification and by infections including HPV 16 and 18, singly or in combination with other HPV genotypes.

CONTEXTO E OBJETIVO: Diferentes tipos de papilomavírus humano (human papillomavirus, HPV) podem ser correlacionados com a capacidade biológica e risco de invasão das neoplasias intra-epitelial de alto grau cervical (NIC 2 e NIC 3). O objetivo deste estudo foi determinar a relação de diferentes tipos de HPV com a gravidade da NIC. TIPO DE ESTUDO E LOCAL: Estudo transversal na Universidade Estadual de Campinas (Unicamp). MÉTODOS: Foram avaliados os genótipos específicos de HPV da amostra cervical de 106 mulheres com NIC 2 (18) ou NIC 3 (88), utilizando Roche Linear Array® (LA) HPV genotyping assay. Foram comparadas as proporções de NIC 2 e NIC 3 em grupos de mulheres infectadas com tipos de HPV dos grupos filogenéticos A7 e A9. Três grupos foram formados: mulheres com infecção simples; infecção múltipla; e infecção simples e múltipla. RESULTADOS: Infecções múltiplas foram detectadas em 68 (64,7 por cento) das amostras. Os genótipos de alto risco mais frequentemente detectados em infecção simples ou múltipla foram HPV 16 (57,1 por cento), HPV 58 (24,7 por cento), HPV 33 (15,2 por cento), HPV 52 (13,3 por cento), HPV 31 (10,4 por cento), HPV 51 (7,6 por cento) e HPV 18 (6,6 por cento). A probabilidade de mulheres com NIC 3 serem infectadas com HPV que não da espécie Alfa 9 era menor do que com os tipos de HPV da espécie Alfa 9. HPV 16 e ou 18, associado ou não com outros tipos virais eram mais frequentemente encontrados nas mulheres com NIC 3 do que naquelas com NIC 2. CONCLUSÃO: A gravidade da NIC de alto grau pode ser aumentada pela presença de tipos de HPV incluídos na classificação filogenética Alfa 9 e por infecções que incluem HPV 16 e 18 combinados ou não com outros genótipos de HPV.

Recovery of DNA for the detection and genotyping of human papillomavirus from clinical cervical specimens stored for up to 2 years in a universal collection medium with denaturing reagent.

The recovery and stability of DNA for the detection and genotyping of HPV in UCM-containing specimens, after exposure to denaturing reagents and stored for up to 2 years were evaluated. Samples were collected from 60 women who had cervical cytology specimens harboring cervical intraepithelial neoplasia (CIN) 2 or 3. All samples were stored in UCM and had been frozen at -20 degrees C following the addition of the denaturing reagent (sodium hydroxide) and the removal of the aliquot required for Hybrid Capture 2 testing for the identification of HPV DNA. The samples had been stored for 6, 12 and 24 months (20 samples for each storage time). HPV DNA extraction was performed according to a protocol designed specifically and the presence and quality of DNA was confirmed by human beta-globin detection using the consensus primers G73 and G74. HPV DNA was amplified using the consensus primers PGMY09 and PGMY11, and reverse line-blot hybridization was used to detect type-specific amplicons for 37 HPV types. The DNA extracted from the denatured specimen was recovered in 57/60 (95%) of the samples. HPV DNA was detected in 56/57 (98%) of the recovered samples. Twenty-six of the 56 samples recovered (48%) were genotyped successfully.