Competing-risks regression models in analysis of biomarkers as predictors of high-risk human papillomavirus (HPV) infection outcomes and incident CIN in the LAMS cohort.

To assess the prediction potential of a 5-biomarker panel for detecting high-risk human papillomavirus (HR-HPV) infections and/or cervical intraepithelial neoplasia (CIN) progression. Five biomarkers, lipocalin, plasminogen activator inhibitor-2, p300, interleukin-10, and stratifin, were assessed in cervical biopsies from 225 women of the Latin American Screening Study. Competing-risks regression models were constructed to assess their predictive power for (i) HR-HPV outcomes (negative, transient, or persistent infection) and (ii) CIN outcomes (no progression, incident CIN1, CIN2, or CIN3). p300, LCN2, stratifin were significantly associated with prevalent HR-HPV but lost their significance in multivariate analysis. In the multivariate model, only p300 was an independent predictor of CIN3 (odds ratio=2.63; 95% confidence interval, 1.05-6.61; P=0.039). In univariate competing-risks regression, lipocalin predicted permanent HR-HPV-negative status, but in the multivariate model, IL-10 emerged as a independent predictor of HPV-negative status (subhazard ratio=4.04; 95% confidence interval, 1.81-9.01; P=0.001). The clinical value of the panel in predicting longitudinal outcomes of HR-HPV infection and/or incident CIN is limited.

Ten-Year Follow-up of 9-Valent Human Papillomavirus Vaccine: Immunogenicity, Effectiveness, and Safety.

BACKGROUND AND OBJECTIVES: The 9-valent human papillomavirus (9vHPV) vaccine Phase III immunogenicity study in 9- to 15-year-old boys and girls was extended to assess immunogenicity and effectiveness through 10 years after the last vaccine dose (NCT00943722). METHODS: Boys (n = 301) and girls (n = 971) who received three 9vHPV vaccine doses in the base study (day 1, months 2 and 6) enrolled in the extension. Serum was collected through month 126 for antibody assessments by competitive Luminex immunoassay and immunoglobulin G-Luminex immunoassay. For effectiveness analysis starting at age 16 years, genital swabs were collected (to assess HPV DNA by polymerase chain reaction) and external genital examinations conducted every 6 months. Primary analyses were conducted in per-protocol populations. RESULTS: Geometric mean antibody titers peaked around month 7, decreased sharply between months 7 and 12, then gradually through month 126. Seropositivity rates remained ≥81% by competitive Luminex immunoassay and ≥95% by immunoglobin G-Luminex immunoassay at month 126 for each 9vHPV vaccine type. After up to 11.0 (median 10.0) years of follow-up postdose 3, there were no cases of HPV6/11/16/18/31/33/45/52/58-related high-grade intraepithelial neoplasia or condyloma in males or females. Incidence rates of HPV6/11/16/18/31/33/45/52/58-related 6-month persistent infection in males and females were low (54.6 and 52.4 per 10000 person-years, respectively) and within ranges expected in vaccinated cohorts, based on previous human papillomavirus vaccine efficacy trials. CONCLUSIONS: The 9vHPV vaccine demonstrated sustained immunogenicity and effectiveness through ∼10 years post 3 doses of 9vHPV vaccination of boys and girls aged 9 to 15 years.

Elevated aromatase expression correlates with cervical carcinoma progression.

OBJECTIVES: We have previously demonstrated that aromatase mRNA is induced in cervical carcinomas compared to normal tissue, suggesting that in situ aromatase expression leading to elevated local estrogen production may contribute to cervical carcinogensis. Our objectives are to examine 1) whether aromatase protein and activity are induced in cervical carcinomas, 2) aromatase expression correlates with disease stage, and 3) inflammatory cytokines (e.g., IL-6 and TNFalpha) may correlate with aromatase expression. METHODS: RNA and protein were isolated from human cervical carcinomas and normal cervical biopsies to examine aromatase expression, using real-time RT-PCR, Western blot analysis, and immunohistochemistry. Aromatase activity in tissue was measured using the tritiated water release method. IL-6 and TNFalpha expression was also examined. RESULTS: Aromatase protein and activity levels were increased in cervical carcinomas compared to normal tissue. RNA levels correlated significantly with disease progression, with highest aromatase expression detected in stage IV tumors (p<0.001, R(2)=0.77). Aromatase promoters 1.3 and 1.4 were elevated in cervical carcinomas and in cervical cancer cells. The expression of inflammatory cytokines IL-6 and TNFalpha, known to induce aromatase, significantly correlated with aromatase expression (R(2)>0.9). TNFalpha treatment induced aromatase expression in cervical cancer cells. CONCLUSION: Increased aromatase protein and activity in cervical carcinomas and the correlation of its expression with disease stage implicates it in cervical carcinogenesis. The correlation of IL-6 and TNFalpha expression with aromatase suggests that these inflammatory cytokines may induce aromatase expression, which is confirmed by induction of aromatase expression due to TNFalpha treatment of cervical cancer cells.

Elevated expression of the oncogene c-fms and its ligand, the macrophage colony-stimulating factor-1, in cervical cancer and the role of transforming growth factor-beta1 in inducing c-fms expression.

Cervical cancer is the third most common gynecologic cancer in the United States. The presence and possible involvement of several cytokines have been studied in cervical cancer; however, very little data, if any, are available on whether cervical tumors are responsive to stimulation by the macrophage colony-stimulating factor-1 (CSF-1). Given the involvement of c-fms and its ligand CSF-1 in gynecologic cancers, such as that of the uterus and the ovaries, we have examined the expression of c-fms and CSF-1 in cervical tumor (n = 17) and normal cervix (n = 8) samples. The data show that c-fms and its ligand are significantly higher in cervical carcinomas compared with normal samples. Immunohistochemistry not only showed that tumor cells expressed significantly higher levels of c-fms but also c-fms levels were markedly higher in tumor cells than tumor-associated stromal cells. Blocking c-fms activity in cervical cancer cells, which express CSF-1 and c-fms, resulted in increased apoptosis and decreased motility compared with control, suggesting that CSF-1/c-fms signaling may be involved in enhanced survival and possibly invasion by cervical cancer cells via an autocrine mechanism. Combined, the data show for the first time the induction of CSF-1 and c-fms in cervical carcinomas and suggest that c-fms activation may play a role in cervical carcinogenesis. Additionally, our data suggest that transforming growth factor-beta1 may be a factor in inducing the expression of c-fms in cervical cancer cells. The data suggest that c-fms may be a valuable therapeutic target in cervical cancer.

Up-regulation of VEGF, c-fms and COX-2 expression correlates with severity of cervical cancer precursor (CIN) lesions and invasive disease.

OBJECTIVES: To describe the expression of vascular endothelial growth factor (VEGF), proto-oncogene macrophage colony-stimulating factor receptor (c-fms) and cyclooxygenase-2 (COX-2) in cervical carcinogenesis and to analyze the correlation of VEGF with c-fms and COX-2 expression. METHODS: In this study, 26 cases of benign cervix, 28 low-grade cervical intraepithelial neoplasia (CIN; CIN 1), 30 high-grade CIN (CIN 2/3) and 28 squamous cervical carcinomas (SCC) were examined by immunohistochemistry (IHC) and analysis was performed separately for epithelium and stroma. RESULTS: Positive epithelial expressions in normal cervix, low-grade CIN, high-grade CIN and SCC were, respectively: VEGF - 11.5%, 39.3%, 53.3% and 75% (P<0.001); c-fms - 0%, 10.7%, 40% and 67.9% (P<0.001); COX-2 - 7.7%, 39.3%, 80% and 100% (P<0.001). Stromal VEGF expression was higher than epithelial expression in all CIN grades and was also associated with the lesion grade, while c-fms and COX-2 stromal expression was weak. VEGF expression was statistically correlated to c-fms and COX-2 expression in high-grade CIN (P=0.020 and P=0.027, respectively) and SCC (P=0.015 and P=0.005, respectively). CONCLUSIONS: On the basis of our findings, these factors may participate in the development and progression of CIN lesions, with possible interaction of c-fms and COX-2 on VEGF expression, and may be potential molecular targets for studies of cervical cancer prevention and treatment.

Value of conventional pap smear, liquid-based cytology, visual inspection and human papillomavirus testing as optional screening tools among latin american women <35 and > or =35 years of age: experience from the Latin American Screening Study.

OBJECTIVE: To compare performance of conventional Pap test, liquid based cytology (LBC) and visual inspection with acetic acid (VIA), as well as human papillomavirus (HPV) testing in 2 subcohorts of women, <35 and > or =35 years of age. STUDY DESIGN: Performance indicators were calculated using colposcopic biopsies as the gold standard; all results were corrected for verification bias using maximum likelihood. RESULT: Both conventional Pap test n d Hybrid Capture 2 (HC2) assay performed significantly better among older women than younger; no difference was observed in performance of LBC and VIA, both inferior to Pap and HC2. The Pap test was more specific than HC2 in both subcohorts; HC2 had the highest sensitivity. For individual tests, the best balance between sensitivity and specificity was obtained for HC2 assay corrected for verification bias in the older women's subcohort; this was further improved by the combined use of the Pap test and HC2. CONCLUSION: Age of the target population is an important determinant of the performance of different screening tests. The choice of optimal test for women <35 and > or =35 years of age depends on whether the highest positive predictive value (Pap test).

Transvaginal ultrasonographic assessment of the expulsion rate of intrauterine devices inserted in the immediate postpartum period: a pilot study.

OBJECTIVE: To compare the expulsion rates of intrauterine devices (IUDs) inserted in the immediate postpartum after vaginal birth and cesarean section. METHODS: Nineteen patients who had a vaginal birth and 19 patients who had a cesarean section at Hospital de Clínicas de Porto Alegre, Brazil, were selected for copper T 380A IUD insertion. With the aim of detecting clinically unnoticed dislodged devices, ultrasound examinations were performed at 1 month and between 3 and 12 months after delivery. The IUDs were considered completely expelled when found outside the endometrial cavity (e.g., in the cervical canal) or outside the uterus (in the vagina). RESULTS: Expulsion rates were statistically different between the two groups: after a vaginal birth, 50% (ultrasound only) + 27.8% (clinical examination); and post-cesarean section, 0% (p < .001; OR 5.75, 95% CI 2.36-14.01). CONCLUSION: Considering that the contraceptive efficacy of IUDs is associated with their intrauterine location, the high expulsion rates seen when they are inserted immediately after vaginal delivery contraindicate their use in this setting. The use of IUDs immediately after a cesarean section is still a reasonable alternative because its expulsion rate was zero. Ultrasound assessment of IUD positioning performed better than clinical examination, which failed to detect expulsion after postpartum insertion in 75% of the cases (9 from 12 cases).

Up-regulation of lipocalin 2 is associated with high-risk human papillomavirus and grade of cervical lesion at baseline but does not predict outcomes of infections or incident cervical intraepithelial neoplasia.

Our objective was to assess whether neutrophil gelatinase-associated lipocalin (NGAL)/lipocalin 2 (LCN2) expression in cervical human papillomavirus (HPV) lesions has implications on the outcome of HPV infections or disease progression. Cervical biopsy specimens from 225 women in the Latin American Screening study were analyzed for NGAL/LCN2 expression using immunohistochemical analysis, to assess associations with cervical intraepithelial neoplasia (CIN) grade, high-risk HPV, and in predicting outcomes of high-risk (HR)-HPV infections. Expression of NGAL/LCN2 increased with lesion grade (odds ratio [OR], 3.86; 95% confidence interval [CI], 1.53-9.71; P = .001). Up-regulation was also related to HR-HPV detection (OR, 2.21; 95% CI, 1.15-4.24; P = .016) and showed a linear relationship to HR-HPV load (P = .002). NGAL/LCN2 expression was of no value in predicting the outcomes of HR-HPV infections or the surrogate end points (incident CIN 1+ and CIN 2+) of progressive disease. Because the SV40 large T antigen is a powerful up-regulator of this lipocalin, up-regulation of NGAL/LCN2 in CIN is probably induced by HR-HPV E6 oncoprotein, most likely by eliminating its normal transcription repression exerted by wild-type p53.

Up-regulation of 14-3-3sigma (Stratifin) is associated with high-grade CIN and high-risk human papillomavirus (HPV) at baseline but does not predict outcomes of HR-HPV infections or incident CIN in the LAMS study.

To assess whether the potentially high-risk (HR) human papillomavirus (HPV)-related up-regulation of 14-3-3sigma (stratifin) has implications in the outcome of HPV infections or cervical intraepithelial neoplasia (CIN) lesions, cervical biopsy specimens from 225 women in the Latin American Screening Study were analyzed for 14-3-3sigma expression using immunohistochemical analysis. We assessed its associations with CIN grade and HR HPV at baseline and value in predicting outcomes of HR-HPV infections and the development of incident CIN 1+ and CIN 2+. Expression of 14-3-3sigma increased in parallel with the lesion grade. Up-regulation was also significantly related to HR-HPV detection (P = .004; odds ratio, 2.71; 95% confidence interval, 1.37-5.35) and showed a linear relationship to HR-HPV loads (P = .003). 14-3-3sigma expression was of no value in predicting the outcomes (incident, persistent, clearance) of HR-HPV infections or incident CIN 1+ and CIN 2+. 14-3-3sigma is not inactivated in cervical carcinoma and CIN but is up-regulated on transition from CIN 2 to CIN 3. Its normal functions in controlling G(1)/S and G(2)/M checkpoints are being bypassed by HR HPV.

Up-regulation of plasminogen activator inhibitor-2 is associated with high-risk HPV and grade of cervical lesion at baseline but does not predict outcomes of high-risk HPV infections or incident CIN.

Protease inhibitor serpin-B2 (plasminogen activator inhibitor-2 [PAI-2]) protects pRb from degradation in human papillomavirus (HPV)-18+ HeLa cells. Our objective was to assess whether the pRb-mediated HPV-suppressive effect of PAI-2 in cancer cell lines has implications in the outcome of HPV infections. Cervical biopsy specimens from 225 women were analyzed for PAI-2 expression to assess its value as a predictor of cervical intraepithelial neoplasia (CIN) grade, high-risk (HR) HPV at baseline, outcomes of HR-HPV infections, and the development of incident CIN. PAI-2 expression increased in parallel with lesion grade. Nuclear PAI-2 expression was significantly related to HR-HPV detection and had a linear relationship with HR-HPV load. PAI-2 expression was of no value in predicting the outcomes of HR-HPV infections. The same was true for PAI-2 as a predictor of surrogate end points (incident CIN 1+, CIN 2+) of progressive disease. PAI-2 expression is up-regulated on transition from CIN 2 to CIN 3. The HR-HPV suppressive effects of PAI-2 were not related to more favorable outcomes of HR-HPV infections or lower risk of disease progression to CIN.

Value of the International Federation for Cervical Pathology and Colposcopy (IFCPC) Terminology in predicting cervical disease.

OBJECTIVE: To evaluate the colposcopic accuracy according to the International Federation for Cervical Pathology and Colposcopy (IFCPC) 2002 terminology. MATERIALS AND METHODS: A series of 3,040 women derived from a general population were screened by means of Pap smear, unaided visual inspection, and high-risk human papillomavirus testing. All colposcopic examination results with abnormal findings and with biopsy confirmation (n = 468) were recorded, reviewed by 2 blinded colposcopists according to the IFCPC nomenclature, and included in this analysis. RESULTS: The IFCPC terminology was easily reproduced by the 2 observers with excellent interobserver agreement (kappa =.843). Colposcopy had a sensitivity of 86% and a specificity of 30.3% in distinguishing healthy cervix from that with cervical intraepithelial neoplasia (CIN)/carcinoma. In distinguishing healthy cervix/low-grade lesions (CIN 1) from that with high-grade lesions (CIN 2/3)/carcinoma, colposcopy had a sensitivity of 61.1% and a specificity of 94.4%. Colposcopic findings graded as major changes had the highest positive predictive value for detecting high-grade lesion/carcinoma. The colposcopic abnormalities within the transformation zone and large lesions were more closely related to high-grade lesion/carcinoma, whereas a sharp outer border, multiple colposcopic abnormalities, and iodine negativity were not statistically related to severe lesions. CONCLUSIONS: Colposcopy using the new IFCPC classification is a potentially effective screening method; when used for diagnosis, a histological sampling is necessary. The categorization of major changes and minor changes is appropriate. It is important to describe the lesion localization in relation to the transformation zone and the lesion size because these characteristics are related to high-grade lesions.

Macrophages, inflammation and risk of cervical intraepithelial neoplasia (CIN) progression--clinicopathological correlation.

OBJECTIVE: To evaluate the population of macrophages during the cervical malignant transformation and its influence in CIN outcome. METHODS: Biopsies from 26 normal cervix, 28 low-grade (LSIL), 30 high grade squamous intraepithelial lesions (HSIL) and 28 squamous cell carcinomas (SCC) were stained by H&E to assess inflammation and by immunohistochemistry with anti-CD68 to detect macrophages. The macrophage count was corrected for the epithelial and stromal compartments using appropriate software. Clinical and prospective follow-up data were also available. RESULTS: We identified that macrophage count increased linearly with disease progression (median count per case at x200 magnification: normal, 5.1; LSIL, 5.5; HSIL, 9.9; SCC, 14.5; P<0.001), that inflammation also increased (moderate-intense inflammation present in 25%, 46.1%, 58.4% and 89.3% of normal, LSIL, HSIL and SCC, respectively; P<0.001) and that macrophage count was independently associated with the lesion grade (P<0.001). Moreover, macrophages showed an increasing migration into the epithelium along with the progression of CIN to invasive cancer. Of the 24 LSIL cases with information available, followed-up for 805+/-140 days, 16 regressed, 6 persisted and 2 progressed. Age, high-risk HPV or inflammation were not risk factors for persistent/progressed LSIL in our cohort. However, LSIL that persisted or progressed showed a higher macrophage count (median of 10.8) than lesions that regressed (7; P=0.031). CONCLUSIONS: The study on macrophages offers a potential approach for cervical cancer treatment, since macrophages are closely related to progression of CIN, and can be used as an applicable marker of such a risk.