Luciano Serpa Hammes

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-β1 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.

Activin A increases invasiveness of endometrial cells in an in-vitro model of human peritoneum

The aim of this study was to investigate whether activin A has an effect on the attachment and/or invasion of endometrial cells in a modeled peritoneum in vitro. Cultured endometrial stromal cells (ESCs) and endometrial epithelial cells (EECs) were treated with activin A (6.25-50 ng/ml) and with activin A (25 ng/ml) with and without inhibin A or follistatin. Fluorescent labeled cells were added to confluent peritoneal mesothelial cells (PMCs) and to a monolayer of confluent PMCs grown in a Matrigel invasion assay. The rate of endometrial cell attachment and invasion through PMCs was assessed. The expression of cell adhesion proteins N- and E-cadherin was evaluated with real-time RT-PCR. Activin A (25 ng/ml) promoted invasion of the endometrial cells through the modeled peritoneum (>2-fold versus control) and this effect was partially reversed by inhibin A and follistatin. Activin A had no effect on the rate of attachment of the endometrial cells to the PMCs or in the rate of proliferation. In addition, activin A induced a decreased mRNA expression of E-cadherin in cultured EECs. In conclusion, activin A increases invasion of EECs and ESCs into modeled peritoneum. In EECs, this effect may be related to down-regulation of E-cadherin expression. Further studies are warranted to evaluate the role of activin-A in the genesis of the endometriotic lesion.

Conventional pap smear and liquid-based cytology as screening tools in low-resource settings in Latin America : Experience of the Latin American screening study

OBJECTIVE To evaluate the performance of the conventional Pap test and liquid-based cytology (LBC) in an ongoing multicenter trial testing optional screening tools (cytology, screening colposcopy, visual inspection with acetic acid, visual inspection with Lugols Iodine, cervicography and Hybrid Capture II [HCII] (Digene Brazil, São Paulo, Brazil) conventional and self-sampling), for cervical cancer in Brazil and Argentina. STUDY DESIGN A cohort of 12,107 women attending four clinics (Campinas, São Paulo, Porto Alegre, Buenos Aires) were randomized into the 8 diagnostic arms. Women testing positive with any of the tests were referred for colposcopy, and cervical biopsies were used as the gold standard to assess performance characteristics of the diagnostic tests. Conventional Pap smears were sampled by all clinics (n = 10,240), and LBC (Autocyte PREP, [TriPath Imaging, Burlington, North Carolina, U.S.A.], n=320, and DNA-Citoliq [Digene Brazil], n =1,346) was performed by 1 of the clinics. RESULTS Conventional Pap smears showed no squamous intraepithelial lesions (normal) in 8,946 (87.4%) and LBC in 1,373 (82.4%). Using high grade squamous intraepithelial lesions (HSIL) as the cutoff, Pap smears predicted high grade (cervical intraepithelial neoplasia [CIN] 3) with OR 63.0 (95% CI, 36.90-107.70), standard error (SE) 59%, SP 97.8%, positive predictive value (PPV) 68.1% and negative predictive value (NPV) 96.7%. The same figures for Autocyte PREP were: OR 9.0 (95% CI, 2.43-33.24), sensitivity (SE) 33.3%, specificity (SP) 100%, PPV 100% and negative PV (NPV) 88.8%. DNA-Citoliq detected CIN 3 as follows: OR 11.8 (95% CI 2.60-53.26), SE 40.0%, SP 94.6%, PPV 40.0% and NPV 94.6%. Lowering the cutoff to low grade squamous intraepithelial lesions increased SE and NPV but compromised SP and PPV. The detection rates for high grade lesions after an atypical squamous cells of undetermined significance diagnosis were similar with the 3 techniques. In our settings, the 3 methods of cervical cytology were slightly different in performance. The conventional Pap smear had the highest SE, while Autocyte PREP had 100% SP and PPV in detecting CIN3 with the HSIL cutoff. All 3 tests had lower SE but higher SP as compared to HCII.

Increased risk of oncogenic human papillomavirus infections and incident high-grade cervical intraepithelial neoplasia among smokers: experience from the Latin American screening study.

Background: The purpose of this study was to assess the effect of smoking on the prevalence and incidence of high-risk human papillomavirus (hr-HPV) infection and cervical intraepithelial neoplasia (CIN) in a large sample of Latin American women. Methods: The study examines baseline data on over 12,000 women included in the Latin American Screening Study (Brazil and Argentina), and over 1000 women followed-up for a period of 36 months. Three groups were formed: never smokers, current, and past smokers. The prevalence of hr-HPV infection and CIN were compared between the study groups. In the prospective analysis, women were controlled at 6-month intervals to assess the cumulative risk of incident hr-HPV infection, smear abnormalities, and CIN. Results: A higher prevalence (21.7%) of hr-HPV infection was found among current smokers as compared to never smokers (16.5%) or past smokers (13.5%). Being current smoker was significantly (P <0.01) associated with hr-HPV detection (OR = 1.6; 95% CI = 1.2–2.1). Being a current smoker was a significant predictor of incident hr-HPV during the follow-up [Hazards ratio (HR) = 1.4; 95% CI 1.0–1.9]. For incident CIN2+, being a past smoker (HR = 3.6; 95% CI 1.6–9.8) or current smoker (HR = 3.6; 95% CI 1.5–8.6) were the significant independent predictors. Current and past smokers had a significantly increased risk of incident CIN2+ (P <0.01). Conclusions: Smoking increases the risk of contracting hr-HPV infection and modifies the effect of a persistent hr-HPV infection by further increasing the risk of developing CIN2+. It seems that this effect modification persists over several years after smoking cessation.

Hormonal Contraceptives and the Length of Their Use Are Not Independent Risk Factors for High-Risk HPV Infections or High-Grade CIN

Aims: To evaluate the role of hormonal contraceptives as a risk factor of high-risk human papillomavirus (HR-HPV), cervical intraepithelial lesions (CIN) and cervical cancer in our multi-center population-based LAMS (Latin American Screening) study. Methods: A cohort study with >12,000 women from Brazil and Argentina using logistic regression to analyze the covariates of hormonal contraception (HOC – oral, injections, patches, implants, vaginal ring and progesterone intrauterine system) use followed by multivariate modeling for predictors of HR-HPV and CIN2+. Results: HR-HPV infection was a consistent risk factor of high-grade CIN in all three groups of women. The length of HOC use was not significantly related to high-grade squamous intraepithelial lesions (HSIL)+ Pap (p = 0.069), LSIL+ Pap (p = 0.781) or ASCUS+ (p = 0.231). The same was true with the length of HOC use and histology CIN3+ (p = 0.115) and CIN2+ (p = 0.515). Frequently, HOC users have previously shown more HPV-related lesions, as well as lower HPV prevalence if they were current smokers. But HOC use and time of usage were not independent risk factors of either HR-HPV infection or high-grade CIN using multiple logistic regressions. Conclusions: No evidence was found for an association between the use of HOC with an increased risk for HR-HPV infection or high-grade CIN in this cohort.

Smoking worsens the prognosis of mild abnormalities in cervical cytology

Objective. To examine the effect of smoking on the incidence of low‐ and high‐grade cervical intraepithelial neoplasia (CIN) in women with a baseline Pap smear of atypical squamous cells (ASC) or a low‐grade squamous intraepithelial lesion (LSIL). Design. Prospective study in which a cohort of women with normal colposcopy and ASC/LSIL at baseline were followed at 6‐month intervals of up to 36 months. Women were grouped in post‐hoc analysis according to their smoking behavior: never (or past) smokers and current smokers. Setting. This report was based on data from the Latin American Screening Study, conducted in São Paulo, Campinas, Porto Alegre (Brazil) and Buenos Aires (Argentina). Population. A subset of 150 women derived from a cohort of 1,011 women. Methods. Multivariate Cox analysis and Kaplan–Meier curves were used. Main outcome measures. Low‐ and high‐grade CIN during follow‐up. Results. The only factor related to an increased risk of developing CIN was the positive high‐risk (hr) HPV status (hazard ratio (HR) = 3.42; 95% CI: 1.11–9.43). A total of 21 cases of incident CIN were detected during follow‐up. Of these, 11 appeared in the group of 67 smokers and 10 among the 83 non‐smoker women (log‐rank, p = 0.33). Smoking status was not associated with the risk of developing CIN (HR = 0.73; 95% CI: 0.40–1.33). However, when restricting the analysis to high‐grade CIN only (11 cases), the probability of developing the disease was significantly higher among smokers (p = 0.04). Conclusions. Smoking contributes additional risk for developing high‐grade CIN in women with ASC or LSIL cytology but normal colposcopy.

Value of Conventional Pap Smear, Liquid-Based Cytology, Visual Inspection and Human Papillomavirus Testing as Optional Screening Tools Among Latin American Women

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 womens 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).

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.

Computer-assisted immunohistochemical analysis of cervical cancer biomarkers using low-cost and simple software

The study of biomarkers by immunohistochemistry (IHC) for cervical cancer and intraepithelial lesions is a promising field. However, manual interpretation of IHC and reproducibility of the scoring systems can be highly subjective. In this article, we present a novel and simple computer-assisted IHC interpretation method based on cyan-magenta-yellow-black (CMYK) color format, for tissues with diaminobenzidine cytoplasmatic staining counterstained with methyl green. This novel method is more easily interpreted than previous computer-assisted methods based on red-green-blue (RGB) color format and presents a strong correlation with the manual H-score. It is simple, objective, and requires only low-cost software and minimal computer skills. Briefly, a total of 67 microscopic images of cervical carcinoma, normal cervix, and negative controls were analyzed in Corel Photo Paint X3 software in CMYK and RGB color format, and compared with manual H-score IHC assessments. The clearest and best positive correlation with the H-score was obtained using the image in CMYK color format and crude values of magenta color (Spearman correlation coefficient=0.84; agreement of 93.33%, P<0.001). To obtain this value, only 3 steps were necessary: convert the image to CMYK format, select the area of interest for analysis, and open the color histogram tool to visualize the magenta value.