Elevated Expression of T-Cell Immunoglobulin and Mucin Domain 3 on T Cells from Peripheral Blood in Patients with Cervical Carcinoma.

OBJECTIVE: To investigate the expression of T-cell immunoglobulin and mucin domain 3 (TIM-3) on peripheral T cells of cervical carcinoma patients. METHODS: Peripheral blood samples from 15 high-grade cervical squamous intraepithelial lesion (HSIL) patients, 24 cervical carcinoma patients, and 21 healthy controls were collected. TIM-3 expressions on the surface of peripheral CD4+ T cells and CD8+ T cells were analyzed with flow cytometry. RESULTS: There was significantly lower expression of CD4+ T cells and CD8+ T cells in HSIL patients and cervical carcinoma patients compared with healthy controls. We also found that TIM-3 expression on peripheral CD4+ T and CD8+ T cells of both HSIL patients and cervical carcinoma patients was significantly increased compared to the control group. Further analyses revealed that the expression of TIM-3 on peripheral CD4+ T and CD8+ T cells significantly increased in stage III-IV cervical carcinoma patients compared to stages I-II. CONCLUSION: The increased expression of TIM-3 on CD4+ T cells and CD8+ T cells of patients with cervical carcinoma and HSIL suggests the potential role of TIM-3 in the development and progression of cervical carcinoma, which may be a novel therapy target for cervical carcinoma.

Application of CircEIF4G2 in Screening of Cervical Lesions.

BACKGROUND: The aim was to investigate the expression of circulating RNA EIF4G2 (CircEIF4G2) in cervical cancer and its correlation with clinicopathological features. METHODS: Cervical tissue and peripheral blood serum samples were collected from 148 patients with cervical lesions, including 30 patients with low-grade squamous intraepithelial lesions (LSIL group), 35 patients with high squamous intraepithelial lesion (HSIL group), 28 patients with atypical squamous cells (ASC group), and 55 patients with cervical cancer (CC group). At the same time, cervical biopsy specimens and peripheral blood serum were collected from 40 healthy women (Normal group). RT-PCR was used to detect the expression of CircEIF4G2 in cervical tissues and peripheral blood of each group. Electron microscopy was used to observe the distribution of exosomes CircEIF4G2 in cervical tissues. Meanwhile, the correlation between the expression level of CircEIF4G2 and clinical pathological data of patients was analyzed. In vitro, HeLa cells and primary cervical epithelial cells were cultured for 24 hours. Then, the expression levels of CircEIF4G2 in the two kinds of cells were detected by RT-PCR in medium. Furthermore, primary cervical epithelial cells were co-cultured with HeLa cells to observe the effect of exosome CircEIF4G2 on primary cervical epithelial cells. RESULTS: The expression of CircEIF4G2 in the cervical tissue and serum of the normal group was significantly lower than that in the CC group (p < 0.05), but there was no significant difference between the LSIL group and the HSIL group in the cervical tissue and serum (p < 0.05). The distribution and expression of exosomes CircEIF4G2 in each group were consistent with RT-PCR results under an electron microscope. The results of experiments in vitro showed that the expression level of CircEIF4G2 in HeLa cells was significantly higher than that in primary cervical epithelial cells (p < 0.05). The medium in which Hela cells were cultured for 24 hours was added to the culture medium of primary cervical epithelial cells. The process of exosomes CircEIF4G2 entering primary cervical cancer cells was observed by electron microscopy. CONCLUSIONS: The increased expression of CircEIF4G2 in tissues and serum of cervical lesions may be caused by the secretion of exosomes containing CircEIF4G2 by cervical cancer cells. Therefore, CircEIF4G2 can be used as a marker for the diagnosis of cervical lesions.

A prospective cohort study to evaluate immunosuppressive cytokines as predictors of viral persistence and progression to pre-malignant lesion in the cervix in women infected with HR-HPV: study protocol.

BACKGROUND: Cervical cancer (CC) is caused by a persistent infection of high-risk human papillomavirus (HR-HPV). While most HPV infections are transient, persistent HPV infections are a significant health problem in Mexico. With an estimated HPV prevalence of 10% among women in reproductive age, approximately 25% of these women present at least a positive result in triage test, which according to previous studies is expected to be confirmed as positive CIN-2/3. The immune system has a key role in the natural history of HPV infection; alterations in the cellular immune response are responsible for the failure to eliminate HPV. The objective of this project is to assess the prognostic value of detecting immune markers (IL-10, IL-4, TGFß1, IFNγ, IL-6, and TNFα), the expression of HPV-HR E6/E7 proteins, and the viral load at the cervical level with respect to the persistence or clearance of HR-HPV infection, and the regression or progression of a cervical premalignant lesion. METHODS: A dynamic cohort study is being conducted in women with colposcopic, cytological, and histopathological results negative for squamous intraepithelial lesion (SIL) in the cervix and a positive HPV test; the subjects will be followed-up for 5 years, period from which 3 years have already elapsed, with yearly studies (colposcopy, cytology, and histopathology diagnosis, along with molecular HPV test, quantification of viral load and of IL-10, IL-4, TGFß1, INFγ, IL-6, and TNFα levels, along with the expression of the HR-HPV E6/E7 proteins in the cervix as a viral marker. The outcome will be categorized as viral persistence or clearance; and as SIL persistence, progression, or regression. Binomial and/or multinomial regression models adjusted for potential confounders will be used, associating the relative risk of the outcome with the immune and viral markers evaluated. DISCUSSION: This research will generate knowledge about immune markers with predictive value for the persistence and clearance of HPV, which will improve the triage of positive HPV women and thus reduce the economic burden for the Mexican health system imposed by the management of high-grade SIL and CC cases, which are still detected in late stages.

Human papillomavirus infection and cervical cancer precursor lesions in women living by Amazon rivers: investigation of relations with markers of oxidative stress.

Objective To investigate the relation between oxidative stress markers, human papillomavirus infection and cervical cancer precursor lesions. Methods The study comprised women aged 14 to 60 years living in communities located by Amazon rivers in the state of Pará (Itaituba, Limoeiro do Ajuru and Bragança, 126, 68 and 43 women respectively). Papanicolau smears and polymerase chain reaction tests for human papillomavirus DNA detection were performed. Blood samples were collected to test malondialdehyde, total and oxidized glutathione levels. Results Malondialdehyde, total and oxidized glutathione concentrations did not differ significantly (p>0.05) between women with and without low-grade squamous intraepithelial lesions across communities. Malondialdehyde levels (8.02nmols/mL) were almost five times higher in human papillomavirus-positive compared to human papillomavirus-negative women (1.70nmols/mL) living in Itaituba (statistically significant difference; p<0.05). Malondialdehyde levels did not differ significantly (p>0.05) between human papillomavirus-positive and human papillomavirus-negative women living in remaining communities. Significant (p<0.05) differences in total glutathione levels between human papillomavirus-positive and human papillomavirus-negative women (8.20µg/mL and 1.47µg/mL, respectively) were limited to those living in Bragança. Conclusion Malondialdehyde and total glutathione levels were significantly associated with human papillomavirus infection. However, lack of similar associations with squamous lesions suggest oxidative stress alone does not explain correlations with cervical carcinogenesis. Other factors may therefore be involved.

Therapeutic assessment of vulvar squamous intraepithelial lesions with CO2 laser vaporization in immunosuppressed patients / Avaliação terapêutica das lesões intraepiteliais escamosas da vulva com vaporização a laser CO2 em pacientes com imunossupressão

Abstract Objective A vulvar squamous intraepithelial lesion is deemed to be a preceding lesion to vulvar cancer, especially in women aged under 40 years, holders of an acquired or idiopathic immunosuppression. Several treatments have been used to treat these lesions. One of the aesthetically acceptable therapeutic methods is the CO2 laser vaporization. Methods In a transversal study, 46 records of immunosuppressed women bearing a vulvar low grade and/or high grade squamous intraepithelial lesion were selected out of the retrospective analysis, computing age, date of record, date of vulvar lesion treatment with CO2 laser, the time elapsed between the first and the last visit (in months), the number of visits, the presence or absence of condylomatous lesions in other female lower genital tract sites and whether or not recurrences and persistence of intraepithelial lesions have been noticed during the follow-up. Results Patients bearing vulvar high-grade squamous intraepithelial lesion and immunosuppressed (serumpositive forhumanimmunodeficiency virus [HIV] or with solid organs transplantation) have shown a higher level of persistence of lesions and a higher chance of having other areas of the female lower genital tract involved. Conclusion While the CO2 laser vaporization is the most conservative method for the treatment of vulvar high-grade intraepithelial lesions, it is far frombeing the ideal method, dueto the intrinsic infection features considered. The possibility of persistence, recurrences and spontaneous limited regression indicates that a closer surveillance in the long-term treated cases should be considered, in special for immunosuppressed patients.

Resumo Objetivo A lesão intraepitelial escamosa da vulva é considerada uma lesão precursora do câncer vulvar, emespecial emmulheres com idade inferior a 40 anos, portadoras de imunossupressão adquirida ou idiopática. Vários tratamentos têm sido utilizados para tratar esse tipo de lesão. Um dos métodos terapêuticos esteticamente aceitáveis é a vaporização a laser CO2. Métodos Em um estudo transversal, foram selecionados da análise retrospectiva de prontuários arquivados no setor de patologia do trato genital inferior 46 prontuários de mulheres comimunossupressão e portadoras de lesão intraepitelial escamosa de baixo grau e/ou alto grau computando-se: idade, data de registro, data do tratamento da lesão vulvar com laser CO2, tempo entre a primeira e a última consulta (em meses), número de consultas, presença ou ausência de lesões condilomatosas em outros locais do aparelho reprodutor feminino e a ocorrência ou não de recidivas e persistência de lesões intraepiteliais durante o período de acompanhamento. Resultados Pacientes com lesão intraepitelial de alto grau vulvar e imunocomprometidas (soropositivas para HIV ou com transplante de órgãos sólidos) mostraram maior índice de persistência das lesões e maior chance de ter outras áreas do órgão genital feminino comprometidas. Conclusão Embora a vaporização a laser CO2 seja o método mais conservador para o tratamento das lesões intraepiteliais vulvares de alto grau, está longe de ser o método ideal pelas características intrínsecas da afecção em foco. A possibilidade de persistência, recidivas e regressão espontânea limitada recomenda maior vigilância para os casos tratados a longo prazo, em especial para as pacientes com imunossupressão.

Human papillomavirus infection and cervical cancer precursor lesions in women living by Amazon rivers: investigation of relations with markers of oxidative stress / Infecção por papilomavírus humano e lesões precursoras do câncer cervicouterino em Ribeirinhas da Amazônia: avaliação da relação com marcadores de estresse oxidativo

ABSTRACT Objective To investigate the relation between oxidative stress markers, human papillomavirus infection and cervical cancer precursor lesions. Methods The study comprised women aged 14 to 60 years living in communities located by Amazon rivers in the state of Pará (Itaituba, Limoeiro do Ajuru and Bragança, 126, 68 and 43 women respectively). Papanicolau smears and polymerase chain reaction tests for human papillomavirus DNA detection were performed. Blood samples were collected to test malondialdehyde, total and oxidized glutathione levels. Results Malondialdehyde, total and oxidized glutathione concentrations did not differ significantly (p>0.05) between women with and without low-grade squamous intraepithelial lesions across communities. Malondialdehyde levels (8.02nmols/mL) were almost five times higher in human papillomavirus-positive compared to human papillomavirus-negative women (1.70nmols/mL) living in Itaituba (statistically significant difference; p<0.05). Malondialdehyde levels did not differ significantly (p>0.05) between human papillomavirus-positive and human papillomavirus-negative women living in remaining communities. Significant (p<0.05) differences in total glutathione levels between human papillomavirus-positive and human papillomavirus-negative women (8.20μg/mL and 1.47μg/mL, respectively) were limited to those living in Bragança. Conclusion Malondialdehyde and total glutathione levels were significantly associated with human papillomavirus infection. However, lack of similar associations with squamous lesions suggest oxidative stress alone does not explain correlations with cervical carcinogenesis. Other factors may therefore be involved.

RESUMO Objetivo Avaliar a relação de marcadores de estresse oxidativo com a infecção pelo papilomavírus humano e as lesões precursoras do câncer cervical. Métodos Foram incluídas mulheres ribeirinhas da Amazônia (a saber: 126 do município de Itaituba, 68 de Limoeiro do Ajuru e 43 de Bragança), todas do Estado do Pará, de 14 a 60 anos. Foram realizados testes Papanicolau e de reação em cadeia de polimerase para a detecção de DNA do papilomavírus humano. Coleta de amostras de sangue foi realizada para a avaliação do malondialdeído e a determinação da glutationa total e oxidada. Resultados As concentrações oxidativas do malondialdeído e os teores de glutationa total e oxidada não apresentaram diferença significativa (p>0,05) nas mulheres com e sem lesão intraepitelial escamosa de baixo grau, em todas as comunidades pesquisadas. Mulheres com papilomavírus humano em Itaituba apresentaram níveis de malondialdeído (8,02nmols/mL) quase cinco vezes maior quando relacionadas àquelas sem o HPV (1,70nmols/mL), com diferença estatística significativa (p<0,05). As outras comunidades não mostraram diferença estatística significativa entre as concentrações de malondialdeído nas mulheres com e sem papilomavírus humano (p>0,05). Os teores de glutationa total mostraram diferença significativa (p<0,05) somente em Bragança naquelas com (8,20µg/mL) e sem a infecção pelo papilomavírus humano (1,47µg/mL). Conclusão Houve associação significativa da resposta oxidativa do malondialdeído e da glutationa total com a infecção pelo papilomavírus humano, porém não houve diferença quando associada à lesão escamosa, sugerindo que o estresse oxidativo isoladamente não explica a relação com a carcinogênese do colo uterino, que deve ser influenciada ainda por outros fatores.

TLR9 gene polymorphism -1486T/C (rs187084) is associated with uterine cervical neoplasm in Mexican female population.

PURPOSE: The aim of this work was to evaluate the association of single nucleotide polymorphisms in TLR9 (-1486 T/C [rs187084], -1237T/C [rs5743836] and G2848A [rs352140]) with HPV infection, squamous intraepithelial lesions, and uterine cervical neoplasm in a Mexican population. Additionally, the peripheral expression of TLR9 was evaluated to evaluate the differences in the TLR9 expression associated with every genotype in the locus -1486 of the TLR9 gene. The serum concentration of TLR9 was evaluated in a randomly selected subsample. METHODS: Genotyping was performed using predesigned 5' endonuc lease assays and the association of the polymorphisms with the diagnosis groups were assessed by performing multinomial regression models. The relative expression of TLR9 in peripheral blood mononuclear cells was evaluated by real-time polymerase chain reaction and the association of the level of TLR9 expression with the diagnosis was evaluated by performing multinomial regression models. The serum concentration of TLR9 was evaluated in a subsample of patients diagnosed with uterine cervical neoplasm by ELISA. RESULTS: The results showed that genotype TT in the -1486 locus of TLR9 was significantly associated with HPV infection (OR = 3.25, 95% CI 1.12-9.46), squamous intraepithelial cervical lesion (OR = 3.76, 95% CI 1.36-10.41), and uterine cervical neoplasm (OR = 5.30, 95% CI 1.81-15.55). Moreover, the highest level of TLR9 expression was significantly associated with a greater risk for developing squamous intraepithelial cervical lesion and uterine cervical neoplasm. The serum TLR9 concentration was higher in patients with uterine cervical cancer than in controls. CONCLUSION: Our findings indicate that genotype TT in the -1486 locus of the TLR9 gene could comprise a risk genotype for HPV infection, squamous intraepithelial cervical lesion, and uterine cervical neoplasm in Mexican female population. Further studies with larger samples are needed to evaluate if the peripheral expression of TLR9 could be used as a biomarker of uterine cervical neoplasm progression.

The role of oxidative stress in premalignant lesions.

PURPOSE OF INVESTIGATION: The authors aimed to evaluate serum total oxidant status (TOS), total antioxidant status (TAS), and oxida- tive stress index (OSI) in women with abnormal cervical cytology, to determine the association between serum oxidant and antioxidant status of these women, and the progression of abnormal cervical cytology. MATERIALS AND METHODS: A total of 75 women enrolled in the study: 20 women with a determination of atypical squamous cells of undetermined significance (ASCUS), 20 women with low squamous intraepithelial lesions (LSIL), 15 women with high squamous intraepithelial lesions (HSIL) and 20 healthy controls. Serum TOS and TAS were determined and OSI was calculated as the indicator of degree of oxidative stress. RESULTS: Serum TOS levels and OSI were highest in the HSIL group and there was a trend toward increasing serum TOS levels and OSI from ASCUS to HSIL group. CONCLUSION: The authors demonstrated that increased oxidative stress with altered antioxidant level is associated with abnormal cervical cytology. Serum oxidant and antioxidant status may provide guidance as a simple and cost-effective method for follow-up, treatment, and recommendation in all stages of lesions.

Detection of high-risk human papillomavirus using menstrual blood in women with high-grade squamous intraepithelial lesions or high-risk human papillomavirus infections: A pilot study.

AIM: Few studies have reported that human papillomavirus (HPV) tests using menstrual blood (MB) may be a convenient and effective screening modality for cervical cancer. Therefore, we aimed to investigate the efficacy of detecting high-risk (HR)-HPV from MB in women with high-grade squamous intraepithelial lesions or HR-HPV infections dependent on menstrual days. MATERIALS AND METHODS: In this prospective exploratory pilot study, a clinical trial was conducted in 19 women. On enrollment into the study, gynecologists collected cervical cells. On the first and second day of menstruation, MB was self-collected by patients using a sanitary pad with a filter. The distribution of HPVs from MB and the accuracy of menstrual HR-HPV tests were evaluated using HPV genotyping. The agreement rate of detecting HR-HPVs using cervical and MB samples was also investigated. RESULTS: The sensitivity, specificity, positive and negative predictive values of the MB HR-HPV test for detecting cervical intraepithelial neoplasia (CIN) 3 or worse were 87.5%, 45.5%, 53.8%, and 83.3%, respectively, during both menstrual cycle day (MCD) 1 and 2 and MCD 1 only; and 62.5%, 27.3%, 38.5%, and 50.0%, respectively, during MCD 2 only. For CIN 3 or worse, the agreement rate between positive cervical and MB HR-HPV test results was 87.5% during MCD 1 and 62.5% during MCD 2. CONCLUSIONS: We demonstrated the possibility of using the MB HPV test as a screening modality for cervical cancer.

The risk of high-grade squamous intraepithelial lesions in women with low serum levels of vitamin A.

BACKGROUND: The progression of the cervical squamous intraepithelial lesion (SIL) is associated with many cofactors, and nutritional aspects are gradually assuming an important role in understanding the physiopathogenesis. OBJECTIVE: To assess the serum levels of retinol in women from a poor region of Brazil with a histological diagnosis of cervical high-grade SIL (HSIL). METHODS: A cross-sectional study was conducted in 62 women aged 14-48 years who had a cervical biopsy and were treated at the Federal University of Ceara in Fortaleza, Brazil. Serum retinol levels were measured in peripheral blood by a spectrophotometry technique. Values were categorized as subnormal (<20 µg/dl) or normal (≥20 µg/dl). Multivariate logistic regression was used to determine the association between serum levels of retinol and HSIL for a confidence interval of 95%. RESULTS: Twenty-six out of 62 women (42%) had a negative histopathological result for SIL and 36 (58%) were diagnosed with SIL (20 low SIL and 16 high SIL). The level of retinol was more frequently low in women with HSIL, but there was no statistical significance [p = 0.409, OR: 2.26 (0.33-15.59)]. CONCLUSION: There was no association between high SIL and low levels of retinol in peripheral blood.

Evaluation of soluble CD44 protein marker to distinguish the premalignant and malignant carcinoma cases in cervical cancer patients.

The main objective of the present study was to estimate the levels of soluble CD44 in cervical cancer patients by determining whether it consistently discriminates the carcinoma of the cervix from early or premalignant stage of the cervical cancer. Serum concentrations of sCD44s in cervical cancer patients were determined by an enzyme-linked immunosorbent assay from serum of 50 cases of cervical cancer patients and 50 cases of suspected patients with premalignant disease of cervical intraepithelial neoplasia. The sensitivity and specificity of the test for differentiating carcinoma of the cervix from premalignant stage were evaluated by plotting receiver operating characteristic (ROC) curve. Significant increase in the levels of soluble CD44 was observed in cervical cancer patients (664.80 ± 26.58 ng/ml), when compared to healthy (P < 0.001) and suspected (P < 0.05) or premalignant cases (275.19 ± 24.39 and 514.33 ± 54.57 ng/ml, respectively). High-grade squamous intraepithelial lesions, adenocarcinoma in situ and premalignance with dysplasia show significant (P < 0.001) increase in the concentration of soluble CD44 levels when compared to other types. A ROC curve was plotted and estimated the threshold value as 633.11 ng/ml. In conclusion, the data indicated an up-regulation of soluble CD44 protein which detect and differentiates the cervical carcinoma from premalignant cases with 62.6 % sensitivity.

Serum one-carbon metabolites and risk of cervical cancer.

Most cases of cervical cancer are associated with human papilloma virus (HPV) infection of high risk types. In folate deficiency, heterogeneous nuclear ribonucleoprotein E1 (hnRNP-E1) interferes with HPV16 viral capsid protein synthesis. We aimed to study the importance of 1-carbon metabolism in cervical carcinogenesis by examining serum vitamin B12 (cobalamin), homocysteine, folate levels, and the RNA and protein expression of HPV16 L1, L2, E6, E7, and to correlate them with hnRNP-E1 expression and HPV infection in normals, squamous intraepithelial lesions (SILs), and cervical cancer subjects. Serum cobalamin, folate, and homocysteine were estimated using kits, RNA by real time PCR and proteins by Western blotting. We observed that lower folate and vitamin B12 levels were associated with HPV infection. hnRNP-E1 progressively decreased from normals (100%) to SILs (75%) to cervical cancer (52.6%). The findings show that HPV16 E6 and E7 are overexpresed whereas HPV16 L1 and L2 are downregulated at mRNA and protein levels in cervical cancer as compared to normals and SILs. The results indicate that perhaps the reduced expression of hnRNP-E1 might be involved with the cervical cancer pathogenesis, with folate playing a role in the natural history of HPV infection.