High-grade Cervical Intraepithelial Neoplasm Research Articles

P16 expression in uterine cervical lesions and its role as diagnostic markers and clinical management

Background: Cervical cancer is one of the world's third most common gynecologic malignant tumors, especially in developing countries. This cancer is the most common cause of cancer-related death. High-risk human papillomavirus (HPV) is the main cause. p16 is a biological marker that can be used as a surrogate marker to increase diagnostic agreement for high-risk HPV infection. This study proved the differences in p16 expression in various age groups in low-grade cervical intraepithelial neoplasm (CIN), high-grade CIN, and squamous cell carcinoma (SCC). Methods: The research design was a cross-sectional analytic observational study, with a total sample of 40, taken from biopsy and surgery specimen patients diagnosed with CIN and SCC whose tissues were examined at the Anatomical Pathology Laboratory Faculty of Medicine Universitas Udayana/ Prof. dr. I G.N.G. Ngoerah General Hospital Denpasar. Histopathological diagnosis and determination of the type of cervical lesion were performed by Hematoxylin and Eosin (H&E) staining. p16 expression was evaluated by immunohistochemistry. The results were analyzed using the chi-square test to prove differences in p16 expression in various age groups and the Kruskal-Wallis Test followed by the Mann-Whitney Test to prove differences in p16 expression in low-grade CIN, high-grade CIN and SCC, with a significance level of p < 0.05. Results: The mean age of low-grade CIN was younger than high-grade CIN, which was 45.5±8.44 compared to 46.1±9.11. The mean age of low-grade and high-grade CIN is also younger than the mean age of SCC, which is 50.9±10.80. There was no significant difference in the expression of p16 in low grade CIN (p=0.34), high-grade CIN (p=0.45) and SCC (p=0.28) in various age groups. The expression of p16 was found significantly differ in all groups (p=0.011). Low-grade CIN significantly differed from high grade CIN (p=0.012) and SCC (p=0.008). However, we found same p16 expression in high-grade CIN and SCC (p = 0.866). Conclusion: p16 can be used as a screening method for diagnostic markers of high-risk HPV infection and a reference for planning therapy.

Study of Frequency of High Grade Cervical Intraepithelial Neoplasms (CIN 2, 3) in Patients Infected with Uncommon High Risk Human Papillomavirus

Introduction: Despite the significant relationship between human papillomavirus 16 and 18 subtypes and severe cases of cervical cancer, the assessing the frequency of high-grade cervical intraepithelial neoplasms in the patients infected with high-risk human papillomavirus other than 16 and 18 has been less studied in Iranian population.
 Methods: In this cross-sectional study, the patients referred to Oncology Clinic of Shahid Beheshti Hospital in Isfahan, Iran were selected by positive HPV-typing test through Cobas HPV test and high-risk papilloma virus in 2018 by convenience sampling method. Pap smear was first taken from the patients in the usual way, then they underwent colposcopy, and a biopsy was performed on the lesion during colposcopy. If colposcopy was normal, biopsies were randomly taken from 4 quadrants (12, 3, 9, 6 hours) and, if necessary, endocervical curettage was performed. Samplings were taken by the pathologist of the study center and the prevalence of CIN2 and CIN3 was reported. ANOVA test was performed to compare quantitative variables. Chi-square test was used to compare qualitative variables. SPSS version 17 software was used for statistical analysis of data. Significance level was considered less than 0.05.
 Results: In this study, 105 individuals with high-risk HPV other than 16 and 18 were evaluated. In the biopsy performed according to the standard diagnostic method, the prevalence of severe form of precancerous lesions (CIN 2, 3) was 9.6% and the most common types of HPV in the patients with CIN 2, 3 were 52 with a frequency of 40% and type 58 with a frequency 30%.
 Conclusion: Due to the significant prevalence of severe (precancerous) lesions in these patients, it is necessary to review the current protocols.

Effects of hnRNP E1 and both early genes E2 and E6 of HPV16 together with their interactions on cervical carcinogenesis

Objective: To explore the effects of hnRNP E1 and both early genes E2 and E6 of HPV16 as well as their interactions in the progression of cervical carcinogenesis. Methods: Subjects of this study included 56 women with normal cervix (NC), 58 patients with low-grade cervical intraepithelial neoplasm (CINⅠ) and 50 patients with high-grade cervical intraepithelial neoplasm (CINⅡ/Ⅲ) who were all recruited from the 'Cervical Lesions Study Cohort Project' in Jiexiu of Shanxi province from June to September, 2014. Another 40 patients with cervical squamous cell carcinoma (SCC) were from the Shanxi Tumor Hospital during the same period. Information related to cervical lesions were collected, using a structured questionnaire, with cervical tissues and cervical exfoliated cells gathered from all the participants. HPV infection was detected by flow-through hybridization, while the levels of expression on hnRNP E1, HPV16 E2 and E6 protein were measured by Western Blot. Kruskal-Wallis H test, χ(2) test, trend χ(2) test were analyzed by SPSS 22.0 software, while interaction was evaluated by generalized multifactor dimensionality reduction (GMDR). Results: The overall infection rates of HPV16 related to CINⅠ (15.52%, 9/58), CINⅡ/Ⅲ (40.00%, 20/50) and SCC (67.50%, 27/40) groups were all higher than that of the NC group (8.93%, 5/56) and with an increasing trend on the severity of cervical lesions (trend χ(2)=43.613, P<0.001). The levels of expression on hnRNP E1 protein were significantly different in the groups with different cervical lesions (H=9.98, P=0.019), showing a decreasing trend with the severity of cervical lesions (trend χ(2)=9.495, P=0.002). The levels of expression on HPV16 E2 (H=16.20, P=0.001) and HPV16 E6 (H=15.44, P=0.001) were significantly different in groups with different cervical lesions. Results of GMDR showed that the best interaction model in both groups of CINⅡ/Ⅲ and SCC appeared as hnRNP E1 low expression, HPV16 E2 low expression and HPV16 E6 high expression. However, no similar interaction was seen in CINⅠ (P>0.05). Conclusions: Both low expressions of hnRNP E1 and abnormal expression of HPV16 E2 and E6 could increase the risk of high-grade CIN and cervical cancer. It seemed that they might have an important synergistic effect on the progression of cervical cancer.

Meta-analysis on the performance of p16/Ki-67 dual immunostaining in detecting high-grade cervical intraepithelial neoplasm.

Although routine screening contributes to substantial reductions in cervical cancer morbidity and mortality, the low specificity of HPV detection and limited sensitivity of cervical cytology necessitates the application of more optimized markers, such as the newly-introduced p16/Ki-67 dual-staining method. Here we reviewed several studies to evaluate the performance of this method in cervical cancer screening. An electronic database search was performed on PubMed, Web of Science, CNKI and Wanfang Database for studies assessing p16/Ki-67 dual immunostaining in the diagnosis of high-grade cervical intraepithelial neoplasm (HGCIN) with abnormal cytological morphologies. Two reviewers screened literatures, extracted data and assessed the quality of the included studies independently. Meta-analysis was performed using ReV. Man 5.2 and Meta-DiSc 1.2 software packages. The absolute sensitivity of p16/Ki-67 dual staining for diagnosing HGCIN ranged from 80% to 94%, while the sensitivity of triage method with hrHPV testing ranged from 78% to 96%. The specificity of p16/Ki-67 testing and hrHPV detection for predicting absence of CIN2+ ranged from 39% to 79% and 15% to 44%, respectively. Quantitative meta-analysis showed that the pooled sensitivity of p16/ki-67 dual staining is 0.88 [95'CI (0.86-0.90)], the pooled specificity is 0.58 [95'CI (0.56-0.60)]. For hrHPV testing, the pooled sensitivity and pooled specificity is 0.94 [95'CI (0.93-0.96)] and 0.32 [95'CI (0.29-0.34)], respectively. p16/Ki-67 dual immunostaining had comparable sensitivity and improved specificity in screening HGCIN or CC when compared with hrHPV detection. Further studies may be beneficial to assess the efficacy of this novel biomarker, which can be potentially used as one of the initial screening assays.

Cytokeratin7 and cytokeratin19 expression in high grade cervical intraepithelial neoplasm and squamous cell carcinoma and their possible association in cervical carcinogenesis

BackgroundHigh risk human papillomavirus (HR HPV) infects cells at the squamocolumnar junction (SCJ) of the cervix, causing cancer. Cytokeratin (CK)7 is an SCJ marker, and stains cervical neoplasia. CK19 is a binding partner of CK7 and expressed in cervical cancer. Despite this possible association between CK7/CK19 and cervical cancer, not much is known about the mechanism of CK7/CK19 involvement in HR HPV-mediated cervical carcinogenesis.MethodsWe analyzed the expression pattern of CK7, CK19, and p16 by using immunohistochemistry and HPV infection by in situ hybridization in 25 cases of high grade cervical intraepithelial neoplasia (CIN3) and in 30 cases of squamous cell carcinoma (SCC).ResultsCK19, p16, and HPV expression was positive in all CIN3 and SCC cases. CK7 expression was positive in all CIN3 cases and in 20/30 (66%) SCCs. Each protein showed diffuse or patchy staining with topographic distinction. Patchy staining of CK7 and episomal HPV DNA overlapped in the upper layer of CIN3 and central portion of an invasive nest in the SCC, whereas patchy CK19 staining and integrated HPV DNA were usually noted in the lower layer of CIN3 and the periphery of the SCC nest. The p16 staining pattern coincided with that of CK19 in a subset of SCC.ConclusionThese results suggest that CK7 may be more related with viral episomal replication and CK19 with viral integration, contributing to viral replication and malignant transformation in HR HPV infected cells. In addition, coordinate CK7/CK19 staining may be used as a valuable marker for predicting physical status of HR HPV and E7 oncoprotein level in cervical tumor.

S100A7 promotes the migration, invasion and metastasis of human cervical cancer cells through epithelial-mesenchymal transition.

S100A7 is an EF-hand calcium-binding protein that has been suggested to be implicated in cell proliferation, migration, invasion and tumor metastasis. However, its role in cervical cancer has not yet been fully clarified. The present study used immunohistochemistry analysis of S100A7 in clinical specimens of cervical cancer to show that S100A7 expression was significantly upregulated in cervical cancer tissues compared with normal cervical tissues and S100A7 expression in high grade cervical intraepithelial neoplasm (CIN) was significantly higher than cervical cancer. Statistical analysis showed that S100A7 expression was associated with tumor grade (P <0.01) and lymph node metastasis (P <0.05). Functional studies showed that overexpression of S100A7 in cervical cancer cells promoted migration, invasion and metastasis of cervical cancer cells without influencing cell proliferation. Furthermore, S100A7 was found to be secreted into the conditioned media and extracellular S100A7 enhanced cell migration and invasion. Mechanistically, S100A7 bound to RAGE and activated ERK signaling pathway. And S100A7 enhanced cell mesenchymal properties and induced epithelial–mesenchymal transition. In summary, these data reveal a crucial role for S100A7 in regulating cell migration, invasion, metastasis and EMT of cervical cancer and suggest that targeting S100A7 may offer a new targeted strategy for cervical cancer.

Interaction between folate and the expression of human papillomavirus 16 E6/E7 mRNA in the progression of cervix carcinogenesis

To explore the interaction between folate and the expression of HPV16 E6/E7 mRNA in the progression of cervix carcinogenesis. Subjects were selected from the participants who were diagnosed pathologically, including 64 patients with cervical squamous cell carcinoma (SCC), 55 patients with low-grade cervical intraepithelial neoplasm (CIN1), 55 patients with high-grade cervical intraepithelial neoplasm (CIN2+) and 80 with normal cervix (NC). The levels of serum folate and RBC folate were detected by microbiological assay, and the expression levels of HPV16 E6/E7 mRNA were measured, using the real-time polymerase chain reaction (real-time PCR). Data was analyzed by methods as chi-square test, analysis of variance (ANOVA), Welch test, Kruskal-Wallis H test and ordinal logistic regression. Spearman correlation was tested using the SPSS statistical software (version 16.0) while the interaction effects were evaluated by additive model. There was a positive correlation seen between the serum folate and RBC folate (r=0.41, P<0.001). The levels of serum folate and RBC folate decreased gradually along with the severity of cervical lesions (χ(2)=32.71, P<0.001; χ(2)=16.32, P<0.001). The expression levels of HPV16 E6/E7 mRNA increased gradually with the severity of cervical lesions (χ(2)=30.11, P<0.001; χ(2)=38.99, P<0.001). There was a negative correlation between the levels of RBC folate, expression levels of HPV16 E6 (E6: r=-0.14, P=0.009) and HPV16 E7 mRNA (E7: r=-0.21, P=0.001), respectively. Both RBC folate deficiency and HPV16 E6/E7 mRNA high expression showed additive interaction in CIN1,CIN2+ and SCC. Folate deficiency and high expression of HPV16 E6/E7 mRNA might increase the risk of cervical cancer and cervix precancerous lesions, and having a synergistic action in the progression of cervix carcinogenesis.

Differences in the ARID-1 alpha expressions in squamous and adenosquamous carcinomas of uterine cervix.

AT-rich interactive domain 1A (ARID1A) is a tumor suppressor gene involved in chromatin remodeling which encodes ARID1A (BAF250a) protein. Recent studies have shown the loss of ARID1A expression in several types of tumors. This retrospective study was designed to evaluate the differences in tissue expressions of ARID1A in a spectrum of cervical neoplasms. Cervical intraepithelial neoplasms, invasive squamous or adenosquamous carcinomas were identified in 100 patients recently diagnosed as cervical neoplasms based on pathology databases. In this series, there were 29 low- and 29 high-grade cervical intraepithelial neoplasms, 27 squamous cell carcinomas, and 15 adenosquamous carcinomas. Mean age of the patients was 47.8±13years (20-80years). It was determined that the expression of ARID1A was statistically significantly down-regulated in adenosquamous carcinomas when compared with non-invasive or invasive squamous cell carcinomas (p=0.015). Lower levels of the ARID1A expression were detected in cases with adenosquamous carcinomas (60%), low- or high-grade squamous intraepithelial lesion (SIL) (31%), and squamous cell carcinomas (18.5%). Our findings have demonstrated the presence of a correlation between ARID1A expression and adenomatous differentiation of uterine squamous cell carcinomas. Therefore, ARID1A gene may suggestively have a role in the pathogenesis of cervical adenosquamous carcinomas.

New frontiers in cancer therapy: the 11th International Meeting of the Asian Clinical Oncology Society (ACOS)

New frontiers in cancer therapy: the 11th International Meeting of the Asian Clinical Oncology Society (ACOS)

Determination of Human Papillomavirus (HPV) Load and Type in High-Grade Cervical Lesions Surgically Resected from HIV-Infected Women during Follow-up of HPV Infection

The role of human papillomavirus (HPV) load and the importance of multiple-strain HPV infections as biomarkers for the development of cervical disease were evaluated in human immunodeficiency virus (HIV)-positive women. A total of 108 samples were analyzed, 64 of which were obtained from 16 HIV-positive women who underwent surgical resection of the cervical cone for treatment of a histologically confirmed high-grade cervical intraepithelial neoplasm (cases) and 44 of which were obtained from 22 HIV-positive women who had high-risk HPV but a negative colposcopy result (controls). Each patient underwent periodic examinations at 6-12-month intervals that included colposcopy, Papanicolaou testing, biopsy (if indicated), and cervical brushing for HPV testing. Viral typing was performed by reverse dot-blot hybridization and quantification of viral load by in-house real-time PCR and commercial assays. Analysis of the cervical-brush samples collected when high-grade squamous intraepithelial lesions were diagnosed revealed that all cases had HPV loads that were significantly higher than those of controls (P=.0004 and P=.0003, by PCR and the Hybrid Capture 2 index [Digene], respectively). Decreasing concentrations of HPV load were observed when comparing samples obtained before and after treatment (P<.0001). The number and type of HPV strains that were detected were not statistically different between cases and controls. The significantly higher HPV load detected in women with high-grade cervical dysplasia, as well as the dramatic decrease in the load after surgical removal of the lesion, suggest that HPV load is a possible prognostic marker of high-grade SIL.

Regulation of aminopeptidase A expression in cervical carcinoma: role of tumor–stromal interaction and vascular endothelial growth factor

We previously demonstrated that aminopeptidase A (APA), a membrane-bound metallopeptidase degrading bioactive peptides such as angiotensin II (Ang II), is expressed in neoplastic lesions of the uterine cervix, and that its expression is upregulated as the lesion progresses from cervical intraepithelial neoplasms (CIN) toward invasive squamous cell carcinomas (SCC). The present study investigated the regulatory mechanisms involved in APA expression and its potential role in cervical carcinoma. Immunohistochemical staining in high-grade CIN and SCC tissues showed that APA was strongly expressed at the edge of lesions adjacent to cervical stromal cells. Fluorescence-activated cell sorting analysis demonstrated that cell surface APA expression was extremely low in three human SCC cell lines, SiHa, TCS and CaSki, under basal conditions. However, both contact and noncontact cocultures with human cervical fibroblasts resulted in the induction of APA expression in these SCC cells. APA expression was also induced in vivo when TCS cells were subcutaneously inoculated into nude mice. Furthermore, APA expression and enzymatic activity were enhanced by addition of the conditioned medium (CM) from fibroblast culture, but not by heat-treated CM. Among the various cytokines tested, vascular endothelial growth factor (VEGF) significantly increased APA activity, and induction of APA by the fibroblast CM was partly inhibited by anti-VEGF neutralizing antibody. Finally, APA cDNA-transfected APA-overexpressing TCS cells significantly reduced the Ang II-induced cell invasion ability as compared with parental or control vector-transfected TCS cells, although there was no significant difference in cellular proliferation among them. These results suggested the importance of tumor–stromal interaction for the regulation of APA expression in the microenvironment of cervical carcinoma and the potential role for this peptidase in regulating tumor invasion through inactivation of Ang II activity.

Immunohistochemical detection of metallothionein and MIB1 in uterine cervical squamous lesions.

Metallothioneins (MTs) are ubiquitous low molecular weight proteins with a high affinity for heavy metal ions such as zinc, copper, cadmium, and platinum. Immunohistochemically detectable MT overexpression has been demonstrated in a variety of cancers, especially breast carcinoma. In this study, the immunohistochemical expression of MT in normal cervical squamous epithelia, cervical intraepithelial neoplasms (CINs), and invasive cervical squamous carcinomas was investigated. Immunohistochemical staining for proliferating cells using the MIB1 antibody was also performed. In normal squamous epithelia (n = 31), positive staining with MT was confined to basal and parabasal cells. In cases of koilocytosis (n = 14) and CIN I (n = 10), staining was also largely confined to basal and parabasal cells, with only occasional cases of CIN I exhibiting positivity within higher cell layers. Cases of CIN II (n = 14) showed positive staining largely confined to basal and parabasal cells, with staining of higher cell layers in a few cases. In the majority of cases of CIN III (n = 29), there was diffuse positive staining throughout the full epithelial thickness and, in almost all cases, positive staining was present above the basal and parabasal layers. Positive staining was present in 19 of 21 invasive squamous carcinomas. With MIB 1, positivity was confined to the parabasal layer in normal squamous epithelia. In cases of CIN, positive cells were present in progressively higher cell layers, in accordance with the grade of CIN. There was widespread positive staining in all cases of invasive squamous carcinoma. Overexpression of MT, demonstrated immunohistochemically, is associated with CIN III and invasive cervical squamous carcinoma, lesions which exhibit the highest proliferative activity, as shown by MIB1 immunostaining. MT overexpression in cervical squamous lesions appears to occur at some point along the spectrum of high grade CIN and may be related to cell proliferation.

Investigation of the Bcl-2 and C-myc expression in relationship to the Ki-67 labelling index in cervical intraepithelial neoplasia.

This is the investigation of the relationship between the neoplastic cell proliferation and the expression of bcl-2 and c-myc in human papillomavirus (HPV)-negative and HPV-positive cervical intraepithelial neoplasms (CIN). The expression of bcl-2 and c-myc was studied using quantitative immunohistochemistry in 20 specimens of normal cervical squamous epithelium and 73 specimens of CIN. To analyze the neoplastic cell proliferation rate, the Ki-67 labelling index was determined; the latter was significantly different between normal epithelium and various grades of CIN (p < 0.0001). The highest proliferation rate was found in high-grade CIN. In precancerous lesions, we found the number of bcl-2 positive cells lower than in normal epithelium, but with a significant difference between low-grade and high-grade CIN (p < 0.0001). The highest percentage of bcl-2 positive neoplastic cells was found in high-grade CIN. C-myc was rarely expressed in normal epithelium. Similar to the Ki-67 labelling index, c-myc immunostaining correlated with the histological grade of CIN, with the highest percentage of c-myc positive nuclei occurring in high-grade CIN (p < 0.0001). In contrast to bcl-2 immunoreactivity, the c-myc significantly was more expressed in high-risk HPV-positive than in HPV-negative lesions. The c-myc expression in CIN is closely related to the neoplastic cell proliferation rate. With progression of intraepithelial neoplasia, bcl-2 production in neoplastic cells increases with high co-expression of c-myc.

Viral and histopathologic correlates of MN and MIB-1 expression in cervical intraepithelial neoplasia

A recently studied tumor antigen, MN, has been associated with cervical carcinomas and cervical intraepithelial neoplasms (CIN), suggesting that it may serve as a marker for cervical cancer or cancer risk. To determine if expression of the MN antigen paralleled parameters reflecting viral or biological events in precursor epithelium, MN expression was correlated with MIB-1 expression, morphological phenotype, and human papillomavirus (HPV) distribution and type in a series of CINs. Seventy-three percent, 62% and 83% of CIN I, II, and III, respectively, were MN antigen positive. The proportion of neoplastic cells immunoreactive for MN did not correlate with the CIN grade or with HPV types stratified by their association with cancer. Evaluation of serial sections showed no correlation between the frequency of MN antigen staining, the proportion of MIB-1 immunoreactive cells, or the proportion of HPV positive cells detected by in situ hybridization (ISH). CINs associated with prototypical high risk (HPV 16) types exhibited increased immunostaining for the MIB-1 antigen and were more often classified as HSIL in contrast to the other types. Thus, although MN expression previously has been associated strongly with squamous carcinoma, it did not emerge as a specific marker for either cancer-associated HPV types or high grade CIN. CIN I lesions associated with low and high risk HPV types were not distinguished by MIB-1 expression and viral replication. This emphasizes the interrelationship between vegetative viral functions (including viral replication) and morphological phenotype, irrespective of HPV type.

HLA class I expression and HPV-16 sequences in premalignant and malignant lesions of the cervix.

A series of 10 normal cervix epithelia, 38 condylomas, 17 CIN (cervical intraepithelial neoplasm) I/II (low-grade CIN), 10 CIN III (high-grade CIN), 27 squamous cell carcinomas and 7 adenocarcinomas of the cervix were studied in paraffin-embedded sections for the expression of MHC class I antigens, using antibodies against HLA antigens and the immunoperoxidase technique. A PCR technique was also used to evaluate the presence of HPV-16 DNA. All samples from normal tissue, benign, premalignant and CIN III lesions expressed HLA class I antigens. However, 15% of the invasive carcinomas completely lacked HLA-B and HLA-C antigen expression, 20% presented a heterogeneous pattern and 2 cases lacked HLA-B and HLA-C heavy chain but retained beta 2-microglobulin. MHC class I antigen expression on tumors was compared with clinical-pathological parameters. The absence of expression of HLA class I molecules was significantly associated with the Glanz histoprognostic index of malignancy. HPV-16 sequences were detected in 60% of the condylomas, 88% of the CIN I/II, 80% of the CIN III and 82% of the cervical carcinomas. Eight-six per cent of the tumors expressing HLA class I antigen presented HPV-16, whereas only 40% of the nonexpressing tumors did. Our results lead us to the following conclusions: a) HLA class I losses occurred when the tumor became invasive, and in tumors of a more aggressive histological type; b) The presence of HPV-16 was associated with tumors expressing HLA class I antigens.