Low-grade Squamous Intraepithelial Lesion Samples Research Articles

Detection of Complement C1q B Chain Overexpression and Its Latent Molecular Mechanisms in Cervical Cancer Tissues Using Multiple Methods.

Aim The aim of this study is to demonstrate the expression and clinicopathological significance of complement C1q B chain (C1QB) in cervical cancer. Methods In total, 120 cervical cancer tissues, as well as 20 samples each of high-grade squamous intraepithelial lesions (HSILs), low-grade squamous intraepithelial lesions (LSILs), and benign cervical tissue, were collected to evaluate the expression of C1QB protein via immunohistochemical staining. We conducted an integrated analysis of C1QB mRNA expression in cervical cancer using public microarrays and RNA-seq data sets by calculating standard mean differences (SMDs). Simultaneously, we explored the relations of C1QB with clinicopathological parameters and the expression of P16, Ki-67, and P53. Results The expression of C1QB protein was higher in cervical cancer samples than that in benign cervical tissue, LSIL, and HSIL samples (p < 0.05). A combined SMD of 0.65 (95% CI: [0.52, 0.79], p < 0.001) revealed upregulation of C1QB mRNA in cervical cancer. C1QB expression may also be related to the depth of infiltration, lymphovascular invasion, and perineural invasion in cervical cancer (p < 0.05). We also found that C1QB protein expression was positively correlated with P16 and Ki-67 expression in cervical cancer (p < 0.05). The gene set enrichment analysis showed that C1QB may participate in apoptosis and autophagy. A relationship was predicted between C1QB expression and drug sensitivity to cisplatin, paclitaxel, and docetaxel. Conclusion We confirmed the overexpression of C1QB in cervical cancer at both mRNA and protein levels for the first time. C1QB may serve as an oncogene in the tumorigenesis of cervical cancer, but this possibility requires further study.

Promoter methylation analysis of DKK2 may be a potential biomarker for early detection of cervical cancer.

Dickkopf 2 (DKK2) plays an important role in multiple cancers. Its potential value in the clinical diagnosis of cervical cancer has remained unclear. To investigate the expression and promoter methylation levels of DKK2 in cervical cancer and their clinicopathological associations. We used the Gene Expression Omnibus, Oncomine, Cancer Genome Atlas, and University of ALabama at Birmingham CANcer data analysis databases, reverse transcription-PCR, and methylation-specific PCR analysis to predict and examine the expression of DKK2 mRNA and DKK2 methylation levels in cell lines and cervical cancer tissues from 79 patients with cervical cancer and 63 with cervical precancerous lesions including 25 with low-grade squamous intraepithelial lesions (LSIL) and 38 patients with high-grade squamous intraepithelial lesions (HSIL). DKK2 mRNA expression was downregulated in all cancer cell lines and cervical cancer tissues, whereas hypermethylation of DKK2 was higher in cervical cancer tissue samples. DKK2 methylation in cervical cancer was significantly higher than that in HSIL (χ2 = 8.346, P = 0.004), whereas DKK2 methylation in HSIL was significantly higher than that in normal cervical samples (χ2 = 7.934, P = 0.005) and in LSIL samples (χ2 = 4.375, P = 0.037). DKK2 silencing caused by its promoter hypermethylation was confirmed by treatment with the methyltransferase inhibitor 5-Aza-dC in cell lines. Patients with lymph node metastasis exhibited increased promoter methylation frequency (χ2 = 5.239, P = 0.022) and low DKK2 mRNA expression (χ2 = 3.958, P = 0.047) compared with patients with no lymph node metastasis. Patients with high-risk human papillomavirus infection exhibited increased promoter methylation frequency (χ2 = 6.279, P = 0.015). DKK2 epigenetic changes of DKK2 may play a key role in the development of cervical cancer, suggesting that DKK2 hypermethylation could be used as a triage test for screening, early diagnosis, or risk prediction of cervical cancer.

The CpG island methylator phenotype increases the risk of high-grade squamous intraepithelial lesions and cervical cancer

BackgroundHigh-risk human papillomavirus (HR-HPV) infection is the main cause of cervical cancer, but additional alterations are necessary for its development. Abnormal DNA methylation has an important role in the origin and dissemination of cervical cancer and other human tumors. In this work, we analyzed the methylation of eight genes (AJAP1, CDH1, CDH13, MAGI2, MGMT, MYOD1, RASSF1A and SOX17) that participate in several biological processes for the maintenance of cell normality. We analyzed DNA methylation by methylation-specific PCR (MSP) and HPV infection using the INNO‑LiPA genotyping kit in 59 samples diagnostic of normal cervical tissue (non-SIL), 107 low-grade squamous intraepithelial lesions (LSILs), 29 high-grade squamous intraepithelial lesions (HSILs) and 51 cervical cancers (CCs).ResultsWe found that all samples of LSIL, HSIL, and CC were HPV-positive, and the genotypes with higher frequencies were 16, 18, 51 and 56. In general, the genes analyzed displayed a significant tendency toward an increase in methylation levels according to increasing cervical lesion severity, except for the CDH13 gene. High CpG island methylator phenotype (CIMP) was associated with a 50.6-fold (95% CI 4.72–2267.3)-increased risk of HSIL and a 122-fold risk of CC (95% CI 10.04–5349.7).ConclusionsWe found that CIMP high was significantly associated with HSIL and CC risk. These results could indicate that CIMP together with HR-HPV infection and other factors participates in the development of HSIL and CC.

The significance of ASC-H and LSIL dual interpretation with risk stratification: one institution experience.

The 2014 Bethesda System categorizes squamous lesions as low-grade squamous intraepithelial lesions (LSIL) and high-grade squamous intraepithelial lesions (HSIL). It also includes intermediate morphologic terminology, such as atypical squamous cells of undetermined significance (ASC-US) and atypical squamous cells, cannot rule out a high grade squamous intraepithelial lesion (ASC-H). Consensus is lacking if when ASC-H is present in an unequivocal LSIL (LSIL + ASC-H) versus ASC-H alone predicts a neoplasm with a different biologic behavior and which is its association with high-risk human papillomavirus (HPV). We reviewed the Columbia University Medical Center Pathology department patient's database from October 2012 through December 2014 and found 2498 cytology samples of LSIL, ASC-H, HSIL, and LSIL + ASC-H with both follow-up histologic samples and HPV tests by Roche cobas. Our objective was to identify, if any, differences in biologic behavior and HPV status present in LSIL + ASC-H compared with ASC-H and other lesions. CIN2+ was documented in tissue examination in 102 from 311 LSIL + ASC-H (32.8%), 101 from 219 ASC-H (46.1%), 252 from 326 HSIL+ (77.3%), and 150 from 1642 LSIL (9.08%). HPV distribution shows significant differences between all diagnostic categories. LSIL + ASC-H appears to have a distinctive HPV distribution pattern that clearly differs from ASC-H and LSIL and approaches HSIL; however, the predictive value for CIN2+ appears higher for ASC-H than LSIL + ASC-H. Our literature review identified conflicting findings, probably suggesting a lack of reproducibility in cytologic criteria and the need for consistent inclusion of ASC-H and LSIL when both are present.

Human Papillomavirus Infections in Cervical Samples From HIV-Positive Women: Evaluation of the Presence of the Nonavalent HPV Genotypes and Genetic Diversity.

Non-nonavalent vaccine (9v) Human papillomavirus (HPV) types have been shown to have high prevalence among HIV-positive women. Here, 1444 cervical samples were tested for HPV DNA positivity. Co-infections of the 9v HPV types with other HPV types were evaluated. The HPV81 L1 and L2 genes were used to investigate the genetic variability of antigenic epitopes. HPV-positive samples were genotyped using the HPVCLART2 assay. The L1 and L2 protein sequences were analyzed using a self-optimized prediction method to predict their secondary structure. Co-occurrence probabilities of the 9v HPV types were calculated. Non9v types represented 49% of the HPV infections; 31.2% of the non9v HPV types were among the low-grade squamous intraepithelial lesion samples, and 27.3% among the high-grade squamous intraepithelial lesion samples, and several genotypes were low risk. The co-occurrence of 9v HPV types with the other genotypes was not correlated with the filogenetic distance. HPV81 showed an amino-acid substitution within the BC loop (N75Q) and the FGb loop (T315N). In the L2 protein, all of the mutations were located outside antigenic sites. The weak cross-protection of the 9v types suggests the relevance of a sustainable and effective screening program, which should be implemented by HPV DNA testing that does not include only high-risk types.

Protein Phosphorylation in Serine Residues Correlates with Progression from Precancerous Lesions to Cervical Cancer in Mexican Patients.

Protein phosphorylation is a posttranslational modification that is essential for normal cellular processes; however, abnormal phosphorylation is one of the prime causes for alteration of many structural, functional, and regulatory proteins in disease conditions. In cancer, changes in the states of protein phosphorylation in tyrosine residues have been more studied than phosphorylation in threonine or serine residues, which also undergo alterations with greater predominance. In general, serine phosphorylation leads to the formation of multimolecular signaling complexes that regulate diverse biological processes, but in pathological conditions such as tumorigenesis, anomalous phosphorylation may result in the deregulation of some signaling pathways. Cervical cancer (CC), the main neoplasm associated with human papillomavirus (HPV) infection, is the fourth most frequent cancer worldwide. Persistent infection of the cervix with high-risk human papillomaviruses produces precancerous lesions starting with low-grade squamous intraepithelial lesions (LSIL), progressing to high-grade squamous intraepithelial lesions (HSIL) until CC is generated. Here, we compared the proteomic profile of phosphorylated proteins in serine residues from healthy, LSIL, HSIL, and CC samples. Our data show an increase in the number of phosphorylated proteins in serine residues as the grade of injury rises. These results provide a support for future studies focused on phosphorylated proteins and their possible correlation with the progression of cervical lesions.

The clinical significance of FAM19A4 methylation in high-risk HPV-positive cervical samples for the detection of cervical (pre)cancer in Chinese women

BackgroundTo explore the diagnostic value of FAM19A4 methylation in high-risk human papilloma virus (hrHPV)-positive cervical samples from Chinese women for estimating cervical cancer or its precancerous lesions.MethodsCervical samples from 215 women infected with high-risk HPV were collected by smear testing. We purposely chose 61 patients with cervical cancer, 57 with high-grade squamous intraepithelial lesions (HSIL), 31 with low-grade squamous intraepithelial lesions (LSIL), and 66 without cervical intraepithelial neoplasia (CIN) after histological confirmation. Taqman probe-based quantitative PCR (qPCR) was utilized to detect the methylation status of FAM19A4 in the cervical samples and further evaluate the use of this gene in the diagnosis of cervical cancer.Results(1) An increasing level of FAM19A4 methylation was detected with increasing progression of cervical lesions, with methylation rates of 10.61%(7/66), 35.48%(11/31), 56.14%(32/57) and 93.44%(57/61) in no CIN, LSIL, HSIL and cervical carcinoma samples respectively. (2) In all hrHPV-positive samples, the levels of FAM19A4 methylation in HPV16/18 groups were higher than that in 12 other hrHPV groups (P < 0.05), but there was no significant difference between two groups after grouping cervical lesions into cervical cancer, HSIL, LSIL and no CIN groups (P>0.05). (3)There were no significant differences of FAM19A4 methylation in different clinicopathological parameters of cervical cancer. (4) Though the sensitivity of FAM19A4 methylation test was inferior to that of cytology and FAM19A4 combining with HPV16/18 genotyping, but showed the best specificity with 81.44% both for detection HSIL alone and ≥ HSIL, with favorable youden index (YI) and area under curve (AUC).ConclusionFAM19A4 is a specific biomarker of cancerous lesions of the cervix. FAM19A4 methylation analysis may serve as an auxiliary screening method for diagnosis of cervical (pre)cancer. However, in consideration of the limitations of this retrospective study, prospective population-based studies are necessary for further confirmation of the diagnostic value of FAM19A4 methylation for detection of cervical (pre)cancer in Chinese women.

GENOTYPING OF HUMAN PAPPILOMAVIRUS IN CERVICAL PRECANCEROUS LESION AND SQUAMOUS CELL CARCINOMA AT DR. SOETOMO HOSPITAL, SURABAYA, INDONESIA.

Background:Cervical cancer caused by human papilloma virus (HPV), is the second most common cancer for women. This cancer is distributed worldwide, with ~80% of cases are found in the developing countries. In Indonesia, data of HPV genotypes are still limited and do not represent all regions of the country. Thus, here we report genotyping of HPV samples collected from the Dr. Soetomo Hospital Surabaya Indonesia patients, in 2013.Materials and Method:A cross sectional study was performed using 68 paraffin blocks of low-grade squamous intraepithelial lesion (LSIL), high-grade squamous intraepithelial lesion (HSIL), and squamous cell carcinoma (SCC) cervix.Result:This study showed that HPV genotypes found in LSIL samples are HPV 16, 18, 6/33 or 68/72. Furthermore, those in HSIL are HPV 16, 18, 52, 59, 67, 6/18, 6/45, 16/67, 26/61, or 52/67, while in SCC are HPV 16, 18, 45, 52, 56, 16/18 or 16/45. Single-genotype infection, i.e. by HPV 16, 18, 45, 52, 56, 59, or 67, was observed in 86.77% (59/68) of samples, whereas multiple-genotype infections, i.e. by HPV 6/18, 6/33, 6/45, 16/18, 16/45, 16/67, 26/61, 52/67, or 68/72, was found in 13.23% (9/68) of the samples.Conclutions:The mostly HPV genotype identified in this study is HPV 16 (62.68%), then followed by HPV 18 (20.9%), HPV 45 (5.97%), 52 (5.97%), and 67 (4.48%). HPV 16 and 18 have used as vaccine, and HPV 45 has cross reaction with HPV 18, then HPV 52 and 67 should be considered as the second-generation HPV vaccines.

Paraffin section thickness in immunohistochemical detection of p16 expression in cervical tissue samples

To investigate paraffin section thickness in immunohistochemical detection of p16 expression in cervical tissue samples. p16 immunohistochemical staining was performed in 150 cases of chronic cervicitis, 126 cases of low-grade squamous intraepithelial lesions(LSIL), 96 cases of high-grade squamous intraepithelial lesions (HSIL) and 78 cases of cervical cancer from January 2014 to March 2018 in Zhongshan Boai Hospital. The results of p16 protein expression in paraffin sections with thickness of 2, 3, 4, 5 and 6 μm were compared using Logistic regression analysis. With the increase of slice thickness, the staining of p16 protein in nucleus gradually increased. The thickness of cervical slices in chronic cervicitis and cervical cancer samples had no significant effect on the positive rate of p16 protein(χ2=7.817 and 1.332, both P>0.05), while the thickness of slices in LSIL and HSIL samples had significant effect on the positive rate of p16 protein (χ2=17.688 and 10.182, P<0.05 or P<0.01). The stable and reliable results were obtained when the slices were between 3 and 5 μm thick. Paraffin sections with thickness of 3.0-5.0 μm are recommended for immnohistochemical staining of p16 protein in cervical tissue samples.

Deleted in Malignant Brain Tumor 1 (DMBT1) Expression Pattern in Normal Cervix and at Different Stages of Squamous Intraepithelial Lesions

Background: Cervical cancer is one of the most frequently occurring malignancies in women worldwide, with high mortality rates. Cervical Squamous Cell Carcinoma (SCC) presents previous states of non-invasive precursor lesions, and early stage Low-Grade Squamous Intraepithelial Lesions (LSIL) regress to normal or Atypical Squamous Cells of Undetermined Significance (ASCUS) in approximately 50% of cases. Deleted in Malignant Brain Tumors 1 (DMBT1) is a tumour suppression glycoprotein, which absence is considered a malignancy marker in many epithelial cancers. Objective: To analyse DMBT1’s presence and localization in SCC and precursor lesions. Method: Immunohistochemistry for DMBT1 was performed in cervix biopsies classified as normal, LSIL, HSIL and SCC. Results: DMBT1 was detected at the supranuclear and sometimes infranuclear regions of the endocervix monolayer epithelial cells in normal and HSIL biopsies. In LSIL samples the detection of DMBT1 in endocervix was variable between patients. Also variable was DMBT1 staining in cells of glandular epithelium. The glycoprotein was not detected in the stratified epithelium of the exocervix, regardless of the lesion grade; nor in dysplastic cells. Conclusion: The absence of DMBT1 from endocervix only in some samples of LSIL is promising as a candidate for possible lesion regression potential marker.

Expression analysis of transglutaminase 2 in premalignant lesions of the cervix

BackgroundThe medical management of low-grade squamous intraepithelial lesions (LSIL) is variable, thus a biomarker could assist with the clinical conduct. Type 2 transglutaminase (TG2) has been proposed as a cellular-interfering factor in HPV infection and carcinogenesis. Therefore, this study has the objective of evaluating TG2 expression in LSIL and high-grade squamous intraepithelial lesions (HSIL) and of relating it to the different HPV viral types.MethodsThis study included 146 patients with suspected LSIL or HSIL detected in routine conventional Papanicolaou tests. The presence of HPV DNA and viral typing was defined by the polymerase chain reaction method (PCR). TG2 Immunohistochemistry (IHC) was conducted according to the manufacturer's instructions; IHC was carried out in an Autosteiner-Link 48 Dako equipment. IHC quantitation was performed by relative expression and by using the software Image J. Qualitative variables, such as frequencies and proportions, were compared by using the χ2 test for independent samples. For comparison of the qualitative to the quantitative data, nonparametric Mann-Whitney test was used.ResultsThe association between histopathological examination and TG2 was statistically significant (p <0.05). Results showed that patients with normal cervical histopathology and LSIL are locally associated with TG2 expression levels >50% (p <0.05), and patients with HSIL are associated with no TG2 expression (p <0.05). The analysis of the samples with the Image J software shows a significant (p <0,001) decrease in TG2 immunostaining in HSIL if compared to normal and to LSIL samples. This demonstrates a correlation between the relative quantification and the results provided by Image J. Analysis of HPV types showed a significant association with HPV11 (p = 0.031). This indicates that patients with HPV type 11 had higher TG2 values than patients with different types. Image J analysis showed no significant association between TG2 and HPV viral types.ConclusionThe present data suggest that TG 2 has a high expression in LSIL and normal tissues, and decreased in HSIL. We also observed that its expression is associated with HPV11.

Association between human papillomavirus and Epstein - Barr virus DNA and gene promoter methylation of RB1 and CDH1 in the cervical lesions: a transversal study.

BackgroundHuman papillomavirus (HPV) inactivates the retinoblastoma 1 (RB1) gene by promoter methylation and reduces cellular E-cadherin expression by overexpression of DNA methyltransferase 1 (DNMT1). The Epstein-Barr virus (EBV) is an oncogenic virus that may be related to cervical carcinogenesis. In gastric cancer, it has been demonstrated that E-cadherin gene (CDH1) hypermethylation is associated with DNMT1 overexpression by EBV infection. Our aim was to analyze the gene promoter methylation frequency of RB1 and CDH1 and verify the association between that methylation frequency and HPV and EBV infection in cervical lesions.MethodsSixty-five samples were obtained from cervical specimens: 15 normal cervices, 17 low-grade squamous intraepithelial lesions (LSIL), 15 high-grade squamous intraepithelial lesions (HSIL), and 18 cervical cancers. HPV and EBV DNA testing was performed by PCR, and the methylation status was verified by MSP.ResultsHPV frequency was associated with cervical cancer cases (p = 0.005) but not EBV frequency (p = 0.732). Viral co-infection showed a statistically significant correlation with cancer (p = 0.027). No viral infection was detected in 33.3% (5/15) of controls. RB1 methylated status was associated with cancer (p = 0.009) and HPV infection (p = 0.042). CDH1 methylation was not associated with cancer (p = 0.181). Controls and LSIL samples did not show simultaneous methylation, while both genes were methylated in 27.8% (5/18) of cancer samples. In the presence of EBV, CDH1 methylation was present in 27.8% (5/18) of cancer samples. Only cancer cases presented RB1 promoter methylation in the presence of HPV and EBV (33.3%).ConclusionsThe methylation status of both genes increased with disease progression. With EBV, RB1 methylation was a tumor-associated event because only the cancer group presented methylated RB1 with HPV infection. HPV infection was shown to be significantly correlated with cancer conditions. The global methylation frequency was higher when HPV was present, showing its epigenetic role in cervical carcinogenesis. Nevertheless, EBV seems to be a cofactor and needs to be further investigated.Virtual SlidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1159157579149317.

Raman microspectroscopy for the early detection of pre-malignant changes in cervical tissue

Cervical cancer is the third most common cancer affecting women worldwide. The mortality associated with cervical cancer can, however, be significantly reduced if the disease is detected at the pre-malignant stage. The aim of this study was to evaluate the potential of Raman microspectroscopy for elucidation of the biochemical changes associated with the pre-malignant stages of cervical cancer. Formalin fixed paraffin preserved tissue sections from cervical biopsies classified as negative for intraepithelial lesion and malignancy (NILM), low grade squamous intraepithelial lesion (LSIL) or high grade squamous intraepithelial lesion (HSIL) were analysed by Raman spectral mapping. Raman mapping, with K-means cluster analysis (KMCA), was able to differentiate the NILM cervical tissue into three layers including stroma, basal/para-basal and superficial layers, characterised by spectral features of collagen, DNA bases and glycogen respectively. In the LSIL and HSIL samples, KMCA clustered regions of the superficial layer with the basal layer. Using principal components analysis (PCA), biochemical changes associated with disease were also observed in normal areas of the abnormal samples, where morphological changes were not apparent. This study has shown that Raman microspectroscopy could be useful for the early detection of pre-malignant changes in cervical tissue.

Expression of BAG-1 and PARP-1 in Precursor Lesions and Invasive Cervical Cancer Associated with Human Papillomavirus (HPV)

Cervical cancer remains persistently the second most common malignancies among women worldwide, responsible for 500,000 new cases annually. Only in Brazil, the estimate is for 18,430 new cases in 2011. Several types of molecular markers have been studied in carcinogenesis including proteins associated with apoptosis such as BAG-1 and PARP-1. This study aims to demonstrate the expression of BAG-1 and PARP-1 in patients with low-grade squamous intraepithelial lesions (LSILs), high-grade squamous intraepithelial lesions (HSILs) and invasive squamous cell carcinomas (SCCs) of the uterine cervix and to verify a possible association with HPV infection. Fifty samples of LSILs, 50 samples of HSILs and 50 samples of invasive SCCs of the uterine cervix were analyzed by immunohistochemistry for BAG-1 and PARP-1 expression. PCR was performed to detect and type HPV DNA. BAG-1 expression levels were significantly different between LSILs and HSILs (p = 0,014) and between LSILs and SCCs (p = 0,014). In regards to PARP-1 expression, we found significant differences between the expression levels in HSILs and SCCs (p = 0,022). No association was found between BAG-1 expression and the presence of HPV. However, a significant association was found between PARP-1 expression and HPV positivity in the HSILs group (p = 0,021). In conclusion our research suggests that BAG-1 expression could contribute to the differentiation between LSIL and HSIL/SCC whereas PARP-1 could be useful to the differentiation between HSIL HPV-related and SCC. Further studies are needed to clarify the molecular aspects of the relationship between PARP-1 expression and HPV infection, with potential applications for cervical cancer prediction.

Changes in Activities of Caspase-8 and Caspase-9 in Human Cervical Malignancy

The apoptotic process of programmed cell death and its dysfunctions in a variety of human diseases, including cervical cancer, has become the focus of extensive scientific research. Caspases are considered key factors in the execution of apoptosis, although there are many aspects of their role to be elucidated. It has been found that disturbance of initiator caspase-8 and caspase-9 expression or function may contribute to cancer formation/progression, and inactivation of them could promote resistance to current treatment approaches. In our research, the activities of caspase-8 and caspase-9 have been estimated during the progression of human cervical malignancy. The experimental material includes human cervical tissue samples (normal and pathological), in which enzyme activities have been measured colorimetrically. Activities of caspase-8 and caspase-9 presented the highest increase, compared to the controls, in the low-grade squamous intraepithelial lesion samples (statistically significant, P < 0.01 by t test). The activities diminished in the high-grade squamous intraepithelial lesion and even more in the cancer samples but remained higher than the controls. The observed changes in the activities of caspase-8 and caspase-9 could be attributed to their involvement in the cervical tissue's effort to resist malignancy progression.

Promoter methylation of SFRPs gene family in cervical cancer

Objectives Oncogenic activation of the Wnt/β-catenin signaling pathway is common in human cancers, including cervical cancer. The secreted frizzled-related proteins (SFRPs) function as negative regulators of Wnt signaling and play an important role in carcinogenesis. Frequent promoter hypermethylation of SFRPs has been identified in human cancers; however, the precise role of SFRPs in cervical cancer is not clear. Methods The methylation status of SFRPs gene family was analyzed in two cervical cancer cell lines and a full spectrum of cervical neoplasia, including 45 low-grade squamous intraepithelial lesions (LSIL), 49 high-grade squamous intraepithelial lesions (HSIL), 109 squamous cell carcinomas (SCC), and 45 normal controls. Results The SFRP1 promoter was hypermethylated in 33.9% of SCC, 8.2% of HSIL, 2.2% of LSIL, but not in normal tissues. The SFRP2 promoter was hypermethylated in 80.7% of SCC, 16.3% of HSIL, 15.6% LSIL and 4.4% normal tissues. The SFRP4 promoter was hypermethylated in 67.9% of SCC, 36.7% of HSIL, 4.4% of LSIL, but not in normal tissues. The SFRP5 promoter was hypermethylated in 10.1% of SCC, 4.1% of HSIL, 13.3% of LSIL and 4.4% normal tissues. The frequency of SFRP1, SFRP2 and SFRP4 promoter methylation in tumors was significantly higher than in normal, LSIL, and HSIL samples ( P < 0.0001). SFRP5 methylation was significantly different in patients with or without lymph-node metastases (0% vs 15.2%, respectively, P < 0.05). Conclusions Our data suggest that promoter hypermethylation of SFRP1, SFRP2 and SFRP4 is associated with cervical carcinogenesis, which could be used for molecular screening of cervical neoplasias in future.

Binding of human papillomavirus type 16 to heparan sulfate is inhibited by mucosal antibodies from patients with low-grade squamous intraepithelial lesions but not from cervical cancer patients

Mucosal antibodies against human papillomavirus type 16 (HPV16) capsids have been detected in infected women. To determine whether these antibodies recognize and block the receptor site mediating attachment of HPV16 to heparan sulfate, mucus samples from 126 HPV16-associated low-grade squamous intraepithelial lesion (LSIL) and 85 cervical cancer patients, previously found to react to HPV16 virus-like particles (VLP), and 101 normal controls were tested in an inhibition assay, using HPV16 VLP and heparan sulfate proteoglycan-coated plates. Inhibition levels of 9.3-67.2% were mediated by type-specific antibodies in 94.4% of LSIL patients. Cervical cancer cases showed significantly lower levels of inhibition than LSIL samples (P < 0.0001). The potential of antibodies to inhibit infection was explored in a pseudoinfection system using HPV16 pseudovirions. Inhibition of pseudoinfection by LSIL samples was significantly higher than that observed in the controls (P < 0.001) and cervical cancer cases (P < 0.005). These results indicate that mucosal antibodies inhibiting binding of VLP to heparan sulfate are developed in most LSIL patients, but are hardly present in cervical cancer patients.

Chromosomal Biomarkers for Detection of Human Papillomavirus Associated Genomic Instability in Epithelial Cells of Cervical Cytology Specimens

The goal of this study was to compare how accumulation of chromosomal aberrations in human papillomavirus (HPV)-infected cells correlates with the severity of cervical dysplastic lesions. We assessed the frequency of genomic alterations for 35 different loci in a pilot biopsy study and selected two loci (3q26 and 8q24) with the highest frequency of copy number gains found in high-grade dysplasia and cancer. These probes were labeled with gold and red fluorophores and combined with HPV biotin-labeled probes for subsequent detection using a tyramide signal amplification system with a green fluorophore. Cells that were both HPV positive and chromosomally abnormal were designated as "double-positive cells." Cervical cytology specimens from 235 patients were used for this blinded study. The average number of double-positive cells increased from two cells in patients with a cytological interpretation of atypical squamous cells of undetermined significance to 22 cells in low-grade squamous intraepithelial lesion and 99 cells in high-grade squamous intraepithelial lesion, reflecting an accumulation of chromosomal abnormality with disease progression. Using a cutoff of four or more double-positive cells as the criterion for the presence of a cervical intraepithelial neoplasia 2 or 3 lesion, we demonstrated that low-grade squamous intraepithelial lesion and high-grade squamous intraepithelial lesion cytology specimens with underlying cervical intraepithelial neoplasia 2/3 histology showed positive test results in more than 80% of cases. Correlation of 3q26 and 8q24 aneusomy with concurrent HPV infection may thus serve as a biomarker of genetic instability in HPV-infected cells.

Comparison between the Hybrid Capture II Test and an SPF1/GP6+ PCR-Based Assay for Detection of Human Papillomavirus DNA in Cervical Swab Samples

We compared the efficacy of human papillomavirus (HPV) DNA detection between a PCR-based genechip (Easychip HPV Blot [hereafter referred to as HPV Blot]; King Car, Taiwan) method and Hybrid Capture II (HCII; Digene, Gaithersburg, MD) in women with previous normal (n = 146) or abnormal (> or =atypical squamous cells of undetermined significance [ASCUS] [n = 208]) cytology. A total of 354 cervical swab samples were collected for HPV DNA assay by both HCII and SPF1/GP6+ PCR followed by HPV Blot tests. Colposcopy-directed biopsy was performed if clinically indicated. Of the 354 samples, HPV-positive rates by these two methods (HCII and HPV Blot) were 12.6% and 18.2% in 143 normal samples, 36.2% and 45.7% in 105 ASCUS samples, 57.4% and 57.4% in 94 low-grade squamous intraepithelial lesion samples, and 83.3% and 75.0% in 12 high-grade squamous intraepithelial lesion samples, respectively. The concordance of HPV Blot and HCII was 80.8% (286/354), and the agreement between the methods (kappa value, 0.68) was substantial. Discrepancies were further investigated by at least one of the following three methods: direct sequencing, type-specific PCR, and HPV Blot genotyping of cervical biopsy tissue. In the 15 HCII-positive samples, HPV Blot detected only non-HCII HPV genotypes; results of further verification methods were consistent with the latter test in the 15 samples. Of the 20 samples with HCII-negative and HPV Blot-positive results, 18 were found to contain the 13 HCII high-risk genotypes by verification methods. In only 16.7% (3/18) of the HCII-positive but HPV Blot-negative samples, further studies detected the 13 HCII genotypes. We conclude that HPV Blot seemed comparable to HCII for detection of HPV DNA in cervical swab samples.

Distribution of human papillomavirus types in ThinPrep Papanicolaou tests classified according to the Bethesda 2001 terminology and correlations with patient age and biopsy outcomes

A survey of the distribution of human papillomavirus (HPV) types across the spectrum of cervical cytologic categories defined by the Bethesda 2001 guidelines was conducted with the objective of examining how HPV detection by polymerase chain reaction (PCR) analysis may benefit the management of patients who have abnormal Papanicolaou (Pap) test results. DNA samples from women with no intraepithelial lesion or malignancy (NLM) (n = 300 samples); atypical squamous cells of undetermined significance (ASC-US) (n = 200 samples); low-grade squamous intraepithelial lesion (LSIL) (n = 200 samples); atypical squamous cells, cannot rule out high-grade squamous intraepithelial lesion (ASC-H) (n = 200 samples); and high-grade squamous intraepithelial lesion (HSIL) (n = 200 samples) were tested for HPV using a modified general primer (GP)5+/GP6+ PCR assay and dot-blot hybridization with type-specific oligonucleotide probes (PCR assay analytical sensitivity: 1-100 copies of HPV, depending on the HPV type, in a background of 100 ng human DNA). HPV was detected in 27% of NLM samples, in 89.5% of ASC-US samples, in 97.5% of LSIL samples, in 93% of ASC-H samples, and in 96.5% of HSIL samples. Thirty-seven different HPV types were identified in total. One or more of 13 high-risk (HR) HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) were detected in 53% of samples that were diagnosed as ASC-US (59.0% of patients younger than age 30 yrs; 45.5% of patients age 30 yrs and older), in 55.5% of samples that were diagnosed as LSIL (60.0% of patients younger than age 30 yrs; 44.0% of patients age 30 yrs and older), in 80% of samples that were diagnosed as ASC-H, and in 87.5% of samples that were diagnosed as HSIL (P < 0.001). HPV-16 was detected in 17.5% of ASC-US samples, in 15.5% of LSIL samples, in 48.5% of ASC-H samples, and in 49.0% of HSIL samples (P < 0.001). Among abnormal smears, HR HPV was significantly more common in women younger than age 30 years compared with women age 30 years and older (P < 0.002). Follow-up biopsy data were obtained for 359 patients. A "benign" biopsy result was recorded for 47 of 64 women (73.5%) with ASC-US, 30 of 66 women (45.5%) with LSIL, 39 of 87 women (45.0%) with ASC-H, and 26 of 142 women (18.0%) with HSIL and was most common in women age 30 years and older (P < 0.0001). Cervical intraepithelial neoplasia (CIN) Grade I (CIN-I) was found in 14.0% of women with ASC-US, in 39.5% of women with LSIL, in 8.0% of women with ASC-H, and in 7.0% of women with HSIL. CIN-II was diagnosed in 9.5% of women with ASC-US, in 13.5% of women with LSIL, in 19.5% of women with ASC-H, and in 24.0% of women with HSIL. CIN-III was identified in 2 women (3.0%) with ASC-US, in 1 woman (1.5%) with LSIL, in 24 women (27.5%) with ASC-H, and in 71 women (50.0%) with HSIL. HR HPV testing by PCR of samples diagnosed according to the Bethesda 2001 guidelines may benefit the management of patients with ASC-US or patients with LSIL, especially among women age 30 years and older, by allowing exclusion from referral for biopsy of women who are negative for HR HPV types. However, the small numbers of women who had CIN-III detected after a diagnosis of ASC-US or LSIL limited the assessment of test sensitivity.