Heat shock protein 27 and p16 immunohistochemistry in cervical intraepithelial neoplasia and squamous cell carcinoma

Heat shock protein 27 (hsp27) is expressed by squamous cell carcinoma of the uterine cervix. Results from an earlier study by our group indicted that hsp27 may be a diagnostic marker for cervical intraepithelial neoplasia (CIN) and carcinoma. p16 expression is known to be elevated in intraepithelial uterine cervical cancer and grades 2 and 3 lesions (CIN2, CIN3), but has also been reported to be negative in 5-20% of cervical cancer and CIN lesions. The aim of our study was to confirm immunohistochemically the expression of hsp27 and p16 in cervical lesions. Formalin-fixed, paraffin-embedded cervical tissue specimens obtained between 2002 and 2010 were investigated for hsp27 and p16 expression. Positive staining was detected for hsp27 in 63% of normal cervical tissues, 47% of CIN1 lesions, 75% of CIN2 lesions, 92% of CIN3 lesions, and 100% of squamous cell carcinomas (SCC); the corresponding rates for p16 positivity were 29, 47, 67, 92, and 75%, respectively. Positive staining for both hsp27 and p16 was observed in 6% of normal cervical tissues and in 19% of CIN1, 18% of CIN2, 85% of CIN3, and 75% of SCC specimens. Hsp27 or p16 positivity had a sensitivity of 95.6 or 84.7% and a specificity of 37.2 or 70.5%, respectively, for the identification of CIN3 or SCC lesions; when both hsp27 and p16 were assessed, both the sensitivity and specificity were improved. In conclusion, both hsp27 and p16 immunohistochemistry is a useful tool for the diagnosis of CIN3 lesions or cervical SCC.     

Pretreatment plasma levels and diagnostic utility of hematopoietic cytokines in cervical cancer or cervical intraepithelial neoplasia patients

In this study, we compared plasma levels and the diagnostic utility of hematopoietic growth factors (HGFs) with SCC-Ag in cervical cancer patients in relation to control groups and cervical intraepithelial neoplasia (CIN) patients and healthy subjects. Pretreatment plasma levels of HGFs (SCF, GM-CSF, G-CSF and M-CSF) were determined by the use of immunoenzyme assay (ELISA), and SCC-Ag by chemiluminescent microparticle immunoassay (CMIA). Significantly different concentrations of GM-CSF, G-CSF and M-CSF were observed in the group of patients with cervical cancer and CIN compared to the healthy controls. Significant differences in plasma levels of GM-CSF and M-CSF between cervical cancer and benign lesions patients were also found. The HGFs and SCC-Ag diagnostic specificities received high values. The diagnostic sensitivity and the predictive value of a positive and negative test result were higher for M-CSF than for antigen SCC in the cancer group. The M-CSF area under the ROC curve (AUC) was the largest from hematopoietic cytokines and SCC-Ag. These results suggest the potential utility of M-CSF as a good candidate for a marker of cervical cancer as well as benign lesions of this organ (CIN).     

Squamous cell carcinoma antigen for monitoring cervical cancer

The tumour-associated antigen was determined in the plasma of patients with squamous cell carcinoma (SCC) of the uterine cervix by radioimmunoassay. Setting a limit of 2 ng/ml, levels were abnormal in 13.4% of healthy controls, in 14% of patients with carcinoma in situ and in 62% of patients with invasive cervical SCC. The incidence of elevated SCC antigen levels and the absolute antigen plasma concentration were dependent upon the tumour load, increasing significantly with advanced stage disease. Abnormal SCC antigen values in operable cervical cancer declined to normal within one week after radical hysterectomy with pelvic lymphadenectomy. In cases of radiotherapy antigen values took 4-6 weeks after the start of treatment to return to normal. The success of both treatment modalities was announced by an early rise in the SCC antigen in the initial phase of therapy, followed by normalisation. After successful primary treatment and a complete remission during further follow-up SCC antigen in plasma was only increased in 3.8% of the cases. Retrospective evaluations in ten patients with progressive disease showed the reappearance of abnormal SCC titers and further increase preceeding the clinically detectable relapse or progression, with a median interval of 8 weeks. The present study indicates that SCC antigen determination is not useful for the early diagnosis of cervical cancer, but it is a potential means for monitoring the efficacy of individual anticancer therapy of SCC of the uterine cervix and for detecting recurrent disease.     

Plasma-free amino acid profiling of cervical cancer and cervical intraepithelial neoplasia patients and its application for early detection

In this study, plasma-free amino acid profiles were used to investigate pre-cancerous cervical intraepithelial neoplasia (CIN) and cervical squamous cell carcinoma (CSCC) metabolic signatures in plasma. Additionally, the diagnostic potential of these profiles was assessed, as well as their ability to provide novel insight into CSCC metabolism and systemic effects. Plasma samples from CIN patients (n = 26), CSCC patients (n = 22), and a control healthy group (n = 35) were analyzed by high-performance liquid chromatography, and their spectral profiles were subjected to the t test for statistical significance. Potential metabolic biomarkers were identified using database comparisons that examine the significance of metabolites. Compared with healthy controls, patients with CIN and CSCC demonstrated lower levels of plasma amino acids; plasma levels of arginine and threonine were increased in CIN patients but were decreased in cervical cancer patients. Additionally, the levels of a larger group of amino acids (aspartate, glutamate, asparagine, serine, glycine, histidine, taurine, tyrosine, valine, methionine, lysine, isoleucine, leucine, and phenylalanine) were gradually reduced from CIN to invasive cancer. These findings suggest that plasma-free amino acid profiling has great potential for improving cancer screening and diagnosis and for understanding disease pathogenesis. Plasma-free amino acid profiles may have the potential be used to determine cancer diagnoses in the early stage from a single blood sample and may enhance our understanding of its mechanisms.     

Relationship of up-regulation of 67-kd laminin receptor to grade of cervical intraepithelial neoplasia and to high-risk HPV types and prognosis in cervical cancer

OBJECTIVE: To evaluate the 67-kd laminin receptor (67LR) in cervical cancer and its molecular links to oncogenic HPV types. STUDY DESIGN: As part of the HPV-PathogenlSS Study, a series of 150 squamous cell carcinomas (SCCs) and 152 carcinoma in situ (CIN) lesions were examined using immunohistochemical staining for LR67 and tested for HPV using polymerase chain reaction (PCR) with 3 primer sets (MY09/11, GP5+/GP6+, SPF). Followup data were available for all SCC patients, and 67 CIN lesions had been monitored with serial PCR for HPV clearance/persistence after cone treatment. RESULTS: 67LR expression increased in parallel with increasing grade of CIN (p = 0. 0001), with the most dramatic up-regulation upon the transition from CIN 2 to CIN 3 and further to SCC. This increased expression was associated with CIN 3/cancer at OR 17.04 (95% CI 7.28-39.87). The seemingly significant association of 67LR with high-risk HPV (HR-HPV) detection (OR 2.20, 95% CI 1.27-3.80) was due to confounding by the histologic grade (Mantel-Haenszel common OR = 1.118, 95% CI 0.576-2.168). Using performance indicators, 67LR expression was of little value as a marker of HR-HPV type, and it did not predict clearance/persistence of HR-HPV after treatment of CIN. Similarly, 67LR expression was not an independent prognostic factor in cervical cancer. CONCLUSION: In cervical carcinogenesis, both integrin- and nonintegrin-type LRs (67LR) probably have functions complementary to each other, mediating transient early and stable adhesions, respectively. Up-regulated 67LR expression is significantly associated with progression from CIN 2 to CIN 3 as a marker of cell proliferation. 67LR is probably orchestrated by mechanisms independent of HR-HPV oncoproteins, which seem to be more closely associated with integrin-type laminin receptors.     

Expression of p53, bcl-2 and Ki-67 in cervical intraepithelial neoplasia and invasive squamous cell carcinoma of the uterine cervix

OBJECTIVE: To assess the expression of p53, bcl-2 and Ki-67 in the progression of cervical neoplasia. STUDY DESIGN: A total of 131 cervical specimens, consisting of normal cervical epithelium (n = 43), cervical intraepithelial neoplasia (CIN) lesions (n =40) and cervical squamous cell carcinomas (SCCs) (n = 48) were examined immunohistochemically in paraffin sections for expression of p53, bcl-2 and Ki-67. RESULTS: Immunoreactivity of p53 was found in 27% of SCC cases, but it had no significant relationship with SCC staging (p = 0.791). Immunoreactivity of bcl-2 was observed in 33% of CIN 3 cases. We found a significant relationship (chi2 test: p = 0.009) between the expression of bcl-2 and CIN grading. Ki-67 index was higher in high grade CIN (HGCIN: CIN 2 and 3) and SCC lesions compared to normal cervices. Ki-67 index showed a correlation with bcl-2 protein expression (p = 0.030), but not with p53 protein expression (p = 0.239). CONCLUSION: HGCIN is an early stage to demonstrate the alteration of bcl-2 and Ki-67 expressions. Progression of neoplasia in the uterine cervix is accompanied by an increase of antiapoptotic protein, bcl-2 as well as cellular proliferation.     

Proteomic analysis of cervical cancer cells treated with suberonylanilide hydroxamic acid

Suberonylanilide hydroxamic acid (SAHA) is an orally administered histone deacetylase inhibitor (HDACI) that has shown significant antitumour activity in a variety of tumour cells. To identify proteins involved in its antitumour activity, we utilized a proteomic approach to reveal protein expression changes in the human cervical cancer cell line HeLa following SAHA treatment. Protein expression profiles were analysed by 2-dimensional polyacrylamide gel electrophoresis (2-DE) and protein identification was performed on a MALDI-Q-TOF MS/MS instrument. As a result, a total of nine differentially expressed proteins were visualized by 2-DE and Coomassie brilliant blue (CBB) staining. Further, all the changed proteins were positively identified via mass spectrometry (MS)/MS analysis. Of these, PGAM1 was significantly downregulated in HeLa cells after treatment with SAHA. Moreover, PGAM1 has been proven to be downregulated in another cervical cancer cell line (CaSki) by western blot analysis. Together, using proteomic tools, we identified several differentially expressed proteins that underwent SAHA-induced apoptosis. These changed proteins may provide some clues to a better understanding of the molecular mechanisms underlying SAHA-induced apoptosis in cervical cancer.     

Dysregulated microRNAs in the pathogenesis and progression of cervical neoplasm

MicroRNAs (miRNAs) play an important role in a variety of physiological as well as pathophysiological processes, including carcinogenesis. The aim of this study is to identify a distinct miRNA expression signature for cervical intraepithelial neoplasia (CIN) and to unveil individual miRNAs that may be involved in the development of cervical carcinoma. Expression profiling using quantitative real-time RT-PCR of 202 miRNAs was performed on micro-dissected high-grade CIN (CIN 2/3) tissues and compared to normal cervical epithelium. Unsupervised hierarchical clustering of the miRNA expression pattern displayed a distinct separation between the CIN and normal cervical epithelium samples. Supervised analysis identified 12 highly differentially regulated miRNAs, including miR-518a, miR-34b, miR-34c, miR-20b, miR-338, miR-9, miR-512-5p, miR-424, miR-345, miR-10a, miR-193b and miR-203, which distinguished the high-grade CIN specimens from normal cervical epithelium. This miRNA signature was further validated by an independent set of high-grade CIN cases. The same characteristic signature can also be used to distinguish cervical squamous cell carcinoma from normal controls. Target prediction analysis revealed that these dysregulated miRNAs mainly control apoptosis signaling pathways and cell cycle regulation. These findings contribute to understanding the role of microRNAs in the pathogenesis and progression of cervical neoplasm at the molecular level.     

TGF-beta signalling pathway factors in HPV-induced cervical lesions

Human papillomaviruses (HPV) are considered the etiological agents of cervical cancer, especially high-risk genotypes. TGF-beta (transforming growth factor-beta) is well known for its anti-proliferative effects but the neoplastic cells often lose their sensitivity to TGF-beta. A characteristic alteration associated with malignant progression is the loss of responsiveness to TGF-beta1-induced cell growth inhibition. The aim of the present study was to establish the possible role of some members of TGF-beta signalling pathway during cervical cancer development and the possible relationship with HPV infection. In order to establish TGF-beta gene expression levels in cervical oncogenesis, TGF-beta1, TGF-beta1 receptors and Smad2 were investigated in precancerous and cervical cancer samples (Quantitative Real-Time PCR). The study revealed that 84.5% of patients were positive for HPV DNA. The most prevalent HPV genotypes were high-risk HPV 16 and 18 in single or co-infections. Expression of TGF-beta1 decreased as tumor cells progressed from cervical intraepithelial neoplasia to cervical carcinoma. Furthermore, we observed that cervical lesions without HPV infection expressed significantly less TGF-beta1. TGF-betaRI and Smad2 gene expression levels were found to be decreased in SCC and AC samples in contrast with CIN1 and CIN2/3 samples. Our results showed that in human cervical cancer the disruption of TGF-beta/Smad signalling pathway might contribute to the malignant progression of cervical dysplasia. These data emphasize the importance of canonical TGF-beta pathway integrity in carcinogenesis.     

Immunohistochemical Expression of RAGE and Its Ligand (S100A9) in Cervical Lesions

Altered expressions of receptor for advanced glycation end-products (RAGE) and its ligand (S100A9) are observed in many cancers and play a key role in inflammation-associated cancer. In our previous study, by two-dimensional gel electrophoresis followed by mass spectrometry, the expression of S100A9 protein was found to increase in squamous cervical cancer compared with adjacent normal cervical tissues. Therefore, in the present study we observed the expressions of S100A9 and RAGE in 30 chronic cervicitis, 50 cervical intraepithelial neoplasia (CIN), and 40 squamous cervical cancer (SCC) using immunohistochemical analysis and analyzed the differential expression and possible role of S100A9 and RAGE in cancer development. Immunohistochemical findings were as follows: the expressions of S100A9 and RAGE were demonstrated in chronic cervicitis, CIN, and SCC. Moreover, their expressions were gradually increasing as the tumor progressed. In SCC, the staining scores of S100A9 and RAGE were significantly higher in well-differentiated tumors compared to moderately and poorly differentiated tumors. The expression of S100A9 in epithelial cells exhibited a positive correlation to RAGE expression in chronic cervicitis, CIN, and SCC. There were no significant difference of S100A9 immunoreactivity in stromal cells among chronic cervicitis, CIN, and SCC. Moreover, there was no correlation between S100A9 immunoreactivity in stromal cells of SCC and clinicopathological parameters. Finally, double immunohistochemistry illustrated that RAGE and S100A9 co-express in SCC. In conclusion, RAGE binds its ligand (S100A9), which plays an important role in the development of SCC. In addition, the expressions of S100A9 and RAGE in SCC tumor cells were closely associated with histological differentiation.     

Altered expression of cellular membrane molecules of HLA-DR, HLA-G and CD99 in cervical intraepithelial neoplasias and invasive squamous cell carcinoma

OBJECTIVE: The aim of this study was to investigate the role of HLA-DR, HLA-G and CD99 during cervical carcinogenesis and to examine the prognostic significance of these protein expressions in invasive squamous cell carcinoma (SCC). METHODS: Using specific antibodies for HLA-DR, HLA-G and CD99, we examined protein expressions in 19 normal cervix, 15 mild dysplasia (CIN I), 22 moderate dysplasia (CIN II), 23 severe dysplasia (CIN III), and 34 invasive squamous cell carcinoma by immunohistochemistry. And we detected the expression of Ki67 in the same specimens. RESULTS: None of normal cervix and CINs except three cases of CIN III expressed HLA-DR. HLA-DR expression increased progressively with the grade of the tumor, and significant differences could be observed between grade 1 and grade 2 (P<0.01) and between grade 1 and grade 3 (P<0.05). In all normal epithelial control samples, HLA-G expression was seen in ectocervical squamous and endocervical columnar epithelium and the staining was strong and uniform. Only a small proportion of CINs and SCCs showed reduced expression of HLA-G. Compared with the results in the control samples, CINs and SCCs showed significantly reduced expression of HLA-G (P<0.001). SCCs showed significantly increased expression of CD99 when compared with normal cervix and CINs (P<0.05). Ki67 was expressed in all specimens. Significant differences were observed between CINs and normal cervix (P<0.001) and SCCs and controls (P<0.001), but no significant differences could be observed between SCCs and CINs. None of the expressions of these proteins was associated with any of clinicopathological parameters. CONCLUSIONS: These results indicate that increased expression of HLA-DR and CD99 may be related to the evolution of cervical cancer. All protein expressions were not associated with clinicopathological parameters.     

Evaluation of MCM-2 expression in TMA cervical specimens

Background

Minichromosome maintenance proteins (MCM) are highly expressed in actively replicating cells. The need for biological markers for cervical carcinoma and its precursor lesions is emerging. Our main aim was to determine the immunohistochemical expression of MCM-2 in HIV-positive and -negative dysplastic cervical specimens.

Methods

Immunohistochemical analysis of MCM-2 was performed in a total of 352 cervical TMA specimens of normal control, low-grade CIN, high-grade CIN and invasive tumor. 38 specimens were from HIV-positive women. A receiver operating characteristic (ROC) curve was constructed to determine the best cutoff to diagnose high-grade CIN and invasive cervical cancer.

Results

In the progression from normal epithelium to high-grade CIN and invasive tumor we found significant differences in the MCM-2 expression (p<0.05). Based on the ROC curve of 80% with an area under the curve (AUC) of 0.78, expression of MCM-2 to diagnose high-grade CIN and invasive tumor resulted in sensitivity of 81%, specificity of 66%, a positive predictive value (PPV) of 86% and a negative predictive value (NPV) of 57%. HIV-positive cervices revealed a decreasing expression of MCM-2 in both LGCIN and HGCIN compared with HIV-negative specimens (p<0.0001).

Conclusions

The present study suggests that immunohistochemical MCM-2 may not be a promising biomarker for diagnosing high-grade CIN and invasive cancer.     

Expression of toll-like receptor 4 is down-regulated during progression of cervical neoplasia

Chronic infection and inflammation are among the most important factors contributing to cancer development and growth. Toll-like receptors (TLRs) are important families of pattern recognition receptors, which recognize conserved components of microbes and trigger the immune response against invading microorganisms. TLR4 is the signaling receptor for lipopolysaccharide (LPS), the endotoxic component of Gram-negative bacteria. Recent studies demonstrate that TLRs are expressed in some tumor cells, and that the expression of TLRs in these cells is associated with tumorigenesis. Cervical intraepithelial neoplasia (CIN) is a key stage in the development of cervical cancer and human papillomavirus (HPV) infection is an essential factor in cervical carcinogenesis. As the cervix is in constant contact with bacteria, especially Gram-negative bacteria, we hypothesize that TLR4-mediated bacterial stimulation may be involved in the tumorigenesis of cervical cancer. In the present study, the expression and distribution of TLR4 in CIN and cervical squamous carcinoma were investigated by immunohistochemistry. To our surprise, we observed a decrease in the expression of TLR4 during the progression of cervical neoplasia and this down-regulation of TLR4 appeared to be associated with the expression of \textP ^{1 6\textINK4A} , {\text{P}}^{{ 1 6^{\text{INK4A}} }} , which is a crucial marker of HPV integration into host cells. These data offer further insight regarding the association of HPV infection and TLR signaling during the carcinogenesis of cervical cancer.     

HGF和EZH2在宫颈癌组织中的表达及其与临床病理特征的关系

目的:探讨肝细胞生长因子(HGF)、果蝇zeste基因增强子2(EZH2)在宫颈癌组织中的表达及其与临床病理特征的关系。方法:选取2016年10月到2018年1月期间在新疆医科大学附属肿瘤医院接受治疗的宫颈癌患者50例,收集其手术切除的病理组织作为宫颈癌组的检测标本,另选取同期在我院收治的子宫肌瘤患者,收集其行全子宫切除术时切除的宫颈组织,其中上皮内瘤样病变(CIN)组织和正常宫颈组织各50例,CIN组织作为CIN组的检测标本,正常宫颈组织作为对照组的检测标本。比较宫颈癌组、CIN组和对照组标本中HGF和EZH2的阳性表达情况。分析HGF和EZH2的表达与宫颈癌患者临床病理特征的关系,分析宫颈癌组织中HGF、EZH2表达的相关性。结果:各组标本中的HGF和EZH2的阳性表达率整体比较差异均有统计学意义(P0.05),宫颈癌组、CIN组的HGF和EZH2的阳性表达率均明显高于对照组,且宫颈癌组EZH2的阳性表达率高于CIN组(P0.05)。宫颈癌组织中HGF和EZH2的表达与年龄、肿瘤类型、肿瘤大小无关(P0.05),临床分期II期、有淋巴结转移、病理分级G3的宫颈癌组织中HGF和EZH2的阳性表达率高于临床分期I期、无淋巴结转移、病理分级G1+G2的宫颈癌组织(P0.05)。经Spearman相关分析显示,宫颈癌组织中HGF与EZH2表达呈正相关(P0.05)。结论:HGF和EZH2在宫颈癌组织中呈高表达,且其表达水平与临床分期、淋巴结转移、病理分级有关。     

Elevated levels of phosphorylated fibrinogen-alpha-isoforms and differential expression of other post-translationally modified proteins in the plasma of ovarian cancer patients

We evaluated the differentially expressed proteins in the plasma of ovarian cancer (OVC) patients using 2-D SDS-polyacrylamide gel electrophoresis (SDS-PAGE) with post-translational modification (PTM) specific stains after the removal of six high-abundance proteins. The pooled plasma from patients with stage III or IV OVC was compared to a pooled postmenopausal age-matched control. Several proteins were identified as differentially expressed in the plasma of OVC patients. Among them, the phosphorylated fibrinogen-alpha-chain isoform (containing fibrinopeptide-A) was found to be up-regulated. Previously in our laboratory, phosphorylated fibrinopeptide-A was found to be up-regulated in the low molecular weight fraction of serum derived from OVC patients. We examined the levels of phosphorylated fibrinogen-alpha-chain in each patient that constituted the pooled plasma using Western blot, mass spectrometry (MS), and PTM specific stains. Phosphoprotein bands containing fibrinogen-alpha-chain fragments showed up-regulation in all OVC patients.     

Detection of Cervical Cancer Biomarker Patterns in Blood Plasma and Urine by Differential Scanning Calorimetry and Mass Spectrometry

Improved methods for the accurate identification of both the presence and severity of cervical intraepithelial neoplasia (CIN) and extent of spread of invasive carcinomas of the cervix (IC) are needed. Differential scanning calorimetry (DSC) has recently been shown to detect specific changes in the thermal behavior of blood plasma proteins in several diseases. This methodology is being explored to provide a complementary approach for screening of cervical disease. The present study evaluated the utility of DSC in differentiating between healthy controls, increasing severity of CIN and early and advanced IC. Significant discrimination was apparent relative to the extent of disease with no clear effect of demographic factors such as age, ethnicity, smoking status and parity. Of most clinical relevance, there was strong differentiation of CIN from healthy controls and IC, and amongst patients with IC between FIGO Stage I and advanced cancer. The observed disease-specific changes in DSC profiles (thermograms) were hypothesized to reflect differential expression of disease biomarkers that subsequently bound to and affected the thermal behavior of the most abundant plasma proteins. The effect of interacting biomarkers can be inferred from the modulation of thermograms but cannot be directly identified by DSC. To investigate the nature of the proposed interactions, mass spectrometry (MS) analyses were employed. Quantitative assessment of the low molecular weight protein fragments of plasma and urine samples revealed a small list of peptides whose abundance was correlated with the extent of cervical disease, with the most striking plasma peptidome data supporting the interactome theory of peptide portioning to abundant plasma proteins. The combined DSC and MS approach in this study was successful in identifying unique biomarker signatures for cervical cancer and demonstrated the utility of DSC plasma profiles as a complementary diagnostic tool to evaluate cervical cancer health.     

Expression of the carbohydrate tumour marker Sialyl Lewis A, Sialyl Lewis X, Lewis Y and Thomsen-Friedenreich antigen in normal squamous epithelium of the uterine cervix, cervical dysplasia and cervical cancer

The carbohydrate molecules Sialyl Lewis X (SLeX), Sialyl Lewis A (SLeA), Lewis Y (LeY) and Thomsen-Friedenreich antigen (TF) are known to mediate the adhesion between tumor cells and endothelium. They are used as serum markers in diagnosis and treatment in a broad spectrum of human carcinomas, but their expression profile and role in the development of cervical cancer remains unclear. The aim of this study was to investigate the expression of SLeX, SLeA, LeY and TF in normal cervical squamous epithelium, cervical dysplasia and cervical cancer. Slides of paraffin-embedded tissue were fixed and incubated with monoclonal antibodies against SLeX, SLeA, LeY and TF. Immunohistochemical staining was evaluated by using a semi-quantitative score (IRS Score). We found a significant difference of SLeA expression in invasive cervical cancer compared to normal epithelium (p=0.006) and all grades of dysplasia (p=0.002). The expression of SLeX in normal epithelium was less intense than in carcinoma in situ (p=0.036). Staining for LeY showed the weakest results of the investigated markers. Significant differences were found when normal epithelium was compared to CIN I (p=0.011), to CIN II (p=0.013) and to invasive cervical cancer (p=0.005). For TF, significant differences were found in normal epithelium compared to CIN I (p=0.011), CIN II (p=0.013) and compared to invasive cervical cancer (p=0.005). This is the first study on the expression of SLeA, SLeX, LeY and TF in normal cervical endothelium, cervical dysplasia, carcinoma in situ and invasive cervical cancer. Further studies and higher numbers are desirable.     

Influence of cytology development on frequency of pre-cancerous lesions and cervical cancer in east Croatia, 1978-2001

We analyzed the influence of cytology development to frequency of precancerosa (Cervical intraepithelial neoplasia grade 3 - CIN 3) and cervical cancer. The number of examined Pap smears increased significantly in the analyzed period. The calculated linear trend shows an average four-year increase of 17,283.35 smears. The number of detected CIN 3 increased accordingly. The calculated linear trend shows an average four-year increase by 45.03 and decrease of the rate of 0.07. A slight increase in cervical cancer was also noticed. The calculated linear trend shows an average four-year increase in cancer by 1.6 and decrease in the rate of 0.51. Analyses of cervical cancer by stages showed an increase in number of stage I and decrease of other stages. The calculated linear trend shows an average four-year increase of stage I by 4.94 or decrease in other stages of cervical cancer of 3.29 respectively. The rate of cervical cancer stage I decreased by 0.11 and of other stages by 0.39. Further analyses of the stage I showed that the linear trend of IA stage had an average four-year increase by 5.40 and decrease in stage IB of 0.48. The rate of the cervical cancer stage IA increased by 0.02, whereas the rate of IB decreased by 0.13. Cytology development in our country has resulted in detection of higher number of CIN 3 and the earliest stage (IA) of cervical cancer whereas the number of other stages (IB, II, III, IV) has decreased. However, a total number of cervical cancers haven't changed over the whole period. Thus, it is obvious that opportunistic program of detection, which has been using in Croatia, could not decrease frequency of cervical cancer. In order to achieve it, well-organized national program of detection is needed.     

Prevalence of human papillomavirus genotypes in cervical cancer and precursor lesions

There are no data obtained in biopsy material on the prevalence of human papillomavirus (HPV) and HPV genotypes in Croatian women with cervical carcinoma and precursor lesions. Therefore, the prevalence of HPVand HPVgenotypes was investigated in archival material of cervical carcinoma and precursor lesions kept at Department of Pathology, School of Medicine, University of Rijeka. DNA was isolated from formalin fixed, paraffin embedded tissue, histologically classified as cervical intraepithelial neoplasia (CIN) III (n =43), squamous cell carcinoma (SCC) (n =54) and adenocarcinoma (ADC) (n =40). HPV testing was performed bypolimerase chain reaction (PCR) using generic and genotype specific primers. The prevalence of HPV DNA was 93.02%, 92.59%, and 92.5% in CIN III, SCC and ADC, respectively. In CIN III and SCC, HPV-16 was the most common high-risk genotype, identified in 65% and 52%, followed by HPV-18 in 22.5% and 28% of cases, respectively. HPV-18 showed a statistically significant prevalence in ADC (67.6%) as compared with SCC (chi(2)=9.924; p_ 0.01). Study results revealed a high prevalence of HPV-DNA in examined cervical lesions (>90%). HPV-16 predominated in SCC and HPV-18 in ADC. Single infection was more frequently present than multiple infections in all three histological groups.