Development of cervical intraepithelial lesions and cervical cancer is not influenced by SOD2 RS4880 polymorhism.

Some of the more than 200 known HPV types are essential for cervical cancer development, the third type of cancer most incident in the female population. However, for the malignant transformation occur, some cofactors are needed, as the reactive oxygen species (ROS), which can be neutralized by the antioxidant system. The SOD2 enzyme, encoded by the same name gene, is found in mitochondria and is part of the first line of defense against oxidative stress damage. Genetic polymorphisms can act by altering the efficiency of the enzyme, among which the most studied is the rs4880. Thus, the purpose of the present study was to evaluate the association of this polymorphism with HPV infection and the development of low and high grade squamous intraepithelial lesions (LSIL and HSIL) and cervical cancer, in 407 women attended by the public health system in Brazil. HPV detection in cervical secretion samples was carried out by polymerase chain reaction (PCR) and blood samples were used for polymorphism genotyping through PCR followed by restriction fragment length polymorphism (RFLP). PCR and restriction products were subjected to 10% polyacrylamide gel electrophoresis. HPV negative group (control) included 158 women and the HPV positive group (case) 249 women. The infected group was divided into No Lesion (n = 90), LSIL (n = 20), HSIL (n = 67) and cervical cancer (n = 72). The data found on socio-epidemiological characteristics and habits corroborated with data found in the literature. The distribution of genotypes in the control group was 51.9% women TC, 29.8% TT and 18.3% CC. In the case group, the distribution was 55.0% women TC, 26.1% TT and 18.9% CC. This is the first study evaluating the influence of SOD2 rs4880 polymorphism on HPV infection, the development of cervical intraepithelial lesions and cervical cancer in a Brazilian population, although additional studies are needed to corroborate the results.

Robust expression of EZH2 in endocervical neoplastic lesions.

The aim of this study was to evaluate the nuclear expression of histone methyltransferase enhancer of zeste homolog 2 (EZH2) in endocervical neoplastic lesions such as invasive endocervical adenocarcinoma (ECA) and cervical in situ adenocarcinoma (AIS) in comparison with normal endocervix and non-neoplastic counterparts. A total of 54 consecutive neoplastic cases (37 ECA, 17 AIS) and 32 non-neoplastic endocervical lesions (15 reactive atypia, 9 microglandular hyperplasia, 3 tuboendometrioid metaplasia, 3 tunnel cluster, 2 endometriosis) were included in the study with adjacent normal endocervix if present. EZH2 immunoreactivity was evaluated semiquantitatively by three independent experts in lesions and adjacent normal glandular epithelium as well. EZH2 expression was defined robust if at least two of the three experts rated partial or diffuse positivity. Robust EZH2 expression was statistically compared among the neoplastic, non-neoplastic, and normal glandular epithelium samples. Diagnostic test capability of robust EZH2 expression was calculated. Fifty-three out of the 54 neoplastic cases (98%) showed robust EZH2 expression. Robust EZH2 expression was significantly less often (4 out of 32 cases, 12.5%) found in the non-neoplastic endocervical lesions (p < 0.0001) and never (0 out of 66 samples) in the adjacent normal glandular epithelium. Robust EZH2 overexpression had a sensitivity and specificity of over 95% in detecting neoplastic lesions versus non-neoplastic lesions or normal glandular epithelium. EZH2 may play a role in the pathogenesis of endocervical neoplasia, and the detection of robust expression of EZH2 might be a useful differential diagnostic tool in problematic endocervical lesions in histology and cytology as well.

Distinct profiles of TERT promoter mutations and telomerase expression in head and neck cancer and cervical carcinoma.

Two recurrent mutations (-124 G > A and -146 G > A) in the core promoter region of the human telomerase reverse transcriptase (TERT) gene create consensus binding sites for ETS transcription factors and cause increased TERT expression in several tumour types. We analyzed TERT promoter mutations and TERT mRNA levels in head and neck cancer, cervical carcinoma and cervical intraepithelial neoplasia (CIN) as well as in C-4I, CaSki, HeLa and SiHa cervical cell lines. Nucleotide sequence analysis of TERT promoter region showed that 33.3% of oral squamous cell carcinoma (SCC) and 16.8% of cervical SCC harboured mutually exclusive G to A transitions at nucleotide position -124 or -146. TERT promoter was mutated at nucleotide -146 (G > A) in SiHa cell line. Other nucleotide changes creating in some cases putative ETS binding sites were more frequent in oral SCC (26.7%) than in cervical carcinoma (4.8%). The frequency of mutations was independent of human papillomavirus (HPV) tumour status in both cervical and oral cancer. Expression of TERT gene was significantly higher in TERT promoter mutated (-124G > A or -146G > A) cervical SCC compared to not mutated SCC irrespective of HPV16 E6 and E7 levels. Such hot spot changes were not detected in oropharyngeal SCC, cervical adenocarcinoma and CIN lesions. Our results suggest that TERT promoter mutations play a relevant role in oral SCC as well as in cervical SCC, besides the already known effect of HPV16 E6 protein on TERT expression.

Nicotinamide Phosphoribosyl Transferase a Reliable Marker of Progression in Cervical Dysplasia.

BACKGROUND/AIM: Nicotinamide phosphoribosyl transferase (Nampt) catalyses the rate-limiting step of the mammalian nicotinamide adenine dinucleotide (NAD) salvage pathway. Nampt is highly expressed in several epithelial and mesenchymal neoplasms, where is promotes cell-cycle progression ans chemotherapy resistance. To our knowledge, alterations in Nampt expression have not been examined in cervical intraepithelial neoplasia (CIN) or squamous cell carcinoma (SCC). MATERIALS AND METHODS: We performed immunohistochemical analysis for Nampt using tissue microarrays on 14 samples of benign cervical squamous epithelium and 15 CIN I, 15 CIN II, and 13 samples of CIN III. The SCCs included 5 low-grade, 67 intermediate-grade, and 81 high-grade tumors. RESULTS: Nampt levels increased with increased CIN grades were compared to benign cervical squamous epithelium. Similarly, Nampt levels increased with increasing SCC grade. CONCLUSION: Nampt expression is a reliable marker of progression in cervical dysplasia and SCC.

Characterization and clinical validation of MCM2 and TOP2A monoclonal antibodies in the BD ProEx™ C assay: An immunoassay which detects aberrant S-phase induction in cervical tissue.

BACKGROUND: The Papanicolaou (Pap) screen has been successful in reducing cervical cancer; but exhibits low sensitivity when detecting cervical dysplasia. Use of molecular biomarkers in Pap tests may improve diagnostic accuracy. DESIGN: Monoclonal antibodies to Minichromosome Maintenance Protein 2 (MCM2) and DNA Topoisomerase II α (TOP2A) were selected for use in IHC based on their ability to differentiate normal from diseased cervical tissues in tissue microarrays. Enhanced Green Fluorescent Protein Western blot analysis was used to help identify binding epitopes specific to MCM2 and TOP2A antibody clones. Antibody affinity was determined by solution phase affinity measurement and immunohistochemistry was performed using high affinity MCM2 or TOP2A antibodies on serial histological sections. RESULTS: Antibody clones to MCM2 and TOP2A clones were selected based on their ability to detect over expression in abnormal cervical epithelia. In IHC, MCM2-27C5.6 and MCM2-26H6.19 demonstrated superior staining in abnormal cervical tissue over the MCM2-CRCT2.1 antibody. A combination of MCM2 and TOP2A antibodies showed greater staining when compared to staining with any of the antibodies alone on serial histological sections. Distinct linear epitopes were elucidated for each of the MCM2 and TOP2A clones. Affinity values (Kd) for MCM2 or TOP2A antibodies had a similar range. In a research study, the MCM2 and TOP2A (BD ProEx™ C) antibody cocktail showed increased epithelia staining with increasing dysplasia. The use of BD ProEx™ C in combination with H&E staining enhanced immunohistochemical discrimination of dysplastic and non-dysplastic FFPE cervical tissue specimens. CONCLUSIONS: BD ProEx™ C containing MCM2 and TOP2A antibodies showed strong specific nuclear staining that correlated with increased dysplasia and lesion severity. Enhanced performance of the antibodies was linked to their unique topography recognition. BD ProEx™ C incorporates antibodies that enhance detection of CIN2+ cervical disease.

SIRT1 overexpression in cervical squamous intraepithelial lesions and invasive squamous cell carcinoma.

Invasive squamous cell carcinoma (SCC) of the cervix involves the progression of premalignant cervical intraepithelial neoplasia (CIN) and is associated with persistent human papillomavirus infection. Most CINs will regress, and the challenge is to identify the lesions likely to progress to invasive cancer. We evaluated Sirtuin 1 (SIRT1) expression in nonneoplastic cervix, CINs, and SCCs as a potential biomarker to predict disease progression. A total of 101 cases were selected including 29 CIN 1s, 32 CIN 2s, 16 CIN 3s, 2 microinvasive SCCs, and 22 invasive SCCs. Cervical nonneoplastic squamous epithelium showed weak positivity of SIRT1 in the basal layer. SIRT1 cytoplasmic overexpression was found in 13.8% of CIN 1s (4/29), 40.6% of CIN 2s (13/32), and 50% of CIN 3s (8/16), and it was statistically significant between CIN 1 and CIN 2/3 lesions (P=.01). All 24 cases of invasive and microinvasive SCC showed SIRT1 overexpression, with 25% (6/24) showing cytoplasmic staining only, 4.2% (1/24) showing nuclear staining only, and 70.8% (17/24) showing both nuclear and cytoplasmic staining. From CIN 1 to SCC, SIRT1 expression showed steady and statistically significant increase (CIN 1 versus CIN 2-3, P=.01; CIN 2-3 versus SCC, P=.0001). Thus, SIRT1 may serve as a potential biomarker for predicting the progression of CIN to invasive SCC.

Human papillomavirus 16 E2 interacts with neuregulin receptor degradation protein 1 affecting ErbB-3 expression in vitro and in clinical samples of cervical lesions.

The ErbB tyrosine kinase receptors play a key role in regulating many cellular functions and human papillomaviruses (HPVs) may interact with transductional pathway of different growth factor receptors. Here, these interactions were analysed in W12 cell line carrying HPV 16 genome and in clinical samples. W12 cells, in which HPV16 becomes integrated during passages, were utilised to detect viral and ErbB family expression at early (W12E) and late passages (W12G). Interestingly, a strong reduction of ErbB-3 expression was observed in W12G. Loss of the E2 and E5 viral genes occurs in W12G and this may affect ErbB-3 receptor expression. E2 and E5 rescue experiments demonstrated that only E2 gene was able to restore ErbB-3 expression. E2 is a transcriptional factor but the expression levels of ErbB3 were unaffected and ErbB-3 promoter did not show any consensus sequence for E2, thus E2 may interact in another way with ErbB3. Indeed, HPV 16 E2 can modulate ErbB-3 by interacting with the ubiquitin ligase neuregulin receptor degradation protein 1 (Nrdp-1) that is involved in the regulation of this receptor, via ubiquitination and degradation. E2 co-immunoprecipitated in a complex with Nrdp-1 leading to hypothesise an involvement of this interaction in ErbB-3 regulation. In addition, 90% of the clinical samples with integrated virus and E2 loss showed no or low ErbB-3 positivity, confirming in vitro results. In conclusion, the new discovered interaction of HPV-16 E2 with Nrdp-1 may affect ErbB-3 expression.

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

BACKGROUND: Human 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. METHODS: Sixty-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. RESULTS: HPV 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%). CONCLUSIONS: The 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 SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1159157579149317 .

Hypermethylation of TFPI2 correlates with cervical cancer incidence in the Uygur and Han populations of Xinjiang, China.

Tissue factor pathway inhibitor 2 (TFPI2) is a Kunitz-type serine proteinase inhibitor, which plays an important role in the etiology of human malignancies. DNA methylation is a common epigenetic modification of the genome that is involved in regulating many cellular processes. In addition to human papilloma virus (HPV) infection, DNA methylation may play a role in the carcinogenesis of cervical cancer. Methylation of 22 CpG sites in the promoter region of the TFPI2 gene was detected by MassARRAY spectrometry and a gene mass spectrogram was drawn using MALDI-TOF MS. HPV16 was detected by PCR. We show that aberrant methylation of TFPI2 is present in a higher proportion of invasive cervical carcinoma (ICC) clinical samples as compared to normal cervical samples in Uygur and Han. Across the four pathologic lesions of the progression of cervical cancer, ICC showed the highest level of aberrant methylation, and with a stronger correlation between CpG site and lesion grade in Uygur than in Han. Moreover, a difference in TFPI2 methylation between Uygur patients positive and negative for HPV16 infection was observed at CpG_6 (P = 0.028) and CpG_15 (P = 0.007). Altogether, these results indicate that DNA methylation of TFPI2 may play an important role in the carcinogenesis of cervical cancer and that the differential methylation of TFPI2 may at least partially explain the disparity in cervical cancer incidence between Uygur and Han women.

Immunosuppression in cervical cancer with special reference to arginase activity.

INTRODUCTION: Cervical cancer is characterized by an immunosuppressive microenvironment and a Th2-type cytokine profile. Expression of arginase (ASE), the enzyme that converts L-arginine into L-ornithine and urea, is stimulated by Th2-type cytokines. OBJECTIVE: To assess the association of ASE activity and L-Arg metabolism products with cervical cancer. METHODS: Sera of 87 and 41 women with histologically confirmed by colposcopy-directed biopsy SCC and CIN3 respectively and 79 with normal cytology or Low-Grade Squamous Intraepithelial Lesion (LSIL), were evaluated. Cytokines were measured using Milliplex Human cytokine/chemokine kit. Arginase (ASE) activity was determined using an enzymatic assay. Levels of L-arginine, L-ornithine, putrescine and spermine were determined by HPLC. RESULTS: Significantly higher levels of ASE activity were observed in women with CIN3 (age-adjusted OR: 24.3; 95%CI: 3.82-155) and SCC (AOR: 9.8; 95%CI: 2.34-40.8). As expected, possibly due to high levels of ASE activity, higher levels of l-Arg were negatively associated with CIN3 (AOR: 0.03; 95%CI: 0.004-0.19) and SSC (AOR: 0.06; 95%CI: 0.02-0.24). Consistent with the role of ASE in the conversion of L-arginine to L-ornithine and polyamine production therefrom, women with cervical cancer had higher levels of spermine and putrescine. A correlation analysis revealed a significant albeit weak relationship between high levels of IL-10 and high levels of ASE (Pearson r=0.32, p-value=0.003) in women with cervical cancer. CONCLUSION: This study indicates that ASE activity and L-Arg degradation mechanisms of immunosuppression are present in cervical cancer. The results foster research in the design of possible strategies to inhibit ASE activity for therapy of cervical cancer.

Differential expression patterns of N-acetylglucosaminyl transferases and polylactosamines in uterine lesions.

Polylactosamine (polyLacNAc) is a fundamental structure in glycoconjugates and it is expressed in specific cells/tissues associated with the development and carcinogenesis. ß1,3-N-acetylglucosaminyl transferases (ß3GnTs) play an important role in polyLacNAc synthesis, however the roles of these glycosyltransferases and their products in cancer progression are still unclear. In this sense, this work aimed to evaluate differential expression pattern of the N-acetylglucosaminyl transferases and polylactosamines in invasive and premalignant lesions of the uterus cervix. The expression of ß3GnT2 and ß3GnT3 were evaluated in normal (n=10) and uterine cervix lesions (n= 120) malignant (squamous carcinoma - SC) and premalignant (cervical intraepithelial neoplasia - CIN - grades 1, 2 and 3) using immunohistochemistry. Besides, lectin histochemistry with Phytolacca americana lectin (PWM) and Wheat germ agglutinin (WGA) was also carried out to observe the presence of polyLacNAc chains and N-acetylglucosamine (GlcNAc), respectively. The ß3GnT3 was expressed in almost all samples (99%) and ß3GnT2 was higher expressed in disease samples mainly in CIN 3, when compared with normal (P=0.002), CIN 1 (P=0.009) and CIN 2 (P=0.03). The expression of polyLacNAc was higher is SC samples, when compared with normal (P=0.03), CIN 1 (P=0.02) and CIN 3 (P=0.004), and was observed only nuclear expression in nearly 50% of the SC samples, showing a statistically significant when compared with normal (P=0.01), CIN 1 (P=0.002), CIN 2 (P=0.007) and CIN 3 (P=0.04). Deferring from transferases and polyLacNAc chains, GlcNAc (WGA ligand) reveals a gradual staining pattern decrease with the increase of the lesion degree, being more expressed in CIN 1 lesions when compared with normal (P<0.0001), CIN 2 (P<0.0001), SC (P<0.0001) and CIN 3 (P=0.0003). Our data reveals ß3GnT2 and polyLacNAc may be involved in the progression of the pre-malignant lesions of human the uterine cervix. In addition, polyLacNAc expression only in the nucleus can be associated a poor prognostic in uterine lesions.

GTPases Rho distribution in intraepithelial and invasive neoplasias of the uterine cervix.

PURPOSE OF INVESTIGATION: To evaluate the distribution of GTPases RhoA, RhoB, and Cdc42 in cervical intraepithelial neoplasias (CIN) and invasive neoplasias of the uterine cervix. MATERIALS AND METHODS: samples of neoplastic lesions of the uterine cervix of 44 patients were classified in: CIN I (n = 10), CIN II (n = 10), CIN III (n = 09), and invasive carcinoma (n = 15). Antibodies anti-RhoA, anti-RhoB, and anti-Cdc42 were used and staining was classified as: negative, mild, moderate, and intense positive. RESULTS: When compared with dysplastic cells, superficial cells showed: higher expression of RhoB in CIN I (p = 0.0018), and lower expression of Cdc42 in CIN I (p = 0.0225). The authors observed higher expression of RhoA (p = 0.0002) and RhoB (p = 0.0046) in CIN dysplastic cells when compared with invasive carcinoma cells. CONCLUSIONS: GTPases Rho may be involved with the regulation of biological processes, important to the progression of cervical neoplasias. Probably, RhoA is important for maintenance of cell differentiation and RhoB protects cells from malignant cervical neoplasia.

Evaluation of microbial enzymes in normal and abnormal cervicovaginal fluids of cervical dysplasia: a case control study.

The aim of the present study was to evaluate the role of microbial enzymes in normal and abnormal cervicovaginal fluids of cervical dysplasia. The cervicovaginal infections were evaluated through the estimation of microbial enzymes in patients with and without abnormal cervical cytology like bacterial and fungal infections. The patients were categorized based on infection caused by organism and stages of dysplasia. The pH, Whiff test, and Pap smear tests were conducted for normal and abnormal cervical swabs based on standard protocols. Microbial enzymes include mucinase, sialidases, and proteases of the cervical swabs and are estimated according to standard methods. The results of abnormal cervical cytological smears showed increased pH and the presence of amines with different levels of Pap smear test. Increased levels of microbial enzymes were observed in patients with abnormal cytology than normal cytology. Three microbial enzymes mucinase, sialidase, and protease were significantly (P < 0.01) more elevated in patients with bacterial infections (8.97 ± 0.64, 10.39 ± 0.28, 8.12 ± 0.64) than without dysplasia (2.02 ± 0.8, 1.98 ± 0.3, 1.96 ± 0.8). The results reinforce that the microbial infection seems to be more prone to cervical dysplasia and may act as risk-factor for the development of cervical cancer along with HPV infection.

MTHFR/p53 polymorphisms as genetic factors for cervical intraepithelial neoplasia and cervical cancer in HPV-infected Mexican women.

We performed a case-control association study to evaluate the association between common polymorphisms in MTHFR (C677T and A1298C) and the Arg72Pro polymorphism in the p53 gene and the risk for cervical intraepithelial neoplasia (CIN) or invasive cervical cancer (ICC) in Mexican HPV-infected women. We included 131 women with diagnosis of CIN grade I-II and 78 with CIN III or ICC; as controls we also included 274 women with normal Pap smear and negative HPV test. Genotyping for MTHFR and p53 polymorphisms was performed by PCR-RFPLs. HPV was tested by Hybrid Capture II. Odds ratios and 95% confidence intervals were estimated. Genotype frequencies for the 3 studied polymorphisms were distributed according to the Hardy-Weinberg equilibrium. The A1298C-MTHFR polymorphism showed significant differences for the heterozygous AC genotype and the C allele, whereas the AA genotype and A allele resulted to be genetic risk factors for CIN or ICC (p<0.03). The Arg72Pro-p53 polymorphism showed for the genotypes Arg/Pro and Pro/Pro, and for the Pro allele, a significant association only to the risk for CIN (p<0.03). The MTHFR/p53 interaction showed that the genotype combinations AA/ArgArg and AA/ArgPro were associated, respectively, to the risk of ICC and CIN (p<0.05). This study suggests that the A1298C-MTHFR polymorphism contributes to the genetic risk for both CIN and ICC, whereas the Arg72Pro-p53 polymorphism only contributes to the risk for CIN. The MTHFR/p53 genetic combinations AA/ArgArg and AA/ArgPro are associated genetic risk factors for ICC and CIN in Mexican HPV-infected women.

Methylation of human papillomavirus 16, 18, 31, and 45 L2 and L1 genes and the cellular DAPK gene: Considerations for use as biomarkers of the progression of cervical neoplasia.

During progression of cervical cancer, human papillomavirus genomes and cellular tumor suppressor genes can become methylated. Toward a better understanding of these biomarkers, we studied 104 samples with HPV16, 18, 31, and 45 representing five pathological categories from asymptomatic infection to cancer. We grouped all samples by HPV type and pathology and measured the overall methylation of informative amplicons of HPV late genes and the cellular DAPK gene. Methylation of all four HPV types as well as of the DAPK gene is lowest in asymptomatic infection and increases successively in all four pathological categories during progression to cancer. 27 out of 28 cancer samples showed methylation both in the L2/L1 genes as well as in DAPK, but a much lower fraction in all other pathological categories. We discuss the problem to develop diagnostic tests based on complex methylation patterns that make it difficult to classify amplicons as "methylated" or "unmethylated".

Overexpression of p16(INK4a) in urothelial carcinoma in situ is a marker for MAPK-mediated epithelial-mesenchymal transition but is not related to human papillomavirus infection.

BACKGROUND: The role of human papillomavirus (HPV) in bladder carcinogenesis remains controversial. Overexpression of p16(INK4a), a surrogate marker for infection with oncogenic HPV in other tumours, has been described for urothelial carcinoma in situ (UCIS). Our goal was therefore to evaluate whether overexpression of p16(INK4a) is associated with HPV infection and to identify mechanisms of p16(INK4a) upregulation in UCIS. MATERIALS AND METHODS: In 60 tissue specimens from a total of 45 patients (UCIS and controls), we performed p16(INK4a) immunohistochemistry followed by detection and subclassification of HPV DNA. In a subset of samples, we tested for gene amplification of p16(INK4a) applying fluorescence in situ hybridization (FISH). RAS/MAPK signalling and epithelial-mesenchymal transition (EMT) was assessed using immunohistochemistry. Finally, we transfected urothelial carcinoma cells with KRAS and examined the expression of p16(INK4a) as well as markers of EMT. RESULTS: We found overexpression of p16(INK4a) in 92.6% of UCIS and in all cervical intraepithelial neoplasia (CIN) controls. In contrast, we detected high-risk HPV DNA in 80% of CIN, but none in UCIS. There was no gene amplification of p16(INK4a). High levels of phosphorylated kinases and urokinase plasminogen activator (uPA) and loss of membraneous E-cadherin were detected in UCIS. KRAS transfection of urothelial carcinoma cells led to upregulation of p16(INK4a) and uPA accompanied by loss of E-cadherin that could be inhibited by application of the kinase-inhibitor Sorafenib. CONCLUSIONS: Our results show that overexpression of p16(INK4a) in UCIS is neither associated with HPV infection nor p16(INK4a) gene amplification but is a consequence of enhanced RAS/MAPK signalling that promotes EMT, possibly due to Sorafenib-sensitive paracrine secretion of the EMT activator uPA. These findings might open a novel therapeutic option for localized but aggressive urothelial cancer.

Genomic amplification of the human telomerase gene (hTERC) associated with human papillomavirus is related to the progression of uterine cervical dysplasia to invasive cancer.

BACKGROUND: Human papillomavirus (HPV) infection plays an etiological role in the development of cervical dysplasia and cancer. Amplification of human telomerase gene (hTERC) and over expression of telomerase were found to be associated with cervical tumorigenesis. This study was performed to analyze genomic amplification of hTERC gene, telomerase activity in association with HPV infection in different stages of cervical intraepithelial neoplasia (CIN) and cervical cancer. We were studying the role of hTERC in the progression of uterine cervical dysplasia to invasive cancer, and proposed an adjunct method for cervical cancer screening. METHODS: Exfoliated cervical cells were collected from 114 patients with non neoplastic lesion (NNL, n=27), cervical intraepithelial neoplasia (CIN1, n=26, CIN2, n=16, CIN3, n=24) and cervical carcinoma (CA, n=21), and analyzed for amplification of hTERC with two-color fluorescence in situ hybridization (FISH) probe and HPV-DNA with Hybrid Capture 2.From these patients, 53 were taken biopsy to analyze telomerase activity by telomeric repeat amplification protocol (TRAP) and expression of human telomerase reverse transcriptase (hTERT), with immunohistochemistry (IHC). All biopsies were clinically confirmed by phathologists. RESULTS: Amplification of hTERC was significantly associated with the histologic diagnoses (p<0.05). The positive correlation was found between the level of hTERC amplification and histologic grading of dysplasia (CIN2/3 from CIN1 or normal, P=0.03). A profounding increase in the accumulation of HPV and hTERC positive cases was observed in the CIN3 subgroup compared with the CIN2 group, 25% versus 62.96%, respectively (p=0.007). CONCLUSIONS: hTERC ampliffication can be detected with FISH technique on exfoliated cervical cells. Amplification of hTERC and HPV infection are associated with more progressive CIN3 and CA. The testing of hTERC amplification might be a supplementary to cytology screening and HPV test, especially high-risk patients. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1857134686755648.

Predictive role of telomerase activity in the clinical outcome of patients with benign lesions of the uterine cervix or CIN.

Telomerase, a fundamental marker of neoplastic transformation, is widely expressed in both premalignant intraepithelial lesions and in most malignant lesions of the uterine cervix. We determined telomerase activity (TA) in uterine cervix by Repeat Amplification Protocol (TRAP) in a series of 62 cases, 44 with benign diseases (inflammation and/or metaplasia and/or acanthosis) and 18 with cervical intraepithelial neoplasia (CIN). No significant differences in TA were observed between benign lesions (median AEU value 36, range 0-119) and CIN (median AEU value 30, range 0-65). Conversely, TA was significantly higher in subjects who showed CIN evolution (65 range 45-119) than in disease-free individuals (34 range 0-95, ρ = 0.017) and in 1 patient with a CIN2 lesion who relapsed after 5 years. Our results suggest that TA of the uterine cervix is capable of predicting CIN evolution or relapse, thus indicating its potential usefulness as a prognostic marker in clinical surveillance programs.

Role of the purinergic system in patients with cervical intraepithelial neoplasia and uterine cancer.

INTRODUCTION: Cervical cancer remains the second leading cause of death among women. Intraepithelial neoplasias and uterine invasive cancer are frequently associated with disturbances in coagulation and changes in the concentrations of adenine nucleotides. This work intended to analyze changes in extracellular adenine nucleotide hydrolysis and blood platelet aggregation in patients diagnosed for cervical intraepithelial neoplasia in different stages as well as uterine invasive cancer. PATIENTS AND METHODS: NTPDase, E-NPP, 5'-nucleotidase, total ADA and its isoforms (ADA1 and ADA2), as well as the platelet aggregation from patients with different stages of cervical intraepithelial neoplasia (NICs I, NIC II, NIC III) and uterine invasive cancer were verified. RESULTS: Neither ATP hydrolysis nor E-NPP activity was changed by the neoplasia stage. On the other hand, ADP and AMP hydrolysis as well as ADA activity were enhanced in NIC I group. AMP hydrolysis was also increased in the cancer group. ADA 1 was the ADA isoform found in platelets from both control and patient groups. CONCLUSION: Our results showed for the first time that NTPDase, 5'-nucleotidase, E-NPP and ADA are not sensible regarding the grade of neoplasia development, since no significant difference was found between the groups studied. Only ADP hydrolysis and ADA activity showed a significant enhancement in NIC I group related to the other stages possibly as a result of the beginning of the neoplasic transformation. This increase could be reflecting a body's reaction against the probable high adenosine levels. We propose for the first time that the ADA isoform present in platelets is ADA 1.