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.

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.

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.

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.

Carbonic anhydrase IX (CA-IX) and high-risk human papillomavirus (H-HPV) as diagnostic biomarkers of cervical dysplasia/neoplasia in Japanese women with a cytologic diagnosis of atypical glandular cells (AGC): a Gynecologic Oncology Group (GOG) Study.

BACKGROUND: High-risk human papillomavirus (H-HPV) infection is linked to cervical neoplasia but its role in detecting cervical glandular lesions (GLs) is unclear. Carbonic anhydrase IX (CA-IX) is a hypoxic biomarker that is highly expressed in neoplastic cervical GLs. The diagnostic utility of these biomarkers was evaluated by the Gynecologic Oncology Group in Japanese women with a cytological diagnosis of atypical glandular cells. METHODS: Immunostaining was used to detect CA-IX in a conventional Pap smear. Immunoreactivity of CA-IX was interpreted by a panel of pathologists blinded to the histological diagnosis. Polymerase chain reaction was used to detect H-HPV in a liquid-based cytology specimen. RESULTS: Significant cervical lesions (SCLs), defined as cervical intraepithelial neoplasia (CIN2, CIN3), adenocarcinoma in situ or invasive carcinoma, were observed in 37/88 (42%) of women. CA-IX testing alone (n=88) had a sensitivity of 89, 100 or 73% for SCLs, GLs or significant squamous lesions (SLs), respectively, with a false negative rate (FNR) of 14%. Testing for H-HPV (n=84) had a sensitivity of 65, 53 or 80% for SCLs, GLs or SLs, respectively, with a FNR of 22%. The combination of CA-IX and H-HPV testing had a sensitivity of 97, 100 or 93% for SCLs, GLs or SLs, respectively, with a FNR of 5%. Among eight H-HPV-negative GLs, six (75%) had a diagnosis of lobular endocervical glandular hyperplasia (LEGH). CONCLUSION: The combination of CA-IX and HPV testing improved the diagnostic accuracy. The low rate of H-HPV positivity in the GLs was associated with coexisting LEGH independent of H-HPV.

Common polymorphisms in methylenetetrahydrofolate reductase gene are associated with risks of cervical intraepithelial neoplasia and cervical cancer in women with low serum folate and vitamin B12.

OBJECTIVE: We evaluated associations between folate, vitamin B12, and the methylenetetrahydrofolate reductase (MTHFR) gene, and risk of cervical intraepithelial neoplasia (CIN) and cervical cancer. METHODS: This multicenter case-control study enrolled 927 Korean women (440 controls, 165 patients with CIN 1, 167 patients with CIN 2/3, and 155 patients with cervical cancer, aged 20-75 years). RESULTS: Patients with cervical cancer had significantly lower median serum folate and vitamin B12 concentrations vs. controls. Higher serum folate was significantly associated with lower cervical cancer risk (p for linear trend = 0.0058) with a trend for a lower CIN risk after multivariate adjustment. Low folate and the MTHFR 677 C > T variant were associated with a higher risk for CIN2/3 and cervical cancer vs. wild-type or heterozygous genotypes with high folate [OR, 2.39 (1.18-4.85) and 3.19 (1.43-7.13)]. Low vitamin B12 and the MTHFR 677 C > T variant also were associated with a higher risk for CIN 2/3 and cervical cancers [OR, 2.52 (1.17-5.42) and 2.40 (1-5.73)] vs. wild-type or heterozygous status with high vitamin levels. CONCLUSION: Serum folate concentration is inversely associated with the risk of cervical cancer, and the MTHFR variant genotype may increase CIN and cervical cancer risk in women with low folate or vitamin B12 status.

Role and predictive strength of transglutaminase type 2 expression in premalignant lesions of the cervix.

The demonstration that type 2 transglutaminase (TG2) can incorporate polyamine into the E7 oncoprotein of human papillomavirus (HPV) type 18 has led to the hypothesis that TG2 can have a role in the host cellular response to HPV infection. The aim of this study was to investigate whether HPV-related pathology, in infected human cervical epithelium, was associated with modulation of TG2 expression. Normal controls and HPV-infected cervical biopsies were analyzed for the expression of TG2, and the findings were compared with lesion grade. The correlation between TG2 expression and p16, a marker for HPV-induced dysplasia, and the retinoblastoma protein (Rb), a target of the E7 protein of HPV, was also investigated. Results obtained showed that TG2 was absent in normal squamous mucosa, whereas it was present in 100% CIN I lesions. Low-grade lesions showed significantly higher TG2 expression than high-grade lesions (P<0.0001). In 94% of CIN I more than 50% of the cells were positive for TG2, with a strong staining intensity (+3), whereas a decreased staining intensity and a low number of positive cells were found in CIN II/III. In CIN I cases, both nuclear and cytoplasmic staining were found in cells exhibiting classical morphological features of HPV infection. In addition, during progression from low-grade squamous intraepithelial lesions to severe dysplasia, TG2 expression was inversely correlated with p16 (Pearson: -0.930), whereas a positive correlation was observed between the expression of TG2 and pRb (Pearson: 0.997). TG2 is expressed in HPV infection as an early phenomenon, not restricted to high-risk genotypes. TG2 upregulation is probably part of host cell reaction against HPV-induced tissue modification. It may act as a cellular antioxidant defense factor, playing an important role in counteracting oxidative damage in neoplastic disease.

Application of hTERC in thinprep samples with mild cytologic abnormality and HR-HPV positive.

OBJECTIVE: Amplification of hTERC is found to be an important genetic event in the progression from cervical dysplasia to cervical cancer. The hTERC value in predicting high-grade cervical intraepithelial neoplasia (CIN) or squamous cell carcinoma (SCC), in high-risk HPV (HR-HPV) positive thinprep samples with atypical squamous cells (ASC) or a low-grade squamous intraepithelial lesion (LSIL) was explored in this study. METHODS: A total of 300 thinprep cytology specimens (129 of ASC-US, 82 of LSIL, and 89 of ASC-H) with positive HR-HPV DNA was detected by a two-probe dual-color FISH panel, targeting hTERC and the centromeric region of chromosome 3 (CSP3). Using >2 signals for hTERC together with ≥2 signals for CSP3 to define abnormal nucleus, and the cutoff value was set at 6.5 per random 200 nuclei displayed increased hTERC signals and/or tumor ploidy. Statistical analyses were based on histologic findings of colposcopy biopsies, allowing CIN2 or worse (CIN2+) as the positive criterion. RESULTS: The FISH results were systematically analyzed among groups, based on histologic diagnosis, cytologic finding, HR-HPV viral load, and age status. hTERC presented good consistency with histology, and had satisfactory sensitivity, specificity, and accuracy among different groups, with less difference intergroup. The individual hTERC positive nuclei ratio was generally increased with severity of the cervical lesions. CONCLUSIONS: hTERC could be a stable predictor in assuring the risk of high-grade CIN in women with mild cytologic abnormality and positive HR-HPV, and the individual positive nuclei ratio of it might be helpful in identifying morbid grade for cervical lesions.

Vascular endothelial growth factor C promotes cervical cancer metastasis via up-regulation and activation of RhoA/ROCK-2/moesin cascade.

BACKGROUND: The elevated expression of vascular endothelial growth factor C (VEGF-C) is correlated with clinical cervical cancer metastasis and patient survival, which is interpreted by VEGF-C functions to stimulate angiogenesis and lymphatic genesis. However, the direct impact of VEGF-C on cervical cancer cell motility remains largely unknown. METHODS: In this study, we investigated the effects of VEGF-C on actin cytoskeleton remodeling and on cervical cancer cell migration and invasion and how the actin-regulatory protein, moesin regulated these effects through RhoA/ROCK-2 signaling pathway. RESULTS: On cervical carcinoma cell line SiHa cells, exposure of VEGF-C triggered remodeling of the actin cytoskeleton and the formation of membrane ruffles, which was required for cell movement. VEGF-C significantly enhanced SiHa cells horizontal migration and three-dimensional invasion into matrices. These actions were dependent on increased expression and phosphorylation of the actin-regulatory protein moesin and specific moesin siRNA severely impaired VEGF-C stimulated-cell migration. The extracellular small GTPase RhoA/ROCK-2 cascade mediated the increased moesin expression and phosphorylation, which was discovered by the use of Y-27632, a specific inhibitor of Rho kinase and by transfected constitutively active, dominant-negative RhoA as well as ROCK-2 SiRNA. Furthermore, in the surgical cervical specimen from the patients with FIGO stage at cervical intra-epithelial neoplasia and I-II cervical squamous cell carcinoma, the expression levels of moesin were found to be significantly correlated with tumor malignancy and metastasis. CONCLUSIONS: These results implied that VEGF-C promoted cervical cancer metastasis by upregulation and activation of moesin protein through RhoA/ROCK-2 pathway. Our findings offer new insight into the role of VEGF-C on cervical cancer progression and may provide potential targets for cervical cancer therapy.

Amplification of the human telomerase gene in liquid-based preparations is associated with cervical dysplasia and carcinoma.

The aim of this study was to analyze the amplification of the human telomerase gene (TERC) in cervical specimens by fluorescence in situ hybridization (FISH), and FISH findings were compared with cytologic and histologic diagnoses. Slides prepared from 123 liquid-based preparations from cervical specimens with cytologic diagnoses of negative for squamous intraepithelial lesion or malignancy (n=20), atypical squamous cells of undetermined significance (n=22), low-grade squamous intraepithelial lesion (n=55), high-grade squamous intraepithelial lesion (n=21), or invasive cervical carcinomas (n=5) were analyzed for the amplification of TERC using a 2-color FISH probe. The results of the cytologic analysis and those of concurrent or subsequent biopsies were compared with the FISH findings. Results showed that amplification of TERC was significantly associated with both cytologic and histologic diagnoses (P<0.05). Patients with high-grade squamous intraepithelial lesion or squamous cell carcinoma cytology diagnoses had significantly higher percentages of cells with the amplification of TERC than did patients with low-grade squamous intraepithelial lesion, ASC-US, and negative for squamous intraepithelial lesion or malignancy (P<0.005). FISH can be performed on cervical liquid-based preparations to detect the amplification of TERC. This test may be an adjunct to cytology screening, early detection of cervix neoplasm, and may determine the progressive potential of individual lesions, especially in high-risk patients.

Carbonic anhydrase IX and human papillomavirus as diagnostic biomarkers of cervical dysplasia/neoplasia in women with a cytologic diagnosis of atypical glandular cells: a Gynecologic Oncology Group study in United States.

High-risk human papillomavirus (H-HPV) infection is strongly linked to cervical neoplasia, but its role in detecting glandular lesions (GLs) is unclear. In the cervix, carbonic anhydrase IX (CA-IX) is expressed in cervical neoplasia, but rarely in the benign cervix. The diagnostic utility of these biomarkers was evaluated in women with a cytologic diagnosis of atypical glandular cells (AGC). H-HPV was detected using hybrid capture 2 (HC2) in liquid-based cytology, and CA-IX immunoreactivity was studied on conventional Pap smears. Of 403 patients, 111 (28%) were positive for significant cervical lesions (SCLs) including CIN2, CIN3, adenocarcinoma in situ or invasive carcinoma. CA-IX testing alone (n = 403) had a sensitivity of 75, 95 or 65% for SCLs, significant GLs or squamous lesions (SLs), respectively, with a specificity of 88% and a false negative rate (FNR defined as 1 minus negative predictive value) of 10%. Testing for H-HPV (n = 122) had a sensitivity of 97, 100 or 96% for SCLs, GLs or SLs, respectively, with a specificity of 87% and a FNR of 1%. The combination of CA-IX and H-HPV testing (n = 122), collectively, had the same sensitivity, specificity and FNR for SCLs, GLs or SLs as H-HPV testing alone. The conclusions of our study are that both H-HPV and CA-IX testing are useful diagnostic markers for GLs. However, H-HPV testing is a better diagnostic marker for SLs. The combination of CA-IX with H-HPV testing does not improve the diagnostic accuracy for cervical neoplasia in women with AGC diagnosis over that of H-HPV testing alone.

High expression of PGE2 enzymatic pathways in cervical (pre)neoplastic lesions and functional consequences for antigen-presenting cells.

Although human papillomavirus (HPV) DNA is detected in the majority of squamous intraepithelial lesions (SIL) and carcinoma (SCC) of the uterine cervix, the persistence or progression of cervical lesions suggest that viral antigens are not adequately presented to the immune system. This hypothesis is reinforced by the observation that most SIL show quantitative and functional alterations of Langerhans cells (LC). The aim of this study was to determine whether prostaglandins (PG) may affect LC density in the cervical (pre)neoplastic epithelium. We first demonstrated that the epithelial expression of PGE(2) enzymatic pathways, including cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase 1 (mPGES-1), is higher in SIL and SCC compared to the normal exocervical epithelium and inversely correlated to the density of CD1a-positive LC. By using cell migration assays, we next showed that the motility of immature dendritic cells (DC) and DC partially differentiated in vitro in the presence of PGE(2) are differentially affected by PGE(2). Immature DC had a lower ability to migrate in the presence of PGE(2) compared to DC generated in vitro in the presence of PGE(2). Finally, we showed that PGE(2) induced a cytokine production profile and phenotypical features of tolerogenic DC, suggesting that the altered expression of PGE(2) enzymatic pathways may promote the cervical carcinogenesis by favouring (pre)cancer immunotolerance.

Local expression of interferon-alpha and interferon receptors in cervical intraepithelial neoplasia.

PURPOSE: The present study evaluated mRNA expression of interferon-alpha (IFN-alpha), IFN-alpha receptor subunits (IFNAR-1 and IFNAR-2) and an IFN-stimulated gene encoding the enzyme 2',5'-oligoadenylate synthetase (2'5'OAS) in biopsies on patients with varying grades of cervical intraepithelial neoplasia (CIN I, II and III). METHODS: Uterine cervix biopsies were collected from women with CIN I, II and III (n = 28) and controls without CIN lesions or human papilloma virus (HPV) infection (n = 17). The presence of high and low-risk HPV DNA was determined using hybrid capture. The mRNA levels of IFNAR-1, IFNAR-2, IFN-alpha and 2'5'OAS were determined by RT-PCR with specific primers. RESULTS: The control group exhibited a greater frequency of IFNAR-1 expression (10/17; 58.3%) than the CIN samples (4/28; 14.2%) (P = 0.0018), while, the expression of IFNAR-2 was also greater in the control samples (11/17; 64.7%) than in the patients with lesions (2/28; 7.1%) (P = 0.0018). Importantly, simultaneous expression of both receptors was observed only in the control group (8/17; 47.0%) (P = 0.0001). Among the CIN samples, there was one case of low expression of mRNA of IFNAR-1 and IFNAR-2. IFN-alpha was present in 14.2% (4/28) of the CIN samples but was not expressed in the control group. mRNA 2'5'OAS were expressed in 28.5% (8/28) of the CIN samples and 11.7% (2/17) of the control samples (not statistically significant). Fifty percent (14/28) of the CIN samples were positive for HPV DNA. CONCLUSIONS: Cervical biopsy samples from control women or those without neoplasia or HPV infection displayed higher IFN-alpha receptor expression than those with CIN, while simultaneous expression of both IFN-alpha receptor subunits was found only in the control group. There was no significant difference in mRNA expression of IFN-alpha and 2'5'OAS between the control and CIN groups. Then we concluded that the samples obtained from patients with CIN present low levels of the IFN-alpha receptor mRNA.

Expression of ADAM9 in CIN3 lesions and squamous cell carcinomas of the cervix.

OBJECTIVE: ADAM9 is a member of the ADAM family which is involved in cellular processes like cell adhesion, migration and signalling [M.L. Moss, J.M. White, M.H. Lambert, R.C. Andrews, TACE and other ADAM proteases as targets for drug discovery. Drug Discov. Today 2001; 6:417-426., G. Murphy, The ADAMS: signalling scissors in the tumour microenvironment. Nat. Cancer Rev. 2008; 8:929-941.]. ADAM9 overexpression has been described in many of solid tumours including prostate, renal, pancreas, lung and gastric cancer [R.J. Grutzmann, J. Luttges, B. Sipos, O. Ammerpohl, F. Dobrowolski, I. Alldinger, et al., ADAM9 expression in pancreatic cancer is associated with tumour type and is a prognostic factor in ductal adenocarcinoma. Br. J. Cancer 2004; 90:1053-1058., C.A. Iacobuzio-Donahue, A. Maitra, M. Olsen, A.W. Lowe, N.T. van Heek, C. Rosty, et al., Exploration of global gene expression patterns in pancreatic adenocarcinoma using cDNA microarrays. Am. J. Pathol. 2003; 162: 1151-1162., Y. Shintani, S. Higashiyama, M. Ohta, H. Hirabayashi, S. Yamamoto,T. Yoshimasu, et al., Overexpression of ADAM9 in non-small cell lung cancer correlates with brain metastasis. Cancer Res. 2004; 64:4190-4196., S. Carl-McGrath, U. Lendeckel, M. Ebert, A. Roessner, C. Rocken, The disintegrin-metalloproteinases ADAM9, ADAM12, and ADAM15 are upregulated in gastric cancer. Int. J. Oncol. 2005; 26:17-24., F.R. Fritzsche, K. Wassermann, M. Jung, A. Tölle, I. Kristiansen, M. Lein, et al., ADAM9 is highly expressed in renal cell cancer and is associated with tumour progression. BMC Cancer 2008; 8:179.]. The involvement of this protease in cervical carcinogenesis has not been yet investigated. The aim of this study was to evaluate the expression of ADAM9 in normal epithelium, CIN3 lesions and squamous cell carcinomas (SCC) of the uterine cervix. METHODS: Archived paraffin-embedded tissue blocks from biopsy or surgery specimens obtained from 50 subjects with CIN3 and squamous cancer of the cervix were studied by immunohistochemistry using heat-induced epitope retrieval for ADAM9 expression. RESULTS: Weak expression of ADAM9 was found in the normal cervical epithelium with weak cytoplasmatic staining but also membrane immunoreactivity. Evident staining for ADAM9 was detected in 31 out of 36 (86%) CIN3 lesions and in 13 out of 14 (93%) squamous cell carcinomas of the uterine cervix. Staining was stronger in SCC compared to CIN3 lesions. Moderate staining was detected in 64% (9/14) of SCC and in 36% (13/36) CIN3 lesions. Weak staining was observed in 50% (18/36) of CIN3 lesions and in 29% (4/14) of SCC. The difference in the ADAM9 protein expression between cervical squamous carcinomas and normal epithelium was highly significant. Statistical significance was also found for the increased expression observed in CIN3 lesions versus normal squamous epithelium. CONCLUSIONS: Our results show for the first time, that ADAM9 expression is low in the squamous epithelium of the cervix, but is increased in CIN3 lesions as well as SCCs being the increase in both cases statistically significant.

Functional polymorphism in manganese superoxide dismutase and antioxidant status: their interactions on the risk of cervical intraepithelial neoplasia and cervical cancer.

OBJECTIVE: Manganese superoxide dismutase (MnSOD), the primary antioxidant enzyme in mitochondria, plays a key role in protecting cells from oxidative stress. Furthermore, the MnSOD rs4880 polymorphism is associated with enzyme activity. The authors evaluated the interaction between MnSOD genotypes and cervical carcinogenesis risk and the modulating effects of serum antioxidant nutrient status (beta-carotene, lycopene, zeaxanthin/lutein, retinol, alpha-tocopherol and gamma-tocopherol). METHODS: Cases and controls for this study were recruited between June 2006 and July 2007 (263 controls, 84 cervical intraepithelial neoplasia (CIN), 94 CIN 2/3, and 99 cases of cervical cancer). The MnSOD polymorphism at rs4880T/C was examined using SNaPshot assays. Serum antioxidant vitamin concentrations were measured by reverse-phase gradient high-pressure liquid chromatography. Odds ratios (OR) and 95% confidence intervals (95%CI) were estimated after adjusting for age, menopause, parity, oral contraceptive use, smoking and alcohol consumption. RESULTS: No association was found between the MnSOD rs4880 polymorphism and cervical cancer. However, genotypes significantly modified the risk of cervical cancer in association with the serum statuses of micronutrients (P(interaction)<0.05 for beta-carotene, lycopene, zeaxanthin/lutein, alpha-tocopherol, and gamma-tocopherol). Decreased CIN1 risk in association with the MnSOD rs4880 variant genotype was also observed particularly for subjects with higher beta-carotene and gamma-tocopherol levels. Similar results were observed for lycopene and alpha-tocopherol in relation to the risk of CIN2/3. CONCLUSION: Our findings suggest that a higher antioxidant micronutrients status may decrease the risk of CIN and cervical cancer and modify the effect of the MnSOD polymorphism on disease risk.

Expression of human peroxiredoxin isoforms in response to cervical carcinogenesis.

Despite considerable progress in understanding the function of peroxiredoxin (Prx) in cancer, its expression patterns have not been extensively studied in response to cervical carcinogenesis. We evaluated the expression of Prx isoforms in normal tissue, cervical intraepithelial neoplasia (CIN1, CIN2, and CIN3), and cervical cancer. We found strong pattern of increased Prx II and III immunostaining with increasing severity of the lesion. No difference in staining intensity by grade of lesion was observed for Prx I, and IV. Therefore, we conclude that Prx II and III are upregulated in response to the development of cervical cancer.

Enhanced sialyltransferases transcription in cervical intraepithelial neoplasia.

Altered sialylation observed during oncogenic transformation, tumor metastases and invasion, has been associated with enhanced sialyltransferases (STs) transcription. Increased mRNA expression of STs (ST6Gal I, ST3Gal III) has been detected in invasive cervical squamous cell carcinoma. A study of the sialic acid concentration in local tissue of cervix and in serum showed a slight elevation in benign inflammatory lesions and a moderate elevation in severe neoplasia, but to date, altered expression of STs in cervical intraepithelial neoplasia has not yet been evaluated. This study investigates the changes in mRNA expression of three STs (ST6Gal I, ST3Gal III, and ST3Gal IV) in cervical intraepithelial lesions (CIN). Alterations of these STs mRNA expression were examined in 35 cervix specimens classified as normal, CIN 1, CIN 2 and CIN 3, by semiquantitative reverse transcription-polymerase chain reaction, mRNA expression of the three STs was enhanced in CIN 1, CIN 2 and CIN 3 with respect to normal tissue, with a significant difference of p < 0.001 (Mann-Whitney U test) for all the enzymes. Our results suggest that altered expression of ST3Gal III, ST3Gal IV and ST6Gal I in CIN could play an important role during malignant transformation and could be related with the enhanced sialic acid expression detected in neoplasic tissues.

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.

Cervix carcinoma is associated with an up-regulation and nuclear localization of the dual-specificity protein phosphatase VHR.

BACKGROUND: The 21-kDa Vaccinia virus VH1-related (VHR) dual-specific protein phosphatase (encoded by the DUSP3 gene) plays a critical role in cell cycle progression and is itself regulated during the cell cycle. We have previously demonstrated using RNA interference that cells lacking VHR arrest in the G1 and G2 phases of the cell cycle and show signs of beginning of cell senescence. METHODS: In this report, we evaluated successfully the expression levels of VHR protein in 62 hysterectomy or conization specimens showing the various (pre) neoplastic cervical epithelial lesions and 35 additional cases of hysterectomy performed for non-cervical pathologies, from patients under 50 years of age. We used a tissue microarray and IHC technique to evaluate the expression of the VHR phosphatase. Immunofluorescence staining under confocal microscopy, Western blotting and RT-PCR methods were used to investigate the localization and expression levels of VHR. RESULTS: We report that VHR is upregulated in (pre) neoplastic lesions (squamous intraepithelial lesions; SILs) of the uterine cervix mainly in high grade SIL (H-SIL) compared to normal exocervix. In the invasive cancer, VHR is also highly expressed with nuclear localization in the majority of cells compared to normal tissue where VHR is always in the cytoplasm. We also report that this phosphatase is highly expressed in several cervix cancer cell lines such as HeLa, SiHa, CaSki, C33 and HT3 compared to primary keratinocytes. The immunofluorescence technique under confocal microscopy shows that VHR has a cytoplasmic localization in primary keratinocytes, while it localizes in both cytoplasm and nucleus of the cancer cell lines investigated. We report that the up-regulation of this phosphatase is mainly due to its post-translational stabilization in the cancer cell lines compared to primary keratinocytes rather than increases in the transcription of DUSP3 locus. CONCLUSION: These results together suggest that VHR can be considered as a new marker for cancer progression in cervix carcinoma and potential new target for anticancer therapy.