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1.  Genomic amplification of the human telomerase gene (hTERC) associated with human papillomavirus is related to the progression of uterine cervical dysplasia to invasive cancer 
Diagnostic Pathology  2012;7:147.
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.
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.
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).
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:
PMCID: PMC3488518  PMID: 23107094
Cervical cancer; Telomerase; hTERT; hTERC; HR-HPV
2.  Cervical Intraepithelial Neoplasia in the "Dr. Salvator Vuia" Clinical Obstetrics and Gynecology Hospital - Arad During the 2000-2009 Period 
Mædica  2012;7(2):138-142.
Objectives: This study intends to analyze some statistical data concerning Cervical Intraepithelial Neoplasia diagnosed in our hospital.
Material and Methods: Our study concerning the incidence of Cervical Intraepithelial Neoplasia (CIN) covers the 2000-2009 time-span, the data being collected from the Histopathology Exams (HPE) registers.
Results: During this period, CIN lesions were discovered in 1256 cases and Cervical Intraglandular Dysplasia (CIGD) in 53 cases. CIN I, CIN II and CIN III lesions represented 65.92%(828 cases), 19.67% (247 cases), and 14.41% (181 cases) of the total CIN cases, respectively. There were 26 cases combined with cervical carcinoma (2.07% of all CIN cases, 3.56% of the 731 cervical cancer cases). The mean patients' age was 44.65± 9.83 years for all cervical dysplasia cases, 44.58± 9.75 years for all CIN cases, 43.81±9.22, 46.50±10.17, and 45.46±11.05 years for CIN I, CIN II, and CIN III, respectively, and 46.45 ± 11.63 years for CIGD. The t-test revealed the following significant differences: all cases versus CIN I (p<0.05) and CIN II (p<0.01), CIGD versus CIN I (p<0.05), all cases versus CIN II (p<0.01), CIN I versus CIN II (p<0.0001) and versus CIN III (p<0.05). The mean age of the 731 cervical cancer cases diagnosed in our hospital during that same period was 52.94±12.96 years,and it was statistically significantly different from the mean ages of patients with CIN I, II and III (p <0.00000001) and with CIGD (p<0.0005).
Conclusions: Early detection of CIN is of utmost importance for preventing cervical cancer, a serious and frequent health problem in Romania.
PMCID: PMC3557421  PMID: 23399814
cervical intraepithelial neoplasia; cervical intraglandular dysplasia; cervical biopsy; cervical cancer
3.  Human papillomavirus and cervical intraepithelial neoplasia in women who subsequently had invasive cancer. 
In a retrospective case-control study biopsy specimens of cervical intraepithelial neoplasia (CIN) lesions from 47 women in whom invasive cancer subsequently developed (cases) and from 94 control subjects in whom CIN was diagnosed within 6 months of the diagnosis for the matched case subject but invasive disease did not develop were tested for human papillomavirus (HPV) DNA with tissue in-situ hybridization. There were no significant differences in the frequency of detection of HPV DNA between the two groups. In a cross-sectional survey the prevalence of HPV DNA was found to be 11% in specimens without CIN, 27% in those with CIN I, 49% in those with CIN II and 56% in those with CIN III. The positivity rates for HPV 16/33 DNA increased with the severity of CIN, but this was not observed for HPV 6/11 and 18 DNA. A comparison of the results of the case-control and cross-sectional studies suggested that the younger cohort of women had higher prevalence rates of HPV DNA than the older cohort.
PMCID: PMC1451821  PMID: 2154306
4.  High expression of astrocyte elevated gene-1 (AEG-1) is associated with progression of cervical intraepithelial neoplasia and unfavorable prognosis in cervical cancer 
Astrocyte elevated gene-1(AEG-1) plays an important role in the development and progression of certain types of human cancers. However, the expression dynamics of AEG-1 in cervical cancer and its clinical/prognostic significance are unclear.
In present study, the methods of tissue microarrays (TMA) and immunohistochemistry (IHC) were utilized to investigate AEG-1 expression in cervical intraepithelial neoplasia (CIN) and cervical cancer. Receiver operating characteristic (ROC) curve analysis, χ2 test, Kaplan-Meier plots, and multivariate Cox regression analysis were used to analyze the data.
The expression level of AEG-1 was increased from CIN I to CIN III. High expression of AEG-1 could be observed in 61.1% (55/90) of cervical cancer. Moreover, high expression of AEG-1 correlated with tumor size and lymph node metastasis (all P <0.05). More importantly, high expression of AEG-1 was closely associated with cervical cancer patient shortened survival time as evidenced by univariate and multivariate analysis (P <0.05).
Our data suggest for the first time that high expression of AEG-1 is associated significantly with progression of cervical cancer. AEG-1 overexpression, as examined by IHC, has the potential to be used as an immunomarker to predict prognosis of cervical cancer patients.
PMCID: PMC3866971  PMID: 24256614
AEG-1; Cervical cancer; Tissue microarrays; Prognosis
5.  Clinical and Pathological Heterogeneity of Cervical Intraepithelial Neoplasia Grade 3 
PLoS ONE  2012;7(1):e29051.
Cervical intraepithelial neoplasia grade 3 (CIN3), the immediate cervical cancer precursor, is a target of cervical cancer prevention. However, less than half of CIN3s will progress to cancer. Routine treatment of all CIN3s and the majority of CIN2s may lead to overtreatment of many lesions that would not progress. To improve our understanding of CIN3 natural history, we performed a detailed characterization of CIN3 heterogeneity in a large referral population in the US.
We examined 309 CIN3 cases in the SUCCEED, a large population-based study of women with abnormal cervical cancer screening results. Histology information for 12 individual loop electrosurgical excision procedure (LEEP) segments was evaluated for each woman. We performed case-case comparisons of CIN3s to analyze determinants of heterogeneity and screening test performance.
CIN3 cases varied substantially by size (1–10 LEEP segments) and by presentation with concomitant CIN2 and CIN1. All grades of CINs were equally distributed over the cervical surface. In half of the women, CIN3 lesions were found as multiple distinct lesions on the cervix. Women with large and solitary CIN3 lesions were more likely to be older, have longer sexual activity span, and have fewer multiple high risk HPV infections. Screening frequency, but not HPV16 positivity, was an important predictor of CIN3 size. Large CIN3 lesions were also characterized by high-grade clinical test results.
We demonstrate substantial heterogeneity in clinical and pathological presentation of CIN3 in a US population. Time since sexual debut and participation in screening were predictors of CIN3 size. We did not observe a preferential site of CIN3 on the cervical surface that could serve as a target for cervical biopsy. Cervical cancer screening procedures were more likely to detect larger CIN3s, suggesting that CIN3s detected by multiple independent diagnostic tests may represent cases with increased risk of invasion.
PMCID: PMC3258246  PMID: 22253702
6.  Cervical glandular atypia associated with squamous intraepithelial neoplasia: a premalignant lesion? 
Journal of Clinical Pathology  1986;39(1):22-28.
Recent studies have described premalignant changes in the endocervical epithelium, but morphological criteria for the diagnosis of cervical glandular atypia of lesser severity than adenocarcinoma in situ have not been established. Adenocarcinoma in situ is often associated with cervical intraepithelial neoplasia (CIN). The endocervical mucosa in 105 cases of CIN grade III was evaluated and compared with that of 100 controls. Sixteen cases of cervical glandular atypia and one case of adenocarcinoma in situ were identified, and it was possible to discriminate between these and a range of benign glandular lesions. Interestingly, the control series included two patients with cervical glandular atypia, one of whom on review had had a cone biopsy for CIN. The progression of cervical glandular atypia through adenocarcinoma in situ to invasive adenocarcinoma is known, but the natural history of cervical glandular atypia is as yet uncertain.
PMCID: PMC499608  PMID: 3950029
7.  Angiogenesis is associated with vascular endothelial growth factor expression in cervical intraepithelial neoplasia. 
British Journal of Cancer  1997;76(11):1410-1415.
Squamous cell carcinoma of the cervix (SCC) is preceded by a premalignant condition known as cervical intraepithelial neoplasia (CIN). The majority of cases of CIN regress spontaneously; however, methods are needed to identify those lesions likely to progress. Increased blood vessel density, signifying angiogenesis, is an independent prognostic indicator in a number of cancers, although little is known about its significance in premalignant lesions. The aim of the present study was to determine the relationship between vessel density, expression of the potent angiogenic factor vascular endothelial growth factor (VEGF) and CIN grade. Using immunohistochemistry, mean vessel density (MVD) and VEGF expression were assessed in samples from 54 patients who had undergone cone biopsy for CIN or hysterectomy for SCC and from 16 patients with no cervical pathology. There were significant increases in MVD and VEGF expression from normal cervix through CIN I to CIN III to invasive SCC, but no difference in mean vessel diameter between groups. There was a strong correlation between mean vessel density and VEGF expression, and both were associated with histological grade of CIN. The original MVDs for a small group of patients later presenting with recurrent disease were found to be equal to or greater than the mean for their histological grade. We conclude that the onset of angiogenesis is an early event in premalignant changes of the cervix due, in part, to enhanced expression of VEGF by the abnormal epithelium.
PMCID: PMC2228179  PMID: 9400935
8.  Local Lymphocytes and Nitric Oxide Synthase in the Uterine Cervical Stroma of Patients with Grade III Cervical Intraepithelial Neoplasia 
Clinics  2010;65(6):575-581.
Precancerous and cancerous cells can trigger an immune response that may limit tumor development and can be used as a prognostic marker. The aims of the present study were to quantify the presence of B and T lymphocytes, macrophages and cells expressing inducible nitric oxide synthase (iNOS) in the cervical stroma of women with grade III cervical intraepithelial neoplasia (CIN III) or in the intratumoral and peritumoral tissue of women with stage I invasive carcinoma.
Cervical tissue specimens were obtained from 60 women (20 each from control tissues, CIN III and invasive carcinomas). The average ages in the control, CIN III and invasive groups were 43.9 (± 4.3), 35.5 (± 9.5), and 50 (± 11.2) years, respectively. The specimens were immunohistochemically labeled with antibodies to identify T lymphocytes (CD3), cytotoxic lymphocytes (CD8), B lymphocytes (CD20), macrophages (CD68) and iNOS. We evaluated the markers in the stroma above the squamocolumnar junction (control), at the intraepithelial lesion (CIN cases), and in the nfiltrating tumor. Two independent observers performed the immunohistochemical analysis.
T lymphocytes, B lymphocytes, macrophages and iNOS were present more frequently (P<0.05) in the stroma of peritumoral invasive tumors compared to the controls and intratumoral invasive cancer samples. CD3+ and CD20+ lymphocytes were present more frequently in CIN III patients compared to samples from patients with intratumoral invasive cancer (P<0.05).
High numbers of T and B lymphocytes, macrophages and iNOS-expressing cells in the peritumoral stroma of the invasive tumors were observed. Cell migration appeared to be proportional to the progression of the lesion.
PMCID: PMC2898547  PMID: 20613932
Cervical cancer; Nitric oxide; CIN III; Lymphocyte; Macrophages
9.  CK17 and p16 expression patterns distinguish (atypical) immature squamous metaplasia from high-grade cervical intraepithelial neoplasia (CIN III) 
Histopathology  2007;50(5):629-635.
Regauer S & Reich O (2007) Histopathology50, 629–635 CK17 and p16 expression patterns distinguish (atypical) immature squamous metaplasia from high-grade cervical intraepithelial neoplasia (CIN III)
Atypical immature metaplasia (AIM) refers to a full-thickness intraepithelial basaloid lesion in the uterine cervix that features both metaplasia and atypia and is therefore difficult to distinguish from high-grade cervical intraepithelial neoplasia (CIN III). p16 is a marker for human papillomavirus (HPV)-induced dysplasia. Cytokeratin (CK) 17 is a marker for cervical reserve (stem) cells, which give rise to metaplasia. The aim was to determine whether AIM can be reclassified into metaplasia and CIN III based on p16 and CK17 immunohistochemistry.
Material and results
Seventy-five cervical biopsy specimens, curettings and cone excisions containing varying proportions of dysplasia and metaplasia and 20 cases regarded as AIM were analysed immunohistochemically with antibodies to CK17, p16 and p63. In immature metaplasia all proliferating cells were immunoreactive with antibodies to CK17 and p63, while p16 was negative. All dysplastic cells of CIN III demonstrated uniform immunoreactivity for p16 and p63, but were CK17–. Based on the reciprocal immunoreactivity of p16 and CK17, 17/20 cases of AIM were reclassified as metaplasia (n = 10) and CIN III (n = 7). Three cases of AIM stained for both CK17 and p16 and were classified as CIN III.
‘AIM’ is a helpful histological descriptor but it should not be used as a final diagnosis. Immunohistochemistry for p16 and CK17 allows distinction between metaplasia and high-grade CIN.
PMCID: PMC1890920  PMID: 17394499
HPV; reactive atypia; squamous intraepithelial lesions
10.  Dynamic behavioural interpretation of cervical intraepithelial neoplasia with molecular biomarkers 
Journal of Clinical Pathology  2006;59(10):1017-1028.
The microscopic phenotype of cervical intraepithelial neoplasia (CIN) reflects a fine balance between factors that promote or reduce CIN development. A shortcoming of the current grading system is its reliance on static morphology and microscopic haematoxylin–eosin features of the epithelium alone. In reality, CIN is a dynamic process, and the epithelium may exhibit differing results over time. Functional biomarkers p16, Ki‐67, p53, retinoblastoma protein cytokeratin (CK)14 and CK13, help in the assessment of an individual CIN's lesion's potential for progression and regression. The aggregate information provided by these biomarkers exceeds the value of the classic grading system. Consequently, many more CINs that will either regress or progress can be accurately identified. These findings agree with known molecular interactions between HPV and the host. For accurate interpretation of a CIN, it is essential that these biomarkers be determined quantitatively and separately in the superficial, middle and deep layers of the epithelium. Such geography‐specific epithelial evaluations of quantitative biomarkers emphasise the dynamic nature of a particular CIN lesion, thereby changing the art of static morphology grading into dynamic interpretation of the diseased tissue, with a strong prognostic effect.
PMCID: PMC1861745  PMID: 16679355
11.  Potential diagnostic value of P16 expression in premalignant and malignant cervical lesions 
The goal of this study was to evaluate the results of the expression of p16INK4a in normal uterine cervical epithelium, low-grade cervical intraepithelial neoplasia (CIN), high-grade CIN, squamous cell carcinoma (SCC), and adenocarcinoma of the cervix, in order to help draw a distinction between low risk and high risk patients with cervical lesions.
Materials ans Methods:
P16INK4a expression was evaluated by immunohistochemistry in 78 paraffin-embedded tissue samples including 39 normal cervical tissues, 11 low-grade CINs, 11 high-grade CINs, 22 cervical SCCs and 8 cervical adenocarcinomas. Two parameters in immunohistochemical p16 expression were evaluated: percentage of p16-positive cells, and reaction intensity.
The p16INK4a expression rate was 81.8% in low-grade CINs, 91% in high-grade CINs, 90% in SCCs and 75% in cervical adenocarcinomas. 10% of normal cervical samples expressed p16. Moreover, there was a significant relationship between the histological diagnoses and percentage of positive cells and reaction intensity of p16 (p < 0.005). The intensity of the reaction was the best parameter to evaluate the positivity of p16.
Over-expression of the p16INK4a was typical for dysplastic and neoplastic epithelia of the uterine cervix. However, p16INK4a-negative CINs and carcinomas did exist. Although negative p16INK4a expression does not definitely exclude the patient with cervical lesion from the high-risk group, immunohistochemical study for p16INK4a may be used as a supplementary test for an early diagnosis of cervical cancers.
PMCID: PMC3634266  PMID: 23626605
P16INK4a; Cervical intraepithelial neoplasia; Immunohistochemistry; Human papilloma virus
12.  Association between HLA DQB1 * 03 and cervical intra-epithelial neoplasia. 
Molecular Medicine  1995;1(2):161-171.
BACKGROUND: Cervical intraepithelial neoplasia (CIN) and cervical cancer have been shown to be strongly associated with infection by human papillomavirus (HPV). However, other factors may be contributory in the progression from normal epithelium to CIN and cervical cancer, since not all women with HPV infection develop disease. Recently, it was demonstrated that there is a high risk for cervical cancer and CIN in women with HLA DQB1 * 03 (RR = 7.1, p < 0.0009) (1). Subsequent reports have been conflicting, due to sample size, genetic heterogeneity and differences in the techniques employed for the detection of HLA DQB1 * 03. MATERIALS AND METHODS: DNA from cervical smears of 178 women with CIN and 420 controls with normal cervical cytology was analyzed by polymerase chain reaction (PCR) with type-specific primers for HPV 16, 18, 31, and 33. The DNA from test and control samples were also analyzed by a novel PCR technique, which mutates the first base of codon 40 (DQ alleles) from T to G to create an artificial restriction site for an enzyme Mlu I that distinguish DQB1 * 03 from other alleles and are confirmed by digestion of amplified DNA with Mlu I. Further analysis of individual DQB1 * 03 alleles was performed using PCR and allele-specific primers. RESULTS: One hundred forty-four (34%) out of 420 controls (all HPV 16, 18, 31, or 33 negative and normal cytology), 37/66 (56%) of CIN I and 72/112 (64%) of CIN III were positive for DQB1 * 03 (trend test, p < 0.001, chi 2 = 37.3). A significant association was observed between DQB1 * 03 and CIN (odds ratio 3.03; 95% CI 2.11-3.45). Of women with CIN, 131/178 (73.5%) had HPV (types 16, 18, 31, or 33) infection. There was a significant association between DQB1 * 03 and presence of HPV (odds ratio 3.43; 95% CI 2.25-5.10). Homozygosity for DQB1 * 03 was more strongly associated with CIN than heterozygosity (odds ratios 4.0 and 2.63, respectively); and for the presence of HPV (odds ratio 4.47; 95% CI 2.58-7.77). HLA DQB1 * 0301 was the most strongly associated allele with CIN and HPV (odds ratios 2.53 and 2.63, respectively). CONCLUSIONS: HLA DQB1 * 03 is associated significantly with CIN and may be permissive for HPV infection. Further analysis of class II HLA typing in CIN is necessary to evaluate this association.
PMCID: PMC2229948  PMID: 8529095
13.  The detection of hTERC amplification using fluorescence in situ hybridization in the diagnosis and prognosis of cervical intraepithelial neoplasia: a case control study 
Currently the routine non-invasive screening methods for cervical intraepithelial neoplasia (CIN) and cervical cancer are Thinprep cytology test (TCT) and human papillomavirus testing. However, both methods are limited by the high false positive and false negative rates and lack of association with patients’ prognosis, especially for the early detection of pro-malignant CIN. The aim of the study was to investigate the role of genomic amplification of human telomerase gene (hTERC) in the diagnosis and prognosis of CIN.
The study group consisted of specimens of exfoliated cervical cells from 151 patients, including 27 with CIN I, 54 with CIN II/III, 17 with carcinoma in situ, and 28 with invasive squamous carcinoma, as well as 25 patients who were at 2-year follow-up after either Loop Electrosurgical Excision treatment (n = 11) or radical surgery (n = 14). hTERC amplification was detected by dual-color interphase fluorescence in situ hybridization (FISH), and the results were compared with TCT and histologic examination. The final diagnosis was determined by the pathological examination. The control group consisted of specimens of exfoliated cervical cells from 40 normal women.
The percentage of cervical exfoliated cells with positive hTERC amplification and incidence rates of hTERC amplification were 9.2% ± 4.6% and 44.4% (12/27) respectively in patients with CIN I; 16.0% ± 14.4% and 85.1% (46/54) in patients with CIN II/III; 19.7% ± 13.3% and 88.3% (15 /17) in patients with carcinoma in situ; 47.0% ± 25.2% and 100% (28/28)in patients with invasive squamous carcinoma. There was statistically significant difference between the control and study group (P <0.01), and between the patients with various diseases within the study group (P <0.05).
The detection of genomic amplification of hTERC using FISH is a non-invasive and effective approach for CIN.
PMCID: PMC3485165  PMID: 22909227
Fluorescence in situ hybridization; Cervical intraepithelial neoplasia; Cervical carcinoma; Telomerase
14.  Using LongSAGE to Detect Biomarkers of Cervical Cancer Potentially Amenable to Optical Contrast Agent Labelling 
Biomarker Insights  2007;2:447-461.
Sixteen longSAGE libraries from four different clinical stages of cervical intraepithelial neoplasia have enabled us to identify novel cell-surface biomarkers indicative of CIN stage. By comparing gene expression profiles of cervical tissue at early and advanced stages of CIN, several genes are identified to be novel genetic markers. We present fifty-six cell-surface gene products differentially expressed during progression of CIN. These cell surface proteins are being examined to establish their capacity for optical contrast agent binding. Contrast agent visualization will allow real-time assessment of the physiological state of the disease process bringing vast benefit to cancer care. The data discussed in this publication have been submitted to NCBIs Gene Expression Omnibus (GEO, and are accessible through GEO Series accession number GSE6252.
PMCID: PMC2717845  PMID: 19662225
longSAGE; cervical cancer; biomarker; optical imaging
15.  EGFR protein expression and gene amplification in squamous intraepithelial lesions and squamous cell carcinomas of the cervix 
The purpose of this study was to evaluate the protein expression and gene amplification of epithelial growth factor receptor (EGFR) in intraepithelial neoplasias and squamous cell carcinoma of the cervix and to determine the value of EGFR in carcinogenesis, progression, and prognosis of cervical cancer. EGFR protein expression and gene amplification involved gene copy number in 75 cases of cervical various lesions were evaluated using immunohistochemistry and by fluorescence in situ hybridization (FISH) techniques. Expression of EGFR was observed in 76.00% of the high-grade CIN and 79.17% of the invasive carcinomas. In contrast, there were low levels of EGFR expression in chronic cervicitis (1/10) and low-grade CIN (7/16). There were statistically significant differences among them (P<0.05). Gene amplification was detected in 20.51% high-grade CIN and invasive carcinoma, but there only 4.35% EGFR gene amplification was observed in chronic cervicitis and low grade CIN. Among the 42 patients with negative or low levels of EGFR expression, 26 patients (61.90%) were found to have diploidy and 11 patients (26.20%) to have balanced triploidy. However, among the 20 patients with an intermediate and high levels of EGFR protein expression, 13 (65.00%) were found to have balanced polyploidy or gene amplification. All cases of EGFR gene amplification involved intermediate and high levels of protein expression. EGFR may be involved in the carcinogenesis of the cervix and may be an early event during the carcinogenesis. Overexpression of EGFR protein may result from gene amplification and increases in gene copy number.
PMCID: PMC3925921  PMID: 24551297
Cervix neoplasms; EGFR; protein expression; gene amplification; gene copy number; FISH
16.  Human Papillomavirus (HPV) DNA Copy Number Is Dependent on Grade of Cervical Disease and HPV Type 
Journal of Clinical Microbiology  1999;37(4):1030-1034.
The association between human papillomavirus (HPV) DNA copy number and cervical disease was investigated. Viral DNA copy number for the most common high-risk HPV types in cervical cancer (types 16, 18, 31, and 45) was determined in cervical cytobrush specimens from 149 women with high-grade cervical intraepithelial neoplasias (CIN II-CIN III), 176 with low-grade CIN (CIN I), and 270 with normal cytology. Quantitative, PCR-based fluorescent assays for each of the HPV genotypes and for the β-globin gene were used. The amount of cellular DNA increased significantly with increasing disease; thus, HPV was expressed as copies per microgram of cellular DNA. The assay had a dynamic range of >107, allowing documentation for the first time of the wide range of HPV copy numbers seen in clinical specimens. Median HPV DNA copy number varied by more than 104 among the viral types. HPV16 was present in the highest copy number; over 55% of HPV16-positive samples contained more than 108 copies/μg. Median copy number for HPV16 showed dramatic increases with increasing epithelial abnormality, an effect not seen with the other HPV types. HPV16 increased from a median of 2.2 × 107 in patients with normal cytology, to 4.1 × 107 in CIN I patients, to 1.3 × 109 copies/μg in CIN II-III patients. Even when stratified by cervical disease and viral type, the range of viral DNA copies per microgram of cellular DNA was quite large, precluding setting a clinically significant cutoff value for “high” copy numbers predictive of disease. This study suggests that the clinical usefulness of HPV quantitation requires reassessment and is assay dependent.
PMCID: PMC88645  PMID: 10074522
17.  HSP70 and ER expression in cervical intraepithelial neoplasia and cervical cancer. 
Journal of Korean Medical Science  1998;13(4):383-388.
Heat shock protein (HSP) is thought to play important roles in the cell cycle and various process of carcinogenesis. This study was performed to evaluate the expression of heat shock protein (HSP70) and estrogen receptor (ER) and Ki-67 and to assess relationship between them in cervical squamous cell neoplasia. The materials were 50 cervical squamous cell lesions, consisted of 30 cervical intraepithelial neoplasia (CIN) (6 moderate dysplasia, 11 severe dysplasia, 13 carcinoma in situ), and 20 invasive squamous cell carcinoma (ISCC) cases. These specimens were immunohistochemically stained for HSP70, ER and Ki-67. The score of HSP70 was significantly higher in ISCC than CIN. Expression rate of the ER was not significantly higher in CIN than in ISCC. Ki-67 labelling index was significantly higher in the ISCC and high HSP70 positive staining group. These results suggested that HSP70 may play an important role in tumor cell proliferation and is related with ISCC than CIN, but ER may be not related with tumor cell proliferation and differentiation. HSP70 may be a useful prognostic factor in cervical dysplasia and cancer.
PMCID: PMC3054429  PMID: 9741542
18.  Indole-3-Carbinol Prevents PTEN Loss in Cervical Cancer In Vivo 
Molecular Medicine  2005;11(1-12):59-63.
Indole-3-carbinol (I3C) is a phytochemical (derived from broccoli, cabbage, and other cruciferous vegetables) with proven anticancer efficacy including the reduction of cervical intraepithelial neoplasia (CIN) and its progression to cervical cancer. In a breast cancer cell line, I3C inhibited cell adhesion, spreading, and invasion associated with an upregulation of the tumor suppressor gene PTEN, suggesting that PTEN is important in inhibition of late stages in the development of cancer. The goal of this study was to determine the expression of PTEN during the development of cervical cancer and whether I3C affected expression of PTEN in vivo. We show diminished PTEN expression during the progression from low-grade to high-grade cervical dysplasia in humans and in a mouse model for cervical cancer, the K14HPV16 transgenic mice promoted with estrogen. The implication is that loss of PTEN function is required for this transition. Additionally, dietary I3C increased PTEN expression in the cervical epithelium of the transgenic mouse, an observation that suggests PTEN upregulation by I3C is one mechanism by which I3C inhibits development of cervical cancer.
PMCID: PMC1449523  PMID: 16557333
19.  Minichromosome maintenance 7 protein is a reliable biological marker for human cervical progressive disease 
This study focused on comparing the expression levels of p16, Ki-67, and minichromosome maintenance 7 (MCM7) protein in normal and affected cervical epithelium to ascertain the biological significance of these markers in detecting progressive cervical disease.
A quantitative and based on-scanning-microscopy analysis of the three markers expression was performed in normal and cervical intraepithelial neoplasia (CIN) I, II, and III tissues. p16 area as well as p16, Ki-67, and MCM7 positive cells or nuclei were evaluated according to their distribution and extent through the cervical epithelium.
A clear p16 over-expression was observed in all the dysplastic epithelium tissue samples. The quantitative analysis of p16 area as well as the number of p16 positive cells was able to better discriminate the CIN lesions grades than the usual semi-quantitative analysis. The average Ki-67 labeling indexes for the normal epithelium, CIN I, CIN II, and CIN III groups were 19.8%, 27.3%, 32.8%, and 37.1%, respectively, whereas the mean MCM7 labeling indexes for the correspondent grades were 27.0%, 30.4%, 50.5%, and 67.2%. The Ki-67 and MCM7 labeling indexes were closely correlated with the CIN histological grade, with higher labeling indexe values obtained from the more severe lesions (p<0.05), being the MCM7 labeling indexes the highest values in all the CIN categories (p<0.05).
We observed a good correlation among the p16, Ki-67, and MCM7 data. In addition, MCM7 demonstrated to be a more efficient and sensitive marker to assess disease progression in the uterine cervix.
PMCID: PMC3280060  PMID: 22355461
Cervical intraepithelial neoplasia; Genes p16; Immuno-fluorescence analysis; Ki-67 antigen; MCM7 protein
20.  Subtype Distribution of Human Papillomavirus in HIV-Infected Women With Cervical Intraepithelial Neoplasia Stages 2 and 3 in Botswana 
Human papillomavirus (HPV) vaccines containing types 16 and 18 are likely to be effective in preventing cervical cancer associated with these HPV types. No information currently exists in Botswana concerning the HPV types causing precancerous or cancerous lesions. Our goal was to determine the prevalence of HPV types associated with precancerous cervical intraepithelial neoplasia (CIN) stages 2 and 3 in HIV-infected women in Gaborone, Botswana. HIV-infected women referred to our clinic with high-grade intraepithelial lesion on the Pap smear were enrolled in the study. HPV typing was only performed if the histopathology results showed CIN stage 2 or 3 disease using linear array genotyping (CE-IVD, Roche Diagnostics).One hundred HIV-infected women were identified with CIN stages 2 or 3 between August 11, 2009 and September 29, 2010. Eighty-two of 100 women enrolled had coinfection by multiple HPV subtypes (range, 2 to 12). Of the remaining 18 women, 14 were infected with a single high-risk subtype and 4 had no HPV detected. Overall, 92 (92%) women were infected with at least 1 high-risk HPV subtype, and 56 were coinfected with more than 1 high-risk HPV type (range, 2 to 5). Fifty-one (51%) women had HPV subtypes 16, 18, or both. HPV 16 and 18 are the most common types in HIV-infected women with CIN 2 or 3 in Gaborone, Botswana, suggesting that the implementation of HPV vaccination programs could have a significant impact on the reduction of cervical cancer incidence. However, given the relative lack of knowledge on the natural history of cervical cancer in HIV-infected women and the significant prevalence of infection and coinfection with other high-risk HPV types in our sample, the true impact and cost-effectiveness of such vaccination programs need to be evaluated.
PMCID: PMC3789124  PMID: 21979597
HIV; HPV; CIN; Cervical cancer
21.  Detection of precancerous cervical lesions is differential by human papillomavirus (HPV) type 
Cancer research  2009;69(8):3262-3266.
Epidemiologic studies have reported the under-representation of cervical precancerous lesions caused by human papillomavirus (HPV) types 18 and 45 (HPV18/45) compared to the proportion of cervical cancers attributed to these HPV types. We investigated the timing of diagnosis of histologic cervical intraepithelial neoplasia grade 3 or worse (CIN3+) using data from the ASCUS-LSIL Triage Study (ALTS). Of the 2,725 women who underwent enrollment colposcopy 412 of 472 (87.3%) diagnosed with histologic CIN3+ over the 2-year duration of ALTS could be assigned to a HPV type or group of types and were included in this analysis. Eighty-four percent of HPV16-positive CIN3+ were diagnosed at enrollment, compared with 57% of HPV18/45-positive CIN3+, and 58% of CIN3 positive for other carcinogenic HPV types at enrollment. In contrast, only 8% of HPV16-positive CIN3+ were diagnosed at exit, while 31% were HPV18/45-positive and 22% were positive for other carcinogenic types at study exit (p<0.001).. These results indicate the under-representation of HPV18/45 in pre-cancers while HPV16-associated CIN3+ is diagnosed much earlier. Whether the under-representation of 18/45 may be due to occult pathology needs further investigation.
PMCID: PMC3155840  PMID: 19351830
HPV genotypes; precancerous cervical lesions; timing of diagnosis
22.  An amino-bisphosphonate targets MMP-9–expressing macrophages and angiogenesis to impair cervical carcinogenesis 
Journal of Clinical Investigation  2004;114(5):623-633.
A mouse model involving the human papillomavirus type-16 oncogenes develops cervical cancers by lesional stages analogous to those in humans. In this study the angiogenic phenotype was characterized, revealing intense angiogenesis in high-grade cervical intraepithelial neoplasias (CIN-3) and carcinomas. MMP-9, a proangiogenic protease implicated in mobilization of VEGF, appeared in the stroma concomitant with the angiogenic switch, expressed by infiltrating macrophages, similar to what has been observed in humans. Preclinical trials sought to target MMP-9 and angiogenesis with a prototypical MMP inhibitor and with a bisphosphonate, zoledronic acid (ZA), revealing both to be antiangiogenic, producing effects comparable to a Mmp9 gene KO in impairing angiogenic switching, progression of premalignant lesions, and tumor growth. ZA therapy increased neoplastic epithelial and endothelial cell apoptosis without affecting hyperproliferation, indicating that ZA was not antimitotic. The analyses implicated cellular and molecular targets of ZA’s actions: ZA suppressed MMP-9 expression by infiltrating macrophages and inhibited metalloprotease activity, reducing association of VEGF with its receptor on angiogenic endothelial cells. Given its track record in clinical use with limited toxicity, ZA holds promise as an “unconventional” MMP-9 inhibitor for antiangiogenic therapy of cervical cancer and potentially for additional cancers and other diseases where MMP-9 expression by infiltrating macrophages is evident.
PMCID: PMC514591  PMID: 15343380
23.  Decreased D2-40 and increased p16INK4A immunoreactivities correlate with higher grade of cervical intraepithelial neoplasia 
Diagnostic Pathology  2011;6:59.
D2-40 has been shown a selective marker for lymphatic endothelium, but also shown in the benign cervical basal cells. However, the application of D2-40 immunoreactivity in the cervical basal cells for identifying the grade of cervical intraepithelial neoplasia (CIN) has not been evaluated.
In this study, the immunoreactive patterns of D2-40, compared with p16INK4A, which is currently considered as the useful marker for cervical cancers and their precancerous diseases, were examined in total 125 cervical specimens including 32 of CIN1, 37 of CIN2, 35 of CIN3, and 21 of normal cervical tissue. D2-40 and p16INK4A immunoreactivities were scored semiquantitatively according to the intensity and/or extent of the staining.
Diffuse D2-40 expression with moderate-to-strong intensity was seen in all the normal cervical epithelia (21/21, 100%) and similar pattern of D2-40 immunoreactivity with weak-to-strong intensity was observed in CIN1 (31/32, 97.2%). However, negative and/or focal D2-40 expression was found in CIN2 (negative: 20/37, 54.1%; focal: 16/37, 43.2%) and CIN3 (negative: 22/35, 62.8%; focal: 12/35, 34.3%). On the other hand, diffuse immunostaining for p16INK4A was shown in 37.5% of CIN1, 64.9% of CIN2, and 80.0% of CIN3. However, the immunoreactive pattern of D2-40 was not associated with the p16INK4A immunoreactivity.
Immunohistochemical analysis of D2-40 combined with p16INK4A may have a significant implication in clinical practice for better identifying the grade of cervical intraepithelial neoplasia, especially for distinguishing CIN1 from CIN2/3.
PMCID: PMC3157410  PMID: 21729305
D2-40; cervical intraepithelial neoplasia; immunohistochemistry; p16INK4A
24.  The prognosis in cervical epithelial changes of uncertain significance is similar to that of cervical intraepithelial neoplasia grade 1 
Journal of Clinical Pathology  2001;54(6):474-475.
Background/Aims—The term epithelial changes of uncertain significance (ECUS) describes a minor degree of nuclear pleomorphism limited to the basal layers of cervical epithelium in the absence of severe inflammation with associated normal mitoses, koilocytosis, or koilocytosis associated features. This study aimed to investigate the long term prognosis of this lesion.
Methods—Slides from 128 women with low grade cervical abnormalities, accessioned consecutively, were reviewed.
Results—In 43 women the initial diagnosis of ECUS was confirmed and in 30 women the initial diagnosis of cervical intraepithelial neoplasia grade 1 (CIN1) was confirmed. Comparison of follow up data from these 73 women revealed a similar prognosis in the two groups in terms of regression to normal, persistence of low grade disease, or progression to high grade CIN.
Conclusions—Low grade cervical disease (ECUS and CIN1) should be managed according to similar treatment protocols.
Key Words: cervical intraepithelial neoplasia • diagnosis • follow up study
PMCID: PMC1731445  PMID: 11376023
25.  Heterogeneity of high grade cervical intraepithelial neoplasia related to HPV16: Implications for natural history and management 
Factors associated with progression from cervical intraepithelial neoplasias (CIN) grade 2 and 3 to invasive cancer are not well understood; most CIN2 and CIN3 do not progress to cancer. Among carcinogenic HPV types, infections with HPV16 have the highest risk of progressing to cancer. We evaluated the heterogeneity of risk factors, lesion size, colposcopic impression, and colposcopic biopsy results in relation to HPV16 status among 627 women with CIN2 or CIN3 in women referred to colposcopy at the University of Oklahoma. Loop excision specimens were evaluated in 12 radial segments to estimate lesion size. The mean age at CIN3 was 27.7 years for HPV16-positive women (n=225) and 33.6 years for HPV16-negative women (n=104), respectively. The average lesion size did not differ by HPV16 status (p=0.83). Among HPV16-positive women with CIN3, lesions were significantly larger in women 30 years and older (p=0.03). Colposcopic impression was worse in women with HPV16 infections (p=0.009), but the detection of CIN3 at the preceding biopsy was not improved in HPV16-positive women. CIN3 is detected at the same lesion size, but at much younger age in women with HPV16 infections, suggesting faster growth. CIN2 lesion size in women without HPV16 peaks below 30 years and then decreases, suggesting frequent regression, while HPV16-related CIN2 is more likely to persist. Lesion size seems to be the most important determinant of colposcopy and biopsy performance. Genotyping for HPV16 in cervical cancer screening can improve risk stratification, but may pose challenges to finding small lesions in colposcopy.
PMCID: PMC3409928  PMID: 22488167
HPV16; CIN3; biopsy; colposcopy; screening

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