Grade Cervical Lesions Research Articles

Diagnosing Cervical Cancer and High-Grade Precursors by HPV16 Transcription Patterns

Infections with high-risk human papillomaviruses (HPV), mainly HPV type 16, can cause malignant transformation of the human cervical epithelium and the development of cervical cancer (CxCa). A rapid and precise diagnosis of the precancerous lesions by conventional cytology or HPV DNA tests remains difficult and often leads to overtreatment. We quantitatively analyzed the HPV16 transcriptome of 80 HPV16 DNA-positive cervical scrapes classified as mild cytologic grade, including no intraepithelial lesion or malignancy (NIL/M; normal, n=25) and low-grade squamous intraepithelial lesion (LSIL; n=24), and severe cytologic grade, including high-grade squamous intraepithelial lesion (HSIL; n=24) and CxCa (n=7), with novel nucleic acid sequence-based amplification-Luminex assays. In severe lesions, HPV16 E6*II and E1C encoding transcripts were strongly upregulated, whereas spliced E1[SYMBOL: SEE TEXT]E4 and L1 encoding transcripts were markedly downregulated. Using a combination of the four marker transcripts, 100% of CxCa and 67% of HSIL cases were correctly identified as severe, and 74% of LSIL and 92% of NIL/M samples as mild cytologic grade. Compared with a commercially available HPV E6/E7 mRNA assay, the specificity of the marker combination for discriminating severe and mild cytologic lesions increased from 23% to 83%. In conclusion, we identified a novel HPV16 RNA pattern for grading of cervical lesions with a potentially high diagnostic value for the primary screening of CxCa precursors and the triage of cervical lesions.

Up-Regulation of Plasminogen Activator Inhibitor-2 Is Associated With High-Risk HPV and Grade of Cervical Lesion at Baseline but Does Not Predict Outcomes of High-Risk HPV Infections or Incident CIN

Protease inhibitor serpin-B2 (plasminogen activator inhibitor-2 [PAI-2]) protects pRb from degradation in human papillomavirus (HPV)-18+ HeLa cells. Our objective was to assess whether the pRb-mediated HPV-suppressive effect of PAI-2 in cancer cell lines has implications in the outcome of HPV infections. Cervical biopsy specimens from 225 women were analyzed for PAI-2 expression to assess its value as a predictor of cervical intraepithelial neoplasia (CIN) grade, high-risk (HR) HPV at baseline, outcomes of HR-HPV infections, and the development of incident CIN. PAI-2 expression increased in parallel with lesion grade. Nuclear PAI-2 expression was significantly related to HR-HPV detection and had a linear relationship with HR-HPV load. PAI-2 expression was of no value in predicting the outcomes of HR-HPV infections. The same was true for PAI-2 as a predictor of surrogate end points (incident CIN 1+, CIN 2+) of progressive disease. PAI-2 expression is up-regulated on transition from CIN 2 to CIN 3. The HR-HPV suppressive effects of PAI-2 were not related to more favorable outcomes of HR-HPV infections or lower risk of disease progression to CIN.

Genetic diversity of human papillomavirus type 16 E6, E7, and L1 genes in Italian women with different grades of cervical lesions

High-risk human papillomaviruses (HPVs) are risk factors for the development of cervical cancer. HPV 16 is the most common type, being present in about 60% of cervical cancers worldwide. Previous studies have reported upon the association between HPV 16 E6 variants and increased risk of cervical intraepithelial neoplasia and invasive cervical cancer. In this study, the presence of HPV 16 polymorphisms in the E6, E7, and L1 genes was investigated in relation to the presence of high-grade lesions. Sequencing of the E6 gene revealed the presence of nucleotide mutations resulting in 15 amino acid changes. Of these, the G134D and C136R fall within the CXXC zinger finger domain important for p53 binding. In the E7 gene, four nucleotide variations were identified with two leading to the amino acid substitutions L15V and S31R. The L1 gene showed 13 nucleotide changes leading to 11 amino acid substitutions. Among these, the R364C and N367D are located at the base of the HI-loop of the L1 protein, considered to be the immunodominant epitope of HPV 16. No significant relationship between HPV 16 variants and high-grade lesions was found. Phylogenetic analysis showed that all the HPV 16 variants identified belonged to the European lineage, except one which was of the Asian-American lineage. The European-350G variant was detected most frequently (22 of 34, 64.7%). The study provides some new data on the genetic diversity of HPV 16 which may help to understand the oncogenic potential of the virus and to improve management of patients.

Staining characteristics of p16INK4a: Is there a correlation with lesion grade or high‐risk human papilloma virus positivity?

The aims of this study were to evaluate the efficiency of p16INK4a in showing cervical lesions and to determine any relationship between lesion grade and high-risk human papilloma virus (HR-HPV) infection and p16INK4a staining characteristics. Immunohistochemical analysis of p16INK4a was performed on 13 low-grade squamous intraepithelial lesions (LSIL), 22 high-grade squamous intraepithelial lesions (HSIL), 23 squamous cell carcinoma (SCC) and 25 normal tissue samples. The distribution, staining pattern and intensity of p16INK4a expression were assessed and correlated with HR-HPV positivity determined by real-time polymerase chain reaction. All HSIL and SCC cases, but only 46.2% of LSIL cases, were positive for p16INK4a. Although positive staining of p16INK4a in showing HR-HPV-positive lesions was statistically significant (P=0.000), we could not find a significant correlation for distribution (P=0.319), staining pattern (P=0.057) or intensity (P=0.057) of p16INK4a in showing HR-HPV in cervical epithelium. These parameters were correlated only with the increasing grade of the lesion (P=0.000). p16INK4a is a highly sensitive marker of cervical intraepithelial neoplasia and cervical cancer. There is a good correlation between p16INK4a expression and cervical lesion grade and HR-HPV positivity. The distribution, staining pattern and intensity of this marker are significantly correlated with the increasing grade of cervical lesions, suggesting that diffuse distribution, full thickness staining pattern and strong intensity of this marker are highly supportive of HSIL and cervical cancer, while focal distribution, scattered/basal staining pattern and weak intensity suggest LSIL. However, these parameters are not correlated with HR-HPV status in cervical lesions.

P16, cyclin D1, and human telomerase reverse transcriptase expression in cervical premalignant and malignant lesions

ABSTRACT Background and aim: Although morphological criteria are important for diagnosis of cervical intraepithelial neoplasia, it is not possible to differentiate those that will regress from those that will progress into invasive cancer. We evaluate the usefulness of p16, cyclin D1, and human telomerase reverse transcriptase (hTERT) as diagnostic and prognostic biomarkers in cervical lesions. Methods: We studied 12 cases of benign lesion, 15 cases of low‐grade squamous intraepithelial lesion, 46 cases of high‐grade squamous intraepithelial lesion, and 13 cases of squamous cell carcinoma by immunohistochemistry. Results: The p16 expression was significantly correlated with high risk‐human papillomavirus (HR‐HPV) status, lesion grade, and decreased cyclin D1 expression. The decreased cyclin D1 expression was significantly correlated with HR‐HPV status, lesion grade, and p16 expression. The positive predictive values for high grade lesion of p16 expression, HR‐HPV status, and decreased cyclin D1 expression were 93.8%, 81.3%, and 51.2%, respectively. The distribution pattern of hTERT positive cells was correlated with morphologically dysplastic cells. Conclusions: A good correlation was observed between cervical lesion grade and p16 expression, decreased cyclin D1 expression, and HR‐HPV status. p16 expression could be used as a diagnostic and prognostic biomarker for risk of developing cervical cancer in women infected with HPV.

The physical state of HPV16 infection and its clinical significance in cancer precursor lesion and cervical carcinoma

Integration of high-risk human papillomavirus (HR-HPVs) into the host DNA has been proposed as a risk for cervical carcinogenesis. HPV-16 is the predominant high-risk type and its integration ration varied largely in different cervical cancer (CC) samples. The aim of this study was to evaluate the correlation between physical state of HPV16 infection and extent of cervical lesion, as well as the clinical significance of virus existing state. A total of 252 cases of paraffin-embedded blocks derived from cancer precursor lesion and cervical carcinoma samples were detected by HC-II for HR-HPV infection. HPV16 infection was confirmed by PCR and immunohistochemistry for HPV16 E7 simultaneously. The physical state of HPV16 infection were assessed by PCR for 3 overlapping fragments in E2 gene and multiple PCR for E2 gene and E7 gene. The infection ratio of HR-HPV in normal cervical tissue, cervical intraepithelial neoplasia (CIN) I, CIN II, CIN III and cervical cancer were 15.0, 32.8, 54.3, 69.7, 93.8%, respectively. HR-HPV positive samples of 62.8% were infected with HPV16. The integration ratio of HPV16 in CIN III and cervical carcinoma were 35.7 and 58.1% respectively, both of which were significantly higher than that of CIN I and normal cervical tissues. The discrepancy was statistically significant (P < 0.05). Furthermore, it was observed that persistent virus infection and progression of cervical lesion were more common in CIN I with integrated HPV16 than that with episomal HPV16. The integration ratio of HPV16 was accompanied by an increase in the grade of cervical lesion. The integrated state of HPV16 infection was strongly associated with persistent HPV infection and progression of cervical lesions.

Human papillomavirus type distribution in women from Asia: a meta-analysis

The aim of this study was to determine human papillomavirus (HPV) type distribution in women with and without cervical neoplasia from Asia and to estimate the potential future impact of an HPV 16/18 prophylactic vaccine in this region. A meta-analysis was conducted including 79 studies using polymerase chain reaction to detect HPV types. A total of 5954, 1653, 958, and 16,803 women with invasive cervical cancer (ICC), high-grade squamous intraepithelial lesions (HSIL), low-grade squamous intraepithelial lesions (LSIL), and normal cytology or histology were included, respectively. Type-specific prevalence of HPV types 6, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 70, 73, and 82 were estimated and stratified by cervical lesion grade. Overall HPV prevalence was 85.9%, 81.0%, 72.9%, and 14.4%, respectively, in women with ICC, HSIL, LSIL, and normal cytology/histology. In ICC, HPV 16 was the predominant type (52.4%), followed by HPV 18, 58, 33, 52, 45, 31, and 35. The estimated HPV 16/18–positive fraction was 66.9%, 40.4%, 26.7%, and 3.3% in women with ICC, HSIL, LSIL, and normal cytology or histology, respectively. In ICC, the estimated HPV 16/18–positive fraction was about 70% in all Asian geographic regions, with the exception of Japan (51.3%). HPV 16/18 vaccines are estimated to provide about 67% protection against ICC in Asia. HPV 58 and 52 were among the five most common types in ICC in eastern and southeastern Asia but not in south central Asia. After HPV 16 and 18, the next most six common HPV types were 58, 33, 52, 45, 31, and 35 that accounted for additional 20% of cervical cancer cases in Asia. For optimal population coverage, these HPV carcinogenic types should be considered for second-generation HPV prophylactic vaccines.

Evidence of HPV16 integration in low- and high-grade cervical lesions that regress demonstrated by multiple displacement amplification and Southern blot hybridisation

The prevalence and significance of human papillomavirus (HPV) integration among different grades of cervical lesions is uncertain. In this study, HPV physical status was examined by the combination of multiple...

Interaction between inflammation and angiogenesis during different stages of cervical carcinogenesis

Objective. The primary aim of this study was to investigate the role of angiogenesis and inflammatory cell response in cervical carcinogenesis. Methods. Formalin-fixed tissue specimens from 58 uterine cervical specimens (8 CIN1, 14 CIN2, 28 CIN3, and 8 SCC), representing the different stages of cervical carcinogenesis, were immunohistochemically analyzed. Normal cervical tissue specimens were also included as controls. The present study assessed the expression of CD31 and CD105 to evaluate microvessel density (MVD), the macrophage marker CD68 and the panleukocyte marker CD45. In addition, expression of iNOS (inducible Nitric Oxide Synthase) was also evaluated. Results. MVD, measured by either CD31 or CD105, increased along the continuum from normal epithelium to squamous cell carcinoma, and a significant correlation between the CD105-MVD and the CD31-MVD was observed ( r = 0.8735; p < 0.0001). Furthermore, the number of infiltrating macrophages was significantly associated with progression to malignancy. Interestingly, there was a close positive correlation between macrophage counts and CD105-MVD ( r = 0.7525; p < 0.0001). In striking contrast to the other angiogenic and inflammatory markers tested, iNOS expression was significantly reduced as cervical lesion grade progressed from low to high. Conclusions. Our findings demonstrated a positive correlation between neovascularity and macrophage counts, whereas iNOS expression displayed an inverse relationship with macrophage density and tumor progression. Low iNOS expression may modify the function of tumor-infiltrating macrophages toward a malignant phenotype that promotes tumor progression rather than an anti-tumor response.

398 POSTER Clinical requirements of “In Silico Oncology” as part of the integrated project ACGT (Advancing Clinico-Genomic Trials on Cancer)

The aim of this study was to analyze aberrant expression of both apoptotic protein p53 and antiapoptotic protein bcl-2 in squamous cell carcinoma (SCC) of the uterine cervix with HPV infection and its significance as a marker for progression of cervical lesions.One hundred and five cervical lesions and 20 normal (age matched) cervical epithelium from patients with complaints other than cervical lesions were investigated immunocytochemically for aberrant expression of p53 and bcl-2 using the streptavidin-biotin-peroxidase method with respective monoclonal antibodies. HPV status was also anlayzed using type-specific primers for HPV 16/18 and HPV 6/11 by polymerase chain reaction (PCR). The statistical correlation analysis was carried out using Spearman's correlation test and univariate analysis by the SPSS system.An abnormal nuclear expression of tumor-suppressor protein p53 and cytoplasmic expression of bcl-2 were observed using immunocytochemistry in biopsies of cervical lesions but not in normal subjects. The intensity of immunoreactivity for both p53 and bcl-2 proteins varied between different histopathological grades of cervical lesions and the correlation analysis showed a highly significant positive correlation for their expression level with different stages from mild dysplasia to invasive cancer with r = 0.88842; P = 0.00001 and r = 0.86929; P = 0.00001, respectively. A highly significant positive correlation was also observed between the expression of both p53 and bcl-2 proteins and HPV infection. The current study indicates a very good significant direct correlation (r = 0.83925; P = 0.00001) between p53 expression and bcl-2 expression in the study population, suggesting the co-overexpression of these proteins in HPV-associated cervical cancer.From the observations it is suggested that the immunodetection of both p53 and bcl-2 proteins in squamous cell carcinoma of the uterine cervix can be used as an independent diagnostic marker for cervical cancer associated with HPV infection. The highly significant association of these proteins with HPV infection suggests that the high-risk HPV infection may be responsible for the co-overexpression of p53 and bcl-2 in cervical cancer even though both of them are antagonistic in their function. This study thus helps to understand the molecular mechanism underlying cervical carcinogenesis and which in turn may improve the therapeutic approach.

Prophylactic vaccination against human papillomavirus infection and disease in women: a systematic review of randomized controlled trials

Human papillomavirus (HPV) is now known to be a necessary cause of cervical cancer, and prophylactic HPV vaccines aimed at preventing genital warts, precancerous cervical lesions and cervical cancer are now available. To gauge the potential impact on disease burden, we performed a systematic review of the evidence from randomized controlled trials. We conducted a systematic search of the literature to identify all randomized controlled trials of prophylactic HPV vaccination. Reports in 5 electronic databases covering 1950 to June 2007 (MEDLINE, MEDLINE in process, EMBASE, the Cochrane Central Registry of Controlled Trials and the Cochrane Library), bibliographies of all included studies and of narrative reviews (2006-2007), clinical trial registries, Google Scholar, public health announcements, selected conference proceedings (2004-2007) and manufacturers' information on unpublished data or ongoing trials were screened against predefined eligibility criteria by 2 independent reviewers. Vaccines had to contain coverage against at least 1 oncogenic HPV strain. The primary outcome of interest was the frequency of high-grade cervical lesions (high-grade squamous intraepithelial lesion, or grade 2 or 3 cervical intraepithelial neoplasia). The secondary outcomes were persistent HPV infection, low-grade cervical lesions (low-grade squamous intraepithelial lesion or grade 1 cervical intraepithelial neoplasia), external genital lesions, adverse events and death. Meta-analysis of the data was done in all cases where adequate clinical and methodological homogeneity existed. Of 456 screened reports, 9 were included in the review (6 were reports of randomized controlled trials and 3 were follow-up reports of initial trials). Findings from the meta-analysis showed that prophylactic HPV vaccination was associated with a reduction in the frequency of high-grade cervical lesions caused by vaccine-type HPV strains compared with control groups: Peto odds ratio 0.14 (95% confidence interval [CI] 0.09-0.21) from combined per-protocol analyses, and 0.52 (95% CI 0.43-0.63) from modified intention-to-treat analyses. Vaccination was also highly efficacious in preventing other HPV-related infection and disease outcomes, including persistent HPV infection, low-grade lesions and genital warts. The majority of adverse events were minor. The incidence of serious adverse events and death were balanced between the vaccine and control groups. Among women aged 15-25 years not previously infected with vaccine-type HPV strains, prophylactic HPV vaccination appears to be highly efficacious in preventing HPV infection and precancerous cervical disease. Long-term follow-up is needed to substantiate reductions in cervical cancer incidence and mortality.

Viral DNA load, physical status and E2/E6 ratio as markers to grade HPV16 positive women for high-grade cervical lesions

Objectives. Cervical intraepithelial neoplasias (CIN) associated with high-risk (HR) human papillomavirus infection, in addition to HR-HPV typing need other viral marker testing to distinguish a subset of lesions with clinical relevant infections. This study has evaluated the significance of viral markers, such as viral load, physical status and E2/E6 ratio, to stratify HPV16 infected women at a single point in time for grade of cervical lesions. Methods. One hundred sixty-six cytological specimens were selected from women with low ( n = 72) and high ( n = 94) grade squamous intraepithelial lesions (SIL), and positive to HPV16. All the 72 LSIL were CINI, 83 of the 94 HSIL were CINII/III and 11 SCC (Squamous Cervical Carcinoma). Cytological specimens were analysed by two different SYBR Green Real-time PCR assays (RT-PCR). Specific primers for both E2 and E6 viral genes and GAPDH cellular gene were designed to determine viral load, physical status and E2/E6 ratio. Results. The viral load was significantly higher in HSIL than in LSIL. In CINI episomal DNA was prevalent (72.2%), mixed forms (episomal and integrated) were 27.8%, suggestive of an early integration of viral DNA into cellular genome, no pure integrated forms were detected. However in CINII/III mixed DNA forms were prevalent (73.5%). In SCC pure integrated DNA was prevalent (81.8%) in absence of episomal forms. E2/E6 ratio decreased significantly from CINI to CINII/III and SCC with a linear trend. The logistic regression analysis showed that viral load higher than 1.38 × 10 6 genome copies per 300 ng of total DNA associated with E2/E6 ratio lower than 0.90 was highly significant in differentiating CINII/III versus CINI, while the only E2/E6 value lower than 0.17 was significant in differentiating SCC from CINI. Conclusions. Viral load higher than 1.38 × 10 6 genome copies per 300 ng of total DNA and E2/E6 ratio values allow HPV16 infected women with high grade cervical intraepithelial lesions to be recognized.

Telomerase Activity as a Tumor Marker in Indian Women with Cervical Intraepithelial Neoplasia and Cervical Cancer

Cervical cancer is the most common cancer in Indian women and is a leading cause of death in women worldwide. Cervical cancer develops from pre-neoplastic cervical intraepithelial neoplasia (CIN). This study was conducted to evaluate telomerase activity as a tumor marker for the detection of cancer in patients with CIN and cervical cancer. The results were compared with human papillomavirus (HPV) status, clinical staging, and histopathologic studies. Telomerase activity was detected using the PCR-based telomeric repeat amplification protocol (TRAP) assay in cervical tissues collected by routine punch biopsy from the uterine cervix of patients with suspected cervical cancer. High-risk (HR) HPV-16 and -18 status was determined in all the study groups, including controls. A total of 125 patients (including 50 patients with CIN and 75 patients with cervical cancer [including nine patients with adeno-squamous disease]) and 22 control subjects were studied. The sensitivity and specificity for detecting CIN and cervical cancer were calculated. Patients with grade I, II, and III CIN showed 17%, 33%, and 57% positivity for telomerase, respectively. In patients with cervical cancer, those at early clinical stages (Ia-IIb) showed 68% positivity and those at later clinical stages showed 92% positivity for telomerase activity. In the present study, telomerase positivity correlated significantly with the detection of HR HPV-16 and -18 (p < 0.001). As a diagnostic test, none of the described analyses combined a sensitivity of > or =90% with a specificity of > or =90%, except in patients with advanced cancer when telomerase activity was used as a diagnostic test. Our findings suggest that telomerase activation is a relatively early event in cervical carcinogenesis and correlates with the grade of cervical lesion, HR-HPV status (16 and 18 subtypes), and clinical staging. Hence, these associations suggest it as a possible target for detection of cervical cancer.

Human papillomavirus type distribution in women from Asia: a meta-analysis

The aim of this study was to determine human papillomavirus (HPV) type distribution in women with and without cervical neoplasia from Asia and to estimate the potential future impact of an HPV 16/18 prophylactic vaccine in this region. A meta-analysis was conducted including 79 studies using polymerase chain reaction to detect HPV types. A total of 5954, 1653, 958, and 16,803 women with invasive cervical cancer (ICC), high-grade squamous intraepithelial lesions (HSIL), low-grade squamous intraepithelial lesions (LSIL), and normal cytology or histology were included, respectively. Type-specific prevalence of HPV types 6, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 70, 73, and 82 were estimated and stratified by cervical lesion grade. Overall HPV prevalence was 85.9%, 81.0%, 72.9%, and 14.4%, respectively, in women with ICC, HSIL, LSIL, and normal cytology/histology. In ICC, HPV 16 was the predominant type (52.4%), followed by HPV 18, 58, 33, 52, 45, 31, and 35. The estimated HPV 16/18-positive fraction was 66.9%, 40.4%, 26.7%, and 3.3% in women with ICC, HSIL, LSIL, and normal cytology or histology, respectively. In ICC, the estimated HPV 16/18-positive fraction was about 70% in all Asian geographic regions, with the exception of Japan (51.3%). HPV 16/18 vaccines are estimated to provide about 67% protection against ICC in Asia. HPV 58 and 52 were among the five most common types in ICC in eastern and southeastern Asia but not in south central Asia. After HPV 16 and 18, the next most six common HPV types were 58, 33, 52, 45, 31, and 35 that accounted for additional 20% of cervical cancer cases in Asia. For optimal population coverage, these HPV carcinogenic types should be considered for second-generation HPV prophylactic vaccines.

Detection of autoantibodies to survivin in cervical mucus from patients with human papillomavirus-associated cervical cancer and precursor lesions

Objectives. To investigate the prevalence of mucosal autoantibodies to survivin in patients with human papillomavirus (HPV)-associated cervical cancer and precursor lesions.Methods. Cervical mucus from 117 HPV-associated cervical cancer, 102 high-grade squamous intraepithelial lesion (HSIL), 107 low-grade SIL (LSIL), and 80 normal controls were tested by ELISA using either full length recombinant survivin or survivin-derived peptides. Survivin expression in cervical tissue biopsies was studied by Western Blotting.Results. Cervical mucus from 33 cervical cancer (28.2%), 17 HSIL (16.6%), and 8 LSIL (7.4%) patients reacted with recombinant survivin. The IgA-class antibody response was significantly higher than that observed in the normal controls. The level of mucosal anti-survivin response was associated to the level and intensity of survivin expression in the different lesions. Finally, reactivity against a survivin Nt-derived peptide was found more frequently than reactivity against a Ct-derived peptide.Conclusions. IgA-class autoantibodies against survivin are present in a substantial proportion of cervical mucus from patients with HPV-associated cervical cancer, and precursor lesions. Mucosal anti-survivin response is positively associated with the level of survivin expression and the grade of cervical lesion.

HPV-induced carcinogenesis of the uterine cervix is associated with reduced serum ATRA level

Objective. In uterine cervical cancer, certain oncogenic HPV types are considered as key etiologic factor. But the progression of HPV associated cervical precancerous lesions depends on many other factors such as oncogenes, immune system, anti-viral factors etc. This study is therefore focused on the effect of an important dietary anti-viral factor called All Trans Retinoic Acid (ATRA) on the development of HPV associated cervical cancer as it is found higher in poor socioeconomic people. Method. We analyzed a total population of 130 including control subjects who have no complaints of uterine cervical lesions and the HPV-6/11, 16/18 infected cases of low grade squamous intraepithelial lesions [SIL], high grade squamous intraepithelial lesions [HSIL], and invasive cancers, for serum ATRA level. This study also focused to find out the association of serum ATRA level with the proliferation status in terms of proliferating cell nuclear antigen (PCNA) expression as it is an anti-proliferation agent and with the grades of cervical lesions, using SPSS statistical package. Results. The results showed a highly significant negative association for serum ATRA level with different stages of cervical lesions ( F = 3.305; P = 0.000) by one-way ANOVA and with intensity of PCNA expression ( r = −0.825; P < 0.01) by Pearson's correlation test. A highly significant association was observed for the PCNA expression with the grades of cervical lesions too ( F = 37.89; P = 0.000). Further, we found from our data that all the invasive cancer cases were infected with HPV-16/18 and none with HPV-6/11. Hence, we analyzed the association of serum ATRA level with HPV-16/18 infected preinvasive cases in developing invasiveness, by Fisher's Exact Test, using Graph Pad Prism as shown in Table 1. The results show an odds ratio (OR) of 36.93 and a relative risk (RR) of 4.99 with an 95% interval being 2.896 to 8.603, which is significant at the level of P = 0.0001 for the reduced [<0.6 μg/ml] serum ATRA level in developing invasive cancer in HPV-16/18 infected preinvasive cases. Conclusion. All these results suggest that the serum ATRA level highly influences the progression of cervical lesions to invasive cancer and can be therefore aimed as a marker for progression in combination with HPV-16/18, which helps to enhance the modalities of therapy towards cost effectiveness.

Evaluation of gene chip technology for high risk type human papillomavirus in cervical cancer screening

To investigate the clinical value of gene chips technology for human papillomavirus (HPV) in cervical cancer screening. A population-based cross-sectional screening study was conducted among 1137 women aged 15-59 in a community, Shenzhen city. Hybrid capture 2 (hc2) and gene chip technology were performed to examine the high risk type human papillomavirus in the exfoliated cervical cells. Liquid-based cytology test (LCT) was also performed at the same time. The HPV-positive women with LCT > or = atypical squamous cells of undetermined sign (ASCUS) and the HPV-negative women with LCT > or = low grade squamous intraepithelial lesion (LSIL) underwent biopsy under colposcopy. The pathological results were used as the gold standard to evaluate the two HPV test methods. Totally 122 biopsy specimens were obtained. Pathological examination showed no cervical cancer case, 3 cases of grade III cervical intraepithelial neoplasia (CIN), 11 cases of grade II CIN, 36 cases of grade I CIN, 69 cases of chronic cervicitis and metaplasia of squamous epithelium, and 3 cases of normal cervix. The HPV-positive rate was 14.0% by hc2 and 9.8% by gene chips with a HPV-positive rate by hc2 higher than that by gene chips (P < 0.001) and an mediocre accordance rate between these methods (kappa = 0.498). The. HPV-positive rate increased along with the increase of the grade of cervical lesions (P < 0.05). The sensitivity, specificity, accuracy, positive prevalue, negative prevalue, positive likelihood ratio and negative likelihood ratio of hc2 for high-risk HPV were 100%, 87.1%, 87.3%, 8.8%, 100%, 7.7 and 0.000, respectively; and those of gene chips were 78.6%, 91.1%, 90.9%, 9.9%, 99.7%, 8.8 and 0.235 respectively. At present hc2 high risk HPV testing is still the better method for cervical cancer screening. Gene chips technology is able to rival hc2 except that its sensitivity for cervical high grade lesions need be improved.

Significance of punch biopsy and endocervical curretage in diagnostics of cervical intraepithelial lesions

Diagnostic procedures during the detection of cervical intraepithelial lesions (CIN) are a combination of cytology, colposcopy, punch biopsy and endocervical curretage. An optimal therapeutic approach according to the distribution, size and grade of cervical lesions is the result of this diagnostic protocol. This study was carried out to assess reliability of the punch biopsy and endocervical curretage in diagnostics of cervical intra-epithelial lesions. Fifty patients undergoing cervical conization were studied retrospectively to evaluate the correlation between the grade of preoperative punch biopsy and endocervical curretage, and the grade of the dysplastic epithelial changes in the cone biopsy. CIN grade was established according to the WHO/ISGYP classification and comparation of the results was performed after that. Out of the total number of patient, 89.36% of them with dysplastic epithelial changes on cone biopsy had also dysplastic changes in the preoperative punch biopsy. An exact correlation between CIN grades was identified in 56% of the cases, 20% in CIN2, and 36% in CIN3 cases. There were 24.14% patients with negative endocervical curretage, while only in one case the cone biopsy was negative, too. Among the remaining 22 patients with CIN changes in endocervical curretage specimens, only one had a negative cone biopsy. An exact correlation of CIN grade was identified in 37.93% of the cases, 13.79% of CIN2, and 24.14% of the CIN3 cases. There was a positive cor relation between the CIN grades in punch and cone biopsy, as well as between the grade on the endocervical curretage and cone biopsy specimen, but with the lower degree than previous.

P16 INK4A expression in cervical premalignant and malignant lesions

p16 INK4a is a cyclin-dependent kinase (CDK) inhibitor which decelerates cell cycle by inactivating CDKs that phosphorylate pRb. Human Papillomavirus persistent infection plays an important role on cervical carcinogenesis, mainly by the action of two viral oncoproteins, E6 and E7, which interact with p53 and pRb, respectively. Increasing expression of E6 and E7 in dysplastic cervical cells might thus be reflected by increased expression of p16 INK4a. Recent studies revealed that p16 INK4a expression could be a marker for dysplastic and neoplastic cervical cells. The aim of this study was to analyze p16 INK4a expression in cervical preneoplastic and neoplastic lesions and correlate with lesion grade. Expression of p16 INK4a was analyzed by immunohistochemistry. A total of 6 low-grade squamous intraepithelial lesion (LSIL), 21 high-grade squamous intraepithelial lesions (HSIL) and 27 cancer samples were studied. In HPV-positive cervical samples ( n = 48), p16 INK4a expression was observed in 1 of 3 LSIL, in 18 of 19 HSIL and in all 26 cancer cases. These results are in accordance with the hypothesis that functional inactivation of pRb by HPV-E7 protein induces p16 INK4a expression in cervical lesions. In our study, a statistically significant association was observed between cervical lesion grade and p16 INK4a expression ( P < 0.001).

Performance of the Roche AMPLICOR® Human papillomavirus (HPV) test in prediction of cervical intraepithelial neoplasia (CIN) in women with abnormal PAP smear

Objectives. To assess the performance of a novel PCR-based assay (Roche AMPLICOR® HPV test) in detection of cervical pathology as a part of management for abnormal PAP smear (MAPS) and in women participating in cervical cancer screening. Study design. Altogether, 504 women comprising 270 patients referred for colposcopy due to an abnormal Pap smear and another 234 women participating in cervical cancer screening (tested for comparison) were analyzed for oncogenic (HR) Human papillomavirus (HPV) types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 using the Roche AMPLICOR® HPV test in cervical samples collected in PreservCyt® liquid media. Colposcopic biopsy and/or LEEP cone biopsy was used as the gold standard in the triage group, while liquid-based cytology (LBC) was the reference test in the screening group. Results. The prevalence of HPV was significantly higher in the MAPS group (65.9%) than in the screening group (31.2%) ( P = 0.0001). There was a poor concordance between the referral PAP and the current LBC, being only moderate in the screening series, ICC (weighted kappa) = 0.291 (95%CI 0.070–0.459) ( P = 0.007), and almost poor in the MAPS Series, with ICC = 0.217 (95%CI 0.04–0.384) ( P = 0.023). AMPLICOR® HPV positivity increased linearly with the increasing grade of cervical lesions. In detecting high-grade (CIN2–3), colposcopy was the most sensitive test (96.5%), very similar to AMPLICOR (95.2%) ( P = 0.731), while LBC with HSIL cutoff was by far the most specific test (99.5%) and showed the highest PPV (96.1%). NPV of colposcopy (97.2%) and AMPLICOR (96.7%) were similar ( P = 0.839). Together with abnormal colposcopy and HSIL cytology, the AMPLICOR® HPV test is a powerful independent predictor of high-grade CIN2–3, and as such suitable to replace cervical cytology in management of women with abnormal PAP test (MAPS). Conclusions. The Roche AMPLICOR® HPV test is comparable to other HPV tests (HCII, PCR) in detecting CIN in MAPS. However, more data are clearly needed on the performance of AMPLICOR test in management of abnormal PAP and particularly as a screening tool.