AgNOR, p16 and human papillomavirus in low-grade squamous intra-epithelial lesions of the uterine cervix. Preliminary report

It has been shown that infection with high-risk human papillomaviruses (HR-HPV) is related to the development of cervical cancer. The persistence of the virus in intra-epithelial lesions of cervix uteri (SILs) is the basis for the application of HPV testing for screening and management of patients. Most infections by HR-HPVs resolve spontaneously, however, and do not progress to dysplasia or cancer. p16INK4a is a useful biomarker of cervical intra-epithelial neoplasia and could be a marker for the progression of low-grade squamous intra-epithelial lesions (LSILs) to high-grade squamous intra-epithelial lesions (HSILs), because it correlates independently with increasing SIL grade. We conducted a preliminary histological study of 28 patients diagnosed with LSIL, HSIL or nondysplastic epithelium (NDE) from whom 28 biopsies of uterine cervix and 28 endocervical brushed biopsies were taken. Argyrophilic nucleolar organizer region (AgNOR) and p16INK4a assays were performed on the biopsies, and endocervical brushings were used for HPV typing. The high risk HPV group showed that the number of patients with AgNOR areas greater than 3.3 μm(2) and with expression of p16INK4a were statistically greater than the number of lower risk patients. None of the biopsies of LR-HPV carriers expressed p16 and AgNOR areas> 3.3 μm(2) simultaneously. Four LSILs and the NDE of this group expressed neither of the two markers. If the correlation between AgNOR areas and p16INK4a is good, we may be able to develop a low cost simple technology for studying patients infected with HR-HPV and diagnosed with LSIL of uncertain behavior.     

Expressions of E2 and E7-HPV16 proteins in pre-malignant and malignant lesions of the uterine cervix

Continuous production of the E7 protein from different types of high risk human papilloma virus (HPV) is required for progression of malignancy. We developed antibodies against HPV type 16 E7 and E2 proteins to evaluate their utility as markers for diagnosis during early stages of cervical cancer. Forty biopsies from uterine cervices were diagnosed as low grade intraepithelial lesion (LSIL), high grade intraepithelial lesion (HSIL), squamous carcinoma (SC), in situ adenocarcinoma (ISA) and invasive adenocarcinoma (AC), all of which were infected with HPV 16. Immunohistochemistry was used to investigate the expressions of E7 and E2 (both from HPV 16) and p16. P16 was expressed in eight of 12 LSILs, in all HSILs, in 16 of 18 SC and in all ACs. E2 was expressed in six of 12 LSILs. E7 was positive in eight of 12 LSILs and in all HSIL and carcinomas. The expressions of E2 and E7 of HPV16 related to p16 expression confirmed the value of the viral oncogenic proteins as complementary to histology and support the carcinogenic model of the uterine cervix, because HPVDNA integration into cellular DNA implies the destruction of the gene encoding E2, which suppresses the expression of the E6 and E7 oncoproteins. E2 from HPV16 could be marker for LSILs, while E7 could be a marker for progression of LSILs to HSILs in patients infected by HPV16, because viral typing has little positive predictive value.     

Procedure for immunocytochemical detection of P16INK4A antigen in thin-layer, liquid-based specimens

OBJECTIVE: To develop a procedure for the immunocytochemical detection of P16INK4A in ThinPrep specimens. STUDY DESIGN: Archived ThinPrep, liquid-based cervical/endocervical cytology specimens (Cytyc Corp., Boxborough, Massachusetts, U.S.A.) diagnosed as LSIL, HSIL and WNL were resampled and fixed in 95% ethanol for at least three days. Rehydration and endogenous peroxidase blocking of both ThinPreps and formalin-fixed, paraffin-embedded tissues were accomplished on a Leica Autostainer (Leica, Deerfield, Illinois, U.S.A.). Microwave antigen retrieval with CitraPlus (Biogenex, San Ramon, California, U.S.A.) was performed using a Panasonic microwave oven (Matsushita Cooking Appliances, Franklin Park, Illinois, U.S.A.) on the high setting twice for five minutes each. After cooling for 20 minutes and undergoing a buffer rinse, the slides were placed in a Dako autostainer (Dako-USA, Carpinteria, California, U.S.A.). The P16INK4A primary antibody, clone E6H4 (MTM Laboratories, Heidelberg, Germany) was diluted 1:200 in antibody diluent buffer. Detection was accomplished with a mouse non-avidin-biotin EnVision+ polymer (Dako). The expression of P16INK4A in ThinPreps and corresponding biopsies were scored by two pathologists. A ThinPrep case was scored as positive if it contained > 10 abnormal cells with nuclear and cytoplasmic immunocytochemical staining. Corresponding biopsies were scored as exhibiting negative, sporadic, focal or diffuse staining, as described by Klaes et al, Overexpression of P16INK4A as specific marker for dysplastic and neoplastic epithelial cells of the cervix uteri (Int J Cancer 2001;92:276-284). RESULTS: The P16INK4A antibody assay was positive in 14 of 19 (73.68%) LSIL ThinPrep cases and in 25 of 26 (96.15%) HSIL ThinPrep cases. Thirty-eight of the 39 (97.44%) biopsies corresponding to the positively stained ThinPreps also were positive, with a staining score of at least focal positivity in the dysplastic regions. The P16INK4A antibody assay was negative in 5 of 19 (26.32%) LSIL ThinPrep cases and negative in 1 of 26 (3.85%) HSIL ThinPrep cases. The six biopsies corresponding to the negative ThinPreps were similarly negative. The two cytologic specimens diagnosed as WNL were negative for P16INK4A, as were two tissue control cases with benign diagnoses. Nondysplastic squamous epithelium, identified in 17 biopsy cases, did not stain, nor did nondysplastic squamous cells identified in ThinPrep cases. Sporadic staining of bacteria, inflammatory cells and occasional endocervical glandular cells was identified. CONCLUSION: P16INK4A expression in ThinPrep specimens correlates with tissue expression of P16INK4A, as implemented in the above protocol. P16INK4A may thus serve as a surrogate marker in gynecologic cytology for high-risk HPV infection and for the development of cervical neoplasia.     

p16(INK4A) immunohistochemical overexpression in premalignant and malignant oral lesions infected with human papillomavirus.

Human papillomavirus (HPV) is believed to promote the oncogenic process, and the correlation between viral oncoproteins and dysfunction of p16(INK4A) tumor suppressor protein in oral lesions is controversial. To test the hypothesis that anogenital HPV types participate in disruption of the regulation of p16(INK4A) suppressor protein in oral lesions, we analyzed 46 oral biopsy specimens for the presence of HPV 6/11 and 16/18 by in situ hybridization (ISH) and for p16(INK4A) expression by immunohistochemistry (IHC). Eighteen (39%) of the 46 oral lesions were HPV-positive and 28 (61%) were HPV-negative. HPV 6/11 DNA was found in 5 (11%) and HPV 16/18 in 13 (28%) of 46 biopsies. Nine of the 18 HPV-positive oral lesions (50%), assessed by catalyzed signal amplification coupled to ISH (CSA-ISH), gave high-intensity p16(INK4A) immunostaining. Focal and diffuse patterns were observed in 11/13 (77%) lesions with HPV 16/18, focal immunopositivity in 3/5 (80%) with HPV 6/11, and negative or sporadic p16-labeling in 18/28 (64%) without the presence of HPV DNA. These results showed a strong association between overexpression of p16 protein and malignant oral lesions, mainly those infected by HPV 16/18. We can conclude that high-risk HPV types are associated with p16 overexpression, and p16 may serve as a biomarker in oral cancer related to high-risk HPV infection.     

Assessment of the association between the frequency of micronucleus and p16INK4a/Ki-67 co-expression in patients with cervical intraepithelial lesions

Human papilloma virus (HPV) infection is the main etiological factor for cervical intraepithelial lesions (CIN). An important characteristic of this process is the loss of genome stability. Therefore, it is imperative to use biomarkers of DNA damage caused by genomic instability to identify high risk individuals. We investigated the frequency of micronuclei (MN) in peripheral blood lymphocytes (PBL) of 20 patients, diagnosed as histologically CIN 1 and 10 healthy controls. We also examined the frequency of other nuclear anomalies including nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) in PBL of patients with CIN 1 and healthy controls, and evaluated the benefits of p16^INK4a and Ki-67 (p16^INK4a/Ki-67) immunohistochemical double staining for identifying cervical squamous cells that express HPV E6/E7 oncogenes. We analyzed the association between the frequency of MN in PBL and the amount of p16^INK4a/Ki-67 co-expression in CIN 1 patients to establish genomic instability. Among CIN 1 subjects, 15% exhibited diffuse p16^INK4a/Ki-67 co-expression and were considered high positive, 25% of the CIN 1 cases exhibited p16^INK4a/Ki-67 co-expression restricted to the lower part of the epithelium and were considered low positive and the remaining 60% of cases were negative. The frequency of MN, NPBs and NBUDs differed significantly among groups. We found a statistically significant positive correlation between p16^INK4a/Ki-67 co-expression and the frequency of MN, NPBs and NBUDs in PBL. Our findings demonstrate the efficacy of p16^INK4a/Ki-67 double immunostaining for histological samples with CIN 1. MN frequency in PBL might be useful for detecting genomic instability in cases of HPV infection and CIN.     

Diagnostic role of p16/INK4A protein in Human Papillomavirus (HPV) induced cervical dysplasia

The p16/INK4A protein is a cellular regulatory polypeptide over-expressed in the presence of high levels of the Human Papillomavirus (HPV) coded E7 protein. This review outlines the use of p16 antigen staining in cervical biopsies as well as in PAP smears summarizing the corresponding literature and commenting the authors’ own experience. The p16 antigen is a reliable marker for dysplastic cells in CINII/CINIII (HSIL) lesions as viewed in cervical biopsies. When PAP smears were examined at large scale screening for p16 antigenreactive and atypical cells, considerable variations could be found especially in ASCUS graded lesions. Therefore, the presence of p16-reactive atypical cells in PAP smears should be interpreted together with the cytological signs of dysplasia, such as the altered N/C ratio. In addition, women revealing p16-positive ASCUS/LSIL specimens should be examined for the presence of HPV DNA. Detection of HPV DNA alone, i.e. in the absence of cytological screening has a low predictive value, since the clearance of HPV may occur even in the absence of morphological alterations. Combined cytological as well as molecular follow up contributes to the efficiency of diagnostic and increases the probability of correct interpretation of the pre-cancerous lesions by non-invasive techniques.     

HPV DNA/RNA detection in various oral and oropharyngeal biomaterials identifies active HPV infections also in non-neoplastic tonsils

Previous studies describe a correlation between HPV-positivity and non-smoking in TSCC; p16^INK4A-expression as surrogate-marker for HPV-DNA/RNA-positivity is discussed controversially. In the present study, these parameters are assessed prospectively. HPV-status of sputum and tonsillar-swabs was analyzed to determine their validity as surrogate-marker for tissue-HPV-status.TSCC- (n = 52) and non-neoplastic tonsillar tissue (n = 163) were analyzed. HPV-DNA- and HPV-RNA-status of total sputum, cellular fraction and supernatants, tonsillar-swabs and -tissue was determined by (RT)-PCR. Immunohistochemistry determined p16^INK4A-expression.23/163 (14.2%) non-neoplastic tonsils were HPV-DNA-positive; five patients (3 HPV16, 2 HPV11) had active HPV-infections (HPV-RNA-positive), in all biomaterials. 140/163 (85.9%) patients were either HPV-DNA-positive or HPV-DNA-negative in all samples. 21/52 (40.4%) TSCC-tonsils were HPV-DNA-positive; 17 patients were HPV-RNA-positive (14 HPV16; 4 HPV18). 40/52 (76.9%) TSCC-patients were congruent in all biomaterials. p16^INK4A-expression alone would have misclassified the HPV-status of 14/52 (26.2%) TSCC-patients.This prospective study confirms the discrepancy between HPV-status and p16^INK4A-expression and the significant correlation between non-smoking and HPV-DNA-positivity. HPV-sputum- and/or swab-results do not consistently match tissue-results, possibly having (detrimental) consequences if those were used to assess tissue-HPV-status. In the 5 patients with active HPV infection in the non-neoplasitic tonsils, tonsillectomy likely prevented subsequent development of TSCC.     

Immunohistochemical expression of p16INK4a and bcl-2 according to HPV type and to the progression of cervical squamous intraepithelial lesions.

Inactivation of the cell cycle inhibitor gene p16MTS1 seems to be involved in human papillomavirus (HPV)-related carcinogenesis because E6 and E7 oncoproteins may impair p16INK4a and, indirectly, bcl-2 functions. In this study, we analyzed the role of immunohistochemical expression of p16INK4a and bcl-2 in HPV-infected cervical biopsies as prognostic markers of the progression of squamous intraepithelial lesion (SIL). Sixty-five cervical biopsies were stratified into two subgroups according to the second biopsy: 27 of them maintained a low-grade (LG)-SIL diagnosis, and 38 progressed from LG-SIL to high-grade (HG)-SIL. p16INK4a and bcl-2 quantitative expression levels were measured by the immunoperoxidase method. PCR-DNA techniques were used to detect and type HPV. The Wilcoxon and Fisher exact tests were employed for the statistical analysis. In the group with an LG-SIL diagnosis at the second biopsy, no significant associations were found between p16INK4a and bcl-2 expression and presence of HPV16/18. In the group that progressed to HG-SIL, a significant association was observed between p16INK4a overexpression and HPV16/18 presence (p=0.021), but none with bcl-2 levels. It is concluded that immunohistochemical bcl-2 expression may not be useful for predicting the progression of HPV-related SIL. In contrast, p16INK4a overexpression seemed to be associated with HPV 16 and 18, suggesting that it may be a good marker for predicting SIL progression.     

Combined analysis of HPV DNA and p16INK4a expression to predict prognosis in ASCUS and LSIL pap smears

Human papillomavirus (HPV) is known to play an important etiological role in the genesis of cervical cancer, but only a very small proportion of infected women develop invasive cervical cancer. The purpose of cervical cancer prevention is early diagnosis of its precursors. The molecular detection of HPV DNA as a diagnostic test to cervical carcinogenesis gave a low positive predictive value as compared to the use of biomarkers. p16INK4a has been proposed as putative surrogate biomarkers that would allow identification of dysplastic cervical epithelia. Serial consecutive cervical smears were test for high-risk HPV stained with immunocytochemistry for p16INK4a and followed-up for 36 months. The aim of the study was to evaluate the immunohistochemical expression of pl6INK4a as a marker of progression risk in low-grade dysplastic lesions of the cervix uteri. In the present series, significant pl6 overexpression was observed in the group that progressed from low to high-grade squamous intraepithelial lesion when compared with the group that did not progress. In conclusion, overexpression of p16INK4a acts as potential biomarkers for cervical cancer progression from premalignant lesions.     

AgNOR technique could be useful for differential diagnosis of squamous carcinomatous cells and atypical cells related to human papillomavirus in cervical smears

The human papillomavirus (HPV)-associated effect can mimic invasive squamous cell carcinoma. Measurements of the argyrophilic nucleolar organizer region (AgNOR) area of cells from smears with HPV infection of the cervix with marked atypia were carried out to differentiate this pathology from keratinizing carcinomas. After destaining, the smears were incubated in the dark for 25 min with a mixture of silver nitrate and gelatin in formic acid. After washing with deionized water and sodium thiosulfate, the slides were dehydrated and mounted with Canada balsam. The average AgNOR area was determined by image cytometry using the immersion oil objective and selecting 100 cells in each smear. Twenty-three patients with a mean AgNOR area of 1.32 µm² among their samples showed normal colposcopies and cervical smears after 18 months. In four patients, whose samples were diagnosed as atypical squamous cells of undetermined significance with viral atypia, the average AgNOR area was 7.70 µm²; biopsies showed keratinizing squamous carcinomas in three of these cases and moderately differentiated squamous carcinoma in one of them. We propose a cutoff of 2.2 µm² for the AgNOR area of cells from smears with HPV infection of the cervix with marked atypia to differentiate this group from keratinizing carcinomas.     

Genetic Variation of Human Papillomavirus Type 16 in Individual Clinical Specimens Revealed by Deep Sequencing

Viral genetic diversity within infected cells or tissues, called viral quasispecies, has been mostly studied for RNA viruses, but has also been described among DNA viruses, including human papillomavirus type 16 (HPV16) present in cervical precancerous lesions. However, the extent of HPV genetic variation in cervical specimens, and its involvement in HPV-induced carcinogenesis, remains unclear. Here, we employ deep sequencing to comprehensively analyze genetic variation in the HPV16 genome isolated from individual clinical specimens. Through overlapping full-circle PCR, approximately 8-kb DNA fragments covering the whole HPV16 genome were amplified from HPV16-positive cervical exfoliated cells collected from patients with either low-grade squamous intraepithelial lesion (LSIL) or invasive cervical cancer (ICC). Deep sequencing of the amplified HPV16 DNA enabled de novo assembly of the full-length HPV16 genome sequence for each of 7 specimens (5 LSIL and 2 ICC samples). Subsequent alignment of read sequences to the assembled HPV16 sequence revealed that 2 LSILs and 1 ICC contained nucleotide variations within E6, E1 and the non-coding region between E5 and L2 with mutation frequencies of 0.60% to 5.42%. In transient replication assays, a novel E1 mutant found in ICC, E1 Q381E, showed reduced ability to support HPV16 origin-dependent replication. In addition, partially deleted E2 genes were detected in 1 LSIL sample in a mixed state with the intact E2 gene. Thus, the methods used in this study provide a fundamental framework for investigating the influence of HPV somatic genetic variation on cervical carcinogenesis.     

P16INK4A as an adjunct test in liquid-based cytology

OBJECTIVE: To assess the utility of P16INK4A as an adjunct test in liquid-based cytology in cases with equivocal morphologic changes of high grade squamous intraepithelial lesion (HSIL). STUDY DESIGN: P16INK4A immunoreactivity was investigated in residual ThinPrep material (Cytyc Corp., Boxborough, Massachusetts, U.S.A.) from 30 cases with equivocal diagnoses of HSIL that had corresponding follow-up biopsies. Two control ThinPrep cases were included: 1 HSIL with biopsy-confirmed cervical intraepithelial neoplasia (CIN) 3 and a negative specimen with a corresponding biopsy of squamous metaplasia. The expression of P16INK4A in ThinPrep specimens and corresponding biopsies was scored as previously described. A ThinPrep case was scored positive if it contained > 10 abnormal cells with nuclear and cytoplasmic immunocytochemical staining. Corresponding biopsies were scored as having negative, sporadic, focal or diffuse staining. RESULTS: The P16INK4A antibody assay was positive in 19 of 30 ThinPrep cases (63.3%). Seventeen of the 19 (89.4%) biopsies corresponding to the positively stained ThinPreps also were positive, with a score of at least focal positivity in the dysplastic regions (2 CIN 1, 4 CIN 2, 11 CIN 3; 2 lesions lost in the tissue recut). The assay was negative in 11 ThinPreps (36.6%) and 10 biopsies (33.3%) with tissue confirmation of chronic cervicitis (5), squamous metaplasia (2), CIN 1 (3) and 1 lesion lost in the tissue recut. Seventeen of 18 (94.4%) ThinPreps confirmed as high grade lesions upon biopsy showed P16INK4A positivity. The control HSIL case with a CIN 3 biopsy was diffusely positive for P16INK4A, and the control negative case with biopsy diagnosis of squamous metaplasia was negative. Nondysplastic squamous and metaplastic epithelium in 7 biopsies and nondysplastic squamous or metaplastic cells in ThinPrep cases were negative. Sporadic staining of bacteria, inflammatory cells and endocervical cells was noted. CONCLUSION: ThinPrep cases in the equivocal cytologic category with the corresponding tissue biopsy assayed for P16INK4A expression showed that there was utility for this type of testing. A larger series comparing corresponding ThinPrep and tissue biopsies will be undertaken. The role of HPV infection in these cases will also be explored.     

宫颈鳞癌演进过程P16INK4a及Hh-Gli信号通路相关蛋白表达及其相关性研究

探讨P16^INK4a及Sonic hedgehog(Hh-Gli)信号通路蛋白在宫颈癌及癌前病变(CIN)中的表达相关性及其意义．采用Western-blot方法检测HPV16阳性及HPV18阳性宫颈癌细胞系P16^INK4a及Hh-Gli信号通路蛋白Smo、Ptch及Gli表达．免疫组化检测组织芯片P16^INK4a、Shh、Smo、Ptch及Gli表达,包括20例正常宫颈、18例癌旁组织、54例CIN及28例宫颈鳞癌组织．分析P16^INK4a与Hh-Gli信号通路蛋白间表达相关性及与临床病理因素的关系．结果显示P16^INK4a、Smo、Ptch及Gli蛋白在HPV16及HPV18阳性宫颈癌细胞系中表达无显著差异(P>0.05)．P16^INK4a、Shh、Smo、Ptch及Gli蛋白在宫颈癌中表达强度显著高于癌旁及正常组织(P<0.05),在CINⅠ与正常组织间差异不显著(P>0.05)．P16^INK4a、Shh、Smo及Gli蛋白,在CINⅠ、CINⅡ与CINⅢ之间均有显著性差异(P<0.05)．相关分析显示,CINⅡ-CINⅢ中P16^INK4a与Shh和Smo蛋白表达正相关,浸润癌中P16^INK4a与Shh、Smo和Gli蛋白正相关．结论认为,P16^INK4a及Hh-Gli信号通路异常激活与宫颈癌发生及演进密切相关,且二者间具有相关性．Hh-Gli信号通路的激活可能是Shh配体增高调控Smo高表达而上调Gli蛋白所致．     

Usefulness of HPV testing in the follow-up of untreated cervical low grade lesions

The aim of the present work was to evaluate the usefulness of high-risk human papillomavirus (HR-HPV) testing for the follow-up of women with untreated low grade cervical squamous cell lesions (LSIL). For that, 412 women with a cytological diagnosis of LSIL at entry were monitored by cytology, HR-HPV testing with the Hybrid Capture II assay (HC-II) and colposcopy. Our primary endpoint was clinical progression defined by the presence of a high grade cervical intraepithelial neoplasia (CIN2 and CIN3) at the biopsy. At baseline, histological control revealed 10 CIN2 and 11 CIN3 only in the cohort of women HR-HPV+. In the follow-up, 4 CIN2 and 8 CIN3 were detected, always in the women initially HR-HPV+. Thus, the recurrence of a HR-HPV+ infection clearly selects a population at high-risk for CIN2-3. The semi-quantitative appreciation of the viral load with HC-II could not be used as a good prognostic factor for the follow-up of women with LSIL. HR-HPV testing reduces the number of cytology and colposcopy examinations in the follow-up of women aged >35 years when HPV testing is initially negative. Thus HR-HPV testing should be reserved for the follow-up of this population of women initially HR-HPV+ and proposed 6 to 12 months after the cytological diagnosis of LSIL.     

Evaluation of p16INK4a in cervical lesion of premenopausal and postmenopausal women

Pap smears of postmenopausal women are often misdiagnosed because of the difficulty in distinguishing atrophic epithelial cells groups only by morphological criteria. In this study we investigated the diagnostic application of immunocytochemical staining of p16INK4a on conventional Pap smear. A total of 137 cervical specimens were enrolled in this study, of which 77 and 60 cervical smears were taken from premenopausal and postmenopausal women, respectively. Two cervical smears were taken simultaneously in 68 women, one for conventional cytology and the other for immunostaining. Additional 69 cervical smears were taken from the archive, decolorized and then used for immunostaining. In premenopausal women 1 out of 14 (7.1%) with negative cytology, 7 out of 24 (29.2%) with low grade squamous intra-epithelial lesion (LSIL), all 35 (100%) with high grade squamous intraepithelial lesion (HSIL) and all 4 (100%) with squamous cell carcinoma (confirmed by histopathology) had positive staining to p16INK4a. In postmenopausal women p16INK4a positivity was observed in 4 out of 7 (57.1%) cases of LSIL, 12 out of 14 (85.7%) cases of HSIL and all 4 out of 5 (80%) different cases of carcinoma (1 cervical adenosquamous carcinoma and 3 cervical squamous cell carcinoma in situ confirmed by histopathology), but none of 34 smears with normal cytology. Twenty smears with normal cytology chosen for the negative control in this study were from the group of postmenopausal women and were as expected negative for p16INK4a immunostaining. In the group of postmenopausal women, 16 out of 60 (26.7%) cases the cytological diagnosis was established on the basis of pl6lNK4a immunostaining as being HSIL. From our preliminary study on a limited number of samples, we can however conclude that pl6INK4a immunostaining is a very useful tool for cytological diagnosis enabling to distinguish HSIL from normal, reactive or inflammatory changes.     

ALDH2 polymorphism for the risk of cervical carcinogenesis

To investigate the clinical significance of ALDH2 genetic polymorphisms in cervical carcinogenesis. ALDH2 polymorphisms together with human papillomavirus (HPV) types were examined in a total of 195 cervical smear in exfoliated cervical cell samples using Real-Time polymerase chain reaction (PCR) System. The frequency for the AG+AA genotype was seven in the normal group (70.0 %), 16 in the LSIL group (57.1 %), and 27 in the HSIL group (90.0 %). A significant difference was found between the LSIL and HSIL groups (P = 0.0064). Patients with HSIL lesions frequently had high-risk HPV infections and concurrently belonged to the AG+AA group. ALDH2 genotype in cervical cell samples may be associated with more severe precancerous lesions of the cervix in a Japanese population.     

Prevalence of Human Papillomavirus (HPV) in Oesophageal Squamous Cell Carcinoma in Relation to Anatomical Site of the Tumour

Background

The prevalence and role of human papillomavirus (HPV) in the aetiology of oesophageal squamous cell carcinoma is uncertain. Based on the presence of HPV in the oral cavity and its causal association with squamous cell carcinoma of the oropharynx, we hypothesised that HPV is more strongly associated with proximal than distal oesophageal squamous cell carcinoma.

Methods

A population-based study comparing HPV infection in relation to tumour site in patients diagnosed with oesophageal squamous cell carcinomas in the Stockholm County in 1999–2006. Multiplex polymerase chain reaction genotyping (PCR) with Luminex was conducted on pre-treatment endoscopic biopsies to identify type specify HPV. Carcinogenic activity of HPV was assessed by p16^INK4a expression. Multivariable logistic regression was used to calculate odds ratios and 95% confidence intervals.

Results

Among 204 patients, 20 (10%) had tumours harbouring HPV DNA, almost all (90%) of HPV high-risk type, mainly HPV16. Tumours containing HPV were not overrepresented in the upper compared to the middle or lower third of the oesophagus (odds ratio 0.6, 95% confidence interval 0.2–1.9). P16^INK4a expression was similarly common (24% and 16%) in the HPV-positive and HPV-negative groups.

Conclusion

This study found a limited presence of HPV in oesophageal squamous cell carcinoma of uncertain oncogenic relevance and did not demonstrate that HPV was more strongly associated with proximal than distal tumours.     

AgNOR counts in cervical smears under normal and other cytopathologic conditions

OBJECTIVE: To investigate the diagnostic value of AgNOR counts in cervical smears in the process of cervical carcinogenesis and in discriminating the different grades of squamous intraepithelial lesion (SIL). STUDY DESIGN: Silver nitrate staining for AgNOR counts was performed in 50 cervical smears of cytologically diagnosed normal, inflammatory, low grade SIL (LSIL) (mild dysplasia), high grade SIL (HSIL) (moderate and severe dysplasia) and squamous cell carcinoma. The smears were derived from the ongoing routine outpatient cytology screening at Queen Mary's Hospital, Lucknow, India. RESULTS: In normal and inflammatory smears, the number of AgNOR dots varied from 1 to 2, in mild dysplasia from 2 to 4, in moderate dysplasia from 4 to 6 and in severe dysplasia from 6 to 8. Frank cervical carcinoma cases revealed 8-10 dots. Thus, a progressive increase in AgNOR counts was observed when the severity of pathologic lesions increased. Statistical analysis revealed a significant difference in AgNOR counts between normal and inflammatory smears, but it was highly significant between inflammatory and LSIL cases, between LSIL and HSIL, and between severe dysplasia and frank malignancy. CONCLUSION: This study underscored the diagnostic importance of AgNOR counts, especially in discriminating between LSIL and HSIL of the cervix. Another study is under way to assess the potentiality of AgNOR counts as tumor markers in cervical carcinogenesis.     

p16INK4a基因的功能及其调控

p16^INK4a蛋白能抑制CDK4和CDK6的活性,使pRb处于非磷酸化或低磷酸化状态而能与转录因子E2Fs结合,从而抑制DNA 的合成,阻止细胞由G1期进入S期.p16^INK4a的表达受Ets1和Ets2的正调控,受Bmi-1的负调控.p16^INK4a基因缺失、突变、甲基化、RNA剪接加工错误可导致细胞周期失控和癌变.应用p16^INK4a对某些肿瘤进行基因治疗的研究正在进行中.     

Association of Trichomonas vaginalis and Cytological Abnormalities of the Cervix in Low Risk Women

Objective

Is Trichomonas vaginalis (TV) an inducing factor for the development of (pre-)cancerous lesions of the cervix?

Design

Cross sectional study.

Setting

Screening healthy Belgian women with low infection risk.

Sample

63,251 consecutive liquid based cervical samples.

Methods

Real time quantitative PCR for presence of TV, 18 HPV types and Pap smear analysis of cytologic abnormalities.

Main Outcome Measures

Association of TV and HPV with cervix dysplasia

Results

The overall prevalence of TV DNA was 0.37%, of low risk HPV 2%, of high risk HPV 13.2%, and 8.8 % had cytological abnormalities. Both LR-HPV and HR-HPV were significantly associated with all cytological abnormalities. Presence of TV was associated with LR- and HR-HPV, ASC-US and HSIL, but not with other abnormalities. All women with TV and HSIL also had HR-HPV, while the latter was present in only 59% of women with TV and ASC-US. Amongst HPV negative women, TV was found in 1.3% of women with ASC-US, but only in 0.03% of women with normal cytology (OR 4.2, CL95% 2.1-8.6). In HR-HPV positive women, presence of TV increased the likelihood of cytological abnormalities somewhat (P=0.05), mainly due to an increase in ASC-US and LSIL, but not HSIL.

Conclusions

We conclude that TV infection is associated with both LR and HR-HPV infection of the cervix, as well as with ASC-US and HSIL. TV is a concomitant STI, but is not thought to be a co-factor in the causation of HSIL and cervical cancer. However, TV may cause false positive diagnoses of ASC-US.