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.     

A thin-layer,liquid-based pap test for mass screening in an area of China with a high incidence of cervical carcinoma. A cross-sectional,comparative study

OBJECTIVE: To confirm the accuracy of the ThinPrep Pap Test (Cytyc Corp., Boxborough, Massachusetts, U.S.A.) on the basis of histologic diagnosis by biopsy and the detection of human papillomavirus (HPV) DNA in mass screening. STUDY DESIGN: A total of 1,997 women residing in Xiangyuan County, Shanxi Province, P.R.C., an area with a high incidence of cervical carcinoma, were enrolled in this study. We collected exfoliative cervical samples from all subjects into a liquid buffer (Preserv-Cyt [Cytyc]) and utilized for both cytologic screening using the ThinPrep Pap Test and HPV DNA testing. Subsequent colposcopic biopsies were taken on all subjects. All the tests were performed in an independent and blinded fashion. We compared the ThinPrep Pap test with colposcopic biopsy and HPV test. RESULTS: High grade squamous intraepithelial lesions (HSIL) (CIN 2/3) were identified in 74 (3.7%) of 1,993 women adequately assessed, and there were 12 cases of squamous cell carcinoma (SCC). The false negative rate of ThinPrep cytology was 3.2% for biopsy-confirmed CIN 3 and 9.3% for CIN 2. Twenty-seven (87%) of the 31 women with biopsy-confirmed CIN 3 and 12 (100%) of 12 with biopsy-confirmed SCC had a diagnosis of either HSIL or greater abnormalities on ThinPrep cytology. In addition, the HPV DNA detection rates offered a good correlation between cytology and biopsy. CONCLUSION: The ThinPrep Pap performed extremely well in this primary screening trial. We found a good correlation between ThinPrep cytology and colposcopic biopsy on detection of HSIL and SCC; cervical specimens collected in ThinPrep liquid buffer serve as a direct test for HPV as well.     

Utility of p16(ink4a) immunocytochemistry in liquid-based cytology specimens from women treated for high-grade squamous intraepithelial lesions

OBJECTIVE: To examine whether p16(ink4a) immunocytochemical (ICC) expression detected intraepithelial disease in liquid-based cytology (LBC) specimens from women with high-grade squamous intraepithelial lesions (HSIL), whose specimen was labeled negative for intraepithelial lesion or malignany (NILM). STUDY DESIGN: Residual LBC specimens from women treated for HSIL (n = 21), whose LBC test was interpreted as NILM including marked benign inflammatory changes (BCC) were used. The control (n = 25) consisted of residual LBC specimens from women with documented HSIL. ICC for p16p(16k4a) was performed on a second ThinPrep (ThinPrep 2000, Cylyl Corporation, Boxborough, Massachusetts, U.S.A.) preparation; the percentage ofpositive cells and intensity of immunostaining were recorded. Standard LBC preparations for p16(ink4a) ICC-positive and ICC-negative control cases were reviewed. RESULTS: Twenty-four of 25 (96%) of the HSIL control group were ICC p16(ink4a) positive. In the NILM/BCC group, 2 of 21 with adequate LBC residua were ICC p16(ink4a) positive; on review both were reclassified as epithelial abnormality--1 HSIL and 1 atypical squamous cells cannot exclude HSIL. In both, subsequent colposcopic biopsy yielded HSIL. CONCLUSION: p16(ink4a) ICC positivity on NILM/BCC LBC residua from patients with HSIL may identify cases that merit cytologic review and possible reclassification. The utility of p16(ink4a) ICC in this situation requires further study.     

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．     

Outcomes of women with atypical squamous cells of undetermined significance and high-risk human papillomavirus DNA

OBJECTIVE: To study the risk of high-grade squamous intraepithelial lesion (HSIL) and cervical intraepithelial neoplasia (CIN) 2 or 3 in women with human papillomavirus (HPV)-positive atypical squamous cells of undetermined significance (ASCUS) cytology over a 2-year period using the 2001 Bethesda System and ThinPrep Paps. STUDY DESIGN: In 2002, 846 patients with ThinPrep cervical cytology having an ASCUS interpretation and positive for high-risk HPV DNA were identified. A cohort of 514 (60.8%) patients with follow-up by repeat cytology, cervical biopsy or both was included in the study. Patient age was 12-81 years, with a median of 25 years. RESULTS: There were 291 women (56.6%) with negative status by cytology, HPV testing or biopsy with a median interval of 8.5 months, and an additional 174 patients (33.9%) had persistent ASCUS, positive HPV DNA or low-grade SIL/CIN 1. Finally, 49 patients (9.5%) had CIN 2 or 3, with a median interval of 8.5 months. CONCLUSION: Our study suggests that HSIL or CIN 2 or 3 will be detected in 1 in 10 women with HPV-positive index ASCUS cervical cytology at initial colposcopy or within a 2-year follow-up period.     

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.     

Significance of high-risk human papillomavirus DNA-positive atypical squamous cells of undetermined significance pap smears in perimenopausal and postmenopausal women

OBJECTIVE: To determine the risk of high-grade squamous intraepithelial lesion (HSIL)/cervical intraepithelial neoplasia (CIN) 2 or 3 in perimenopausal and postmenopausal women with high-risk human papillomavirus (HPV) DNA-positive atypical squamous cells of undetermined significance (ASCUS) on Pap tests. STUDY DESIGN: A total of 752 perimenopausal and postmenopausal women (patients 45 years) with an ASCUS interpretation on ThinPrep Pap tests were identified in 2002 and 2003. High risk HPVDNA was detected in 191 (25.4%) women. The follow-up results in these 136 women were compared with those of women <45 years. RESULTS: Of the 136 perimenopausal and postmenopausal women, 64.7% became negative on cytology or biopsy and 30.9% revealed persistent low-grade SIL (LSIL)/CIN 1. Furthermore, 5 women (3.68%) were diagnosed with HSIL/CIN 2 or 3, and 1 patient had squamous cell carcinoma (0.74%). The rate of HSIL/CIN 2 or 3 or worse detected in perimenopausal and postmenopausal women was significant lower than 10.6% in women <45 years (p = 0.028). CONCLUSION: Our study showed that perimenopausal and postmenopausal women with high-risk HPV positivity and an ASCUS interpretation have a significantly lower risk of HSIL/CIN 2 or 3 detected. Nonetheless, the risk of a significant finding on workup, including invasive carcinoma, indicates the need for colposcopic evaluation of these women.     

Do qualifiers of ASCUS distinguish between low- and high-risk patients?

OBJECTIVE: To evaluate the qualification of a Pap smear classified as atypical squamous cells of undetermined significance (ASCUS) favor reactive or neoplasia as recommended by the Bethesda System. STUDY DESIGN: The smears from 105 concurrent patients with a cytologic diagnosis of ASCUS not otherwise qualified were reviewed and subclassified as ASCUS favor reactive, low grade squamous intraepithelial lesion (LSIL) or high grade squamous intraepithelial lesion (HSIL) based on the Bethesda System criteria. The cervical biopsy diagnoses were correlated. RESULTS: Of the 105 cases classified as ASCUS, 37 were subclassified as favor reactive, 51 as favor LSIL and 17 as favor HSIL on cytologic review. In the ASCUS favor reactive group, 19 (51%) had reactive changes on biopsy, 17 (46%) had cervical intraepithelial neoplasia (CIN) 1, and 1 (2%) had CIN 3. A total of 48% patients had CIN. In the favor LSIL group, there was CIN 1 in 28 cases (55%), CIN 2 or 3 in 12 (23%) and benign changes in 11 (22%) on biopsy. Seventy-eight percent had CIN. In the 17 cases classified as ASCUS favor HSIL group, all had CIN. CONCLUSION: Of the total 105 cases of ASCUS, 71% had CIN, 29% had reactive changes on follow-up biopsies, and 48% of patients in the ASCUS favor reactive group had CIN. Qualifiers of ASCUS have questionable utility in patient management.     

Performance of an imaging system vs. manual screening in the detection of squamous intraepithelial lesions of the uterine cervix

OBJECTIVE: To compare the ThinPrep Imaging System (Cytyc Corp., Boxborough Massachusetts, U.S.A) to manual screening in the detection of cervical squamous epithelial lesion (SIL). STUDY DESIGN: A total of 27,525 manually screened ThinPrep Pap tests were compared with 27,725 imaged ThinPrep Pap tests for: (1) diagnostic rates of atypical squamous cells of undetermined significance; atypical squamous cells of undetermined significance, cannot rule out high grade SIL (ASC-H); low grade SIL and high grade SIL (HSIL); (2) ASC/SIL ratio; (3) high-risk HPV positivity for ASC; and (4) biopsy follow-up for ASC-H and HSIL. RESULTS: There were significant increases in the percentage of cytologic diagnoses in all categories with the imager. The ASC/SIL ratios of both groups were comparable. There was a significant decrease in HPV positivity in the imager group of ASC. Biopsy results confirmed a significant increase in the detection of HSIL in both the ASC-H and HSIL groups of the imaged cohort. CONCLUSION: The ThinPrep Imaging System is significantly better than manual screening in the detection of cervical SIL.     

Accuracy of liquid-based Pap tests: comparison of concurrent liquid-based tests and cervical biopsies on 782 women with previously abnormal Pap smears

OBJECTIVE: To evaluate the diagnostic performance of a liquid-based Pap test, the ThinPrep Pap test (TP) (Cytyc Corp., Boxborough, Massachusetts, U.S.A.), by comparing concurrent TP and cervical biopsy results on 782 patients who were referred for colposcopy because of previously abnormal conventional Pap smears (CPs). STUDY DESIGN: The ability of TP diagnoses of atypical cells of undetermined significance (ASC-US) and squamous intraepithelial lesions (SILs) to predict biopsy diagnoses of cervical intraepithelial neoplasia (CIN) was analyzed using chi2 and McNemar tests. RESULTS: The rate of agreement between diagnoses of SIL by TP and CIN by biopsy was 74.7%. ASC-US accounted for 16.0% of TP diagnoses. ASC-US had biopsy diagnoses of CIN 1 in 60% and CIN 2/3 in 12.8% of cases. For TP diagnosis of low grade SIL, biopsy diagnoses of CIN 2/3 were found in 13.5% of cases. For TP diagnoses of ASC-US and higher, the proportions of TP and cervical biopsies in comparable diagnostic categories were statistically significant (p < 0.001), with TP having sensitivity of 89.4% and positive predictive value of 89.7% for the detection of CIN. The false positive rate for TP was 8.1%, but rescreening confirmed the presence of abnormal cells in 51 of 63 (81.0%) cases of ASC-US or higher having negative biopsies. TP had a false negative rate of 8.3% and negative predictive value of 61.3%. Rescreening showed that most (77.6%) of the false negative TP specimens failed to have abnormal cells on the slides. CONCLUSION: For patients having previously detected cervical abnormalities by CP, concurrent TP demonstrated the following: (1) that it has high diagnostic accuracy for SIL, (2) that ASC-US was diagnostically equivalent to LSIL, and (3) that false negative TP for SIL can be attributed primarily to sampling rather than cytotechnologists' screening errors.     

P16(INK4a) immunocytochemistry in liquid-based cytology samples in equivocal Pap smears: added value in management of women with equivocal Pap smear

OBJECTIVE: To test whether p1l6(INK4a) immunocytochemistry (ICC) in liquid-based cytology (LBC) is useful with colposcopy in abnormal Pap smears. STUDY DESIGN: A series of 248 women with abnormal Pap smear were analyzed for oncogenic (HR) human papillomavirus (HPV) types using the Hybrid Capture II assay and for p16(INK4a) expression using ICC on cervical samples in PreservCyt liquid media. Colposcopic and loop electrosurgical excision procedure (LEEP) cone biopsy were the gold standard. RESULTS: p16(INK4a) ICC did best as predictor of high-grade squamous intraepithelial lesion, with OR 12.18 (2.72-54.57) (p = 0.0001), showing 88.2% sensitivity (SE), 61.9% specificity (SP), 14.6% positive predictive value (PPV) and 98.6% negative predictive value (NPV). In sorting discrepant cases, p16(INK4a) ICC results in 100% SE and 100% NPV in detecting cervical intraepithelial neoplasia (CIN) 2 lesions among Pap+/biopsy- women. In atypical squamous cells undetermined significance (ASCUS) cytology, adding p16(INK4a) ICC improves specificity of colposcopy from 27.3% to 81.8% and PPV from 42.8% to 71.4%. Best performance is obtained with p16(INK4a) ICC and colposcopy: 83.3% SE, 81.8% SP, 71.4% PPV and 90.0% NPV. CONCLUSION p16(INK4a) is useful in sorting clinically relevant discrepant cases, and p16(INK4a) ICC significantly improves SP and PPV of colposcopy in management of ASCUS cytology.     

An evaluation of p16INK4a expression in cervical intraepithelial neoplasia specimens, including women with HIV-1

Although several studies have evaluated the role of p16INK4a as a diagnostic marker of cervical intraepithelial neoplasia (CIN) and its association with disease progression, studies regarding the role of p16INK4a in human immunodeficiency virus (HIV)-infected patients remain scarce. The present study was designed to determine the potential utility of p16INK4a as a diagnostic marker for CIN and invasive cervical cancer in HIV-positive and negative cervical specimens. An immunohistochemical analysis of p16INK4a was performed in 326 cervical tissue microarray specimens. Performance indicators were calculated and compared using receiving operating characteristics curve (ROC)/area under the curve. In HIV-1-negative women, the percentage of cells that was positive for p16INK4a expression was significantly correlated with the severity of CIN (p < 0.0001). A ROC curve with a cut-off value of 55.28% resulted in a sensitivity of 89%, a specificity of 81%, a positive predictive value of 91% and a negative predictive value of 78%. HIV-seropositive women exhibited decreased expression of p16INK4a in CIN2-3 specimens compared with HIV-negative specimens (p = 0.031). The ROC data underscore the potential utility of p16INK4a under defined conditions as a diagnostic marker for CIN 2-3 staging and invasive cervical cancer. HIV-1 infection, however, is associated with relatively reduced p16INK4a expression in CIN 2-3.     

Comparison of manual and automated methods of liquid-based cytology. A morphologic study

OBJECTIVE: To evaluate the morphologic characteristics of gynecologic samples prepared by 3 different methods of liquid-based cytology. STUDY DESIGN: Cytologic samples from representative cases of each diagnostic category of squamous epithelial lesion, prepared by automated and manual liquid-based systems, were analyzed by 3 laboratories in the United States, Portugal and Brazil. The systems included: ThinPrep (automated, U.S. Food and Drug Administration approved; Cytyc Corp., Boxborough, Massachusetts, U.S.A.), Autocyte PREP (South American system, manual; TriPath Imaging, Inc., Burlington, North Carolina, U.S.A.) and DNACITOLIQ (manual; Digene Brazil, S?o Paulo, Brazil). A panel of 16 morphologic parameters was evaluated: cellularity, clean background, uniform distribution, artifacts, cellular overlapping, architectural and cytoplasmic distortion, cytoplasmic vacuolization, cellular elongation, imprecise cytoplasmic borders, folded cytoplasmic borders, nuclear hyperchromasia, coarse chromatin, prominent nucleolus, irregular nuclear borders, atypical mitosis and inflammatory infiltrate. Negative, atypical squamous cells of undetermined significance, low grade squamous intraepithelial lesion (LSIL) and high grade squamous intraepithelial lesion (HSIL) cases were included. Cases without biopsies were confirmed by consensus. RESULTS: Cellularity was adequate in all samples. Clean background was observed in the vast majority of samples with all liquid-based systems. Uniform distribution was frequently found in ThinPrep and Autocyte PREP samples but not in DNACITOLIQ. Artifacts were not present in DNACITOLIQ samples, rare in ThinPrep and observed in 8 (34.7%) Autocyte PREP. Cellular overlapping was observed in all 3 system samples: 11 (31.42%) cases in ThinPrep, 16 (69.56%) in Autocyte PREP and 17 (68%) in DNACITOLIQ System. Architectural and cytoplasmic distortion were present in 3 cases of HSIL (13%) and cytoplasmic vacuolization in 2 cases of LSIL and 1 HSIL of Autocyte PREP. Cellular elongation was found in 13 (56.5%) Autocyte PREP and in 5 (20%) DNACITOLIQ samples. Inflammatory infiltrate was found in all 3 system samples but with more frequency in the Autocyte PREP (69.56%) and DNACITOLIQ System (72%). CONCLUSION: This study clearly indicated that in spite of the different methodologies, the 3 methods adequately preserved cellular structure for morphologic evaluation. The choice of method will depend on price, availability and procedures involved.     

Atypical glandular cells of undetermined significance. Cytologic predictive value for glandular involvement in high grade squamous intraepithelial lesions

OBJECTIVE: To verify the cytologic predictive value of a diagnosis of atypical glandular cells of undetermined significance (AGUS) in high grade squamous intraepithelial lesions (HSIL) cases. STUDY DESIGN: In a retrospective study, 98 cases of HSIL were reviewed. All patients were referred for colposcopy and directed biopsy to confirm the cytologic diagnoses. Loop excision of the transformation zone was performed to treat clinical lesions. Sensitivity, specificity, positive and negative predictive value were evaluated. Kappa statistics and logistic regression analysis were used to evaluate the findings statistically. RESULTS: By logistic regression analysis, we found that the chance of finding squamous intraepithelial lesions involving glands in AGUS smears was 5.32 times higher than in those with no AGUS. It was 5.74 times higher in cervical intraepithelial neoplasia (CIN) 3 lesions than in CIN 2. CONCLUSION: The cytologic predictive value for HSIL involving glands is statistically significant when specific and objective criteria are used for the AGUS diagnosis.     

P16INK4a point mutations and promoter hypermetylation in bronchial preneoplastic lesions

The aim of this study was to determine p16INK4a point mutations and promoter hypermethylation in tumour cells and bronchial preneoplastic lesions in 32 surgically resected lungs due to primary squamous cell carcinoma. P16 point mutations were detected in 1 (3%) and promoter hypermethylation in 12 (31%) of 32 squamous cell carcinomas. The status of p16 was further characterized in 38 premalignant lesions including squamous metaplasias without dysplasia, squamous metaplasias with mild, moderate and severe dysplasias and 4 carcinomas in situ. No p16 point mutations have been found in premalignant or CIS lesions. Methylation of p16 was detected in 1 of 8 (12.5%) cases of squamous metaplasias without dysplasia, in 1 of 10 (10%) cases of squamous metaplasias with mild dysplasia, in 1 of 9 (11%) cases of squamous metaplasia with moderate dysplasia and in 2 of 7 (28.5%) cases of severe dysplasias, as well as in 1 of 4 (25%) carcinomas in situ. This investigation indicates that P16INK4a supressor gene point mutations are rather late event and inactivation of this gene by promoter hypermethylation is early and likely critical in bronchial cancerogenesis.     

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.     

Atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion: cytohistologic correlation study with diagnostic pitfalls

OBJECTIVE: In the current study, we explore the diagnostic parameters and pitfalls in the follow-up of 123 cases of Pap smears diagnosed as high-grade atypical squamous cells (ASC-H) at our institution. STUDY DESIGN: A computer database search was performed from the archives of the Ottawa Hospital Cytopathology Service for cases diagnosed with ASC-H between January 2003 and July 2005. RESULTS: Follow-up of the 123 cases of ASC-H showed high grade squamous intraepithelial lesion (HSIL) in 73 patients (59.4%), low grade squamous intraepithelial lesion (LSIL) in 11 (8.9%), immature squamous metaplasia in 23 (18.7%), reactive squamous cell changes in 12 (9.8%), benign glandular lesions (endocervical atypia, degenerated glandular cells) in 2 (1.6%) and atrophy in 2 (1.6%). In our study, 83 patients were younger than 40 years (67.4%), with biopsy-proven HSIL found in 54 patients (65.1%). The remaining 40 patients (32.6%) were older than 40 years of age, and follow-up biopsies showed HSIL in 19 patients (47.5%). CONCLUSION: In our study, 59.4% of the cases that were diagnosed cytologically as ASC-H were found to have HSIL on subsequent biopsies. This correlation was stronger in patients below the age of 40 years (65.1% vs. 47.5%). The cytopathologic feature most strongly associated with HSIL was the presence of coarse nuclear chromatin (84%).     

Human papillomavirus testing with a liquid-based system: feasibility and comparison with reference diagnoses

OBJECTIVE: To validate the utilization of cervical specimens collected in the fixative liquid used in the CYTO-screen System (SEROA, Monaco) for oncogenic human papillomavirus (HPV) DNA detection by the Hybrid Capture II technique (HCII) (Digene, Gaithersburg, Maryland, U.S.A) by reference to cytologic and/or histologic results. STUDY DESIGN: A technical feasibility study was conducted on 3 modalities of sample preparation before HCII technique, 1 with a proteinase digestion, I with PBS washing and I using the Digene sample conversion kit recommended for ThinPrep medium preparation (Cytyc Corp., Boxborough, Massachusetts, U.S.A.). The stability of cells after storage at days 28, 60 and 90 was tested on 26 positive samples (13 with high initial indices and 13 with low initial indices). Results of HPV testing were compared to cytologic and histologic results on a sample of 98 smears already identified as high grade squamous intraepithelial lesion (HSIL) (48) or low grade squamous intraepithelial lesion (LSIL) (50). A retrospective analysis was then performed on 995 HPV tests perfornmed routinely in 2003 in terms of comparison with the corresponding cytologic and/or histologic results. RESULTS: The HCII technique after direct treatment by proteinase K appeared to be as effective as the Digene sample conversion kit. By using the first technique, all 26 positive cases remained positive at 60 days, but 4 of 13 (30%) with low indices became negative at 90 days. The sensitivity of HPV testing for detecting biopsy- proven cervical intraepithelial neoplasia (CIN) 2 or worse was 100% in the 50 LSIL and 98% in the 48 HSIL samples. In the retrospective study (n = 995), the cytologic diagnoses of atypical squamous cells of undetermined significance (ASC-US) (n=278), LSIL (n = 137) and HSIL (n = 28) were associated with a positive HPVtest in 44%, 75% and 96% of cases, respectively. On a subsample of 156 patients among 278 with a diagnosis of ASC- US, the sensitivity of HPV testingfor detecting CIN 2 or worse was 88%, specificity 57%, positive predictive value 10% and negative predictive value 99%. Performing HPV testing by the HCII technique for cervical specimens collected in the fixative liquid used in the CYTO-screen System is feasible in the context of an ASC-US cytologic diagnosis.     

Benign cellular changes in Pap smears. Causes and significance

OBJECTIVE: We reviewed consecutive cases classified as benign cellular changes (BCC) over a four-month period. STUDY DESIGN: Cases classified as BCC were retrieved from the cytology files. A search was carried out to identify the previous Pap smears and concomitant cervical biopsies. RESULTS: One thousand one hundred three cases (23% of our gynecologic smears) were classified as BCC. Ninety-two patients (8.3%) underwent concurrent cervical biopsies. Specific infections accounted for 8% of BCC cases; reactive changes accounted for 92%. Of the biopsy specimens, 8.3% had no significant pathologic change. The most common biopsy diagnoses were cervicitis (31.5%), immature squamous metaplasia (16.3%) and reserve cell hyperplasia (10.8%). Miscellaneous benign diagnoses accounted for 21.7%. Cervical intraepithelial neoplasia (CIN) 1/human papillomavirus (HPV) was present in 14% of cases. All patients with biopsy diagnoses of CIN 1 had at least two previous abnormal Pap smears. Previous biopsy reports were available for review in 127 (12%) of the 1,103 patients. Of these 127 cases, 53.5% had a previous diagnosis of CIN/HPV; 9.4% had invasive carcinoma. A benign diagnosis was reported in 36.5%. CONCLUSION: The majority of BCC cases are due to reactive and inflammatory processes. In patients with a previous history of CIN, BCC may be of some significance. In patients with no significant prior cervical abnormalities, a Pap smear classified as BCC represents a reactive process.     

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.