CINtec® PLUS dual immunostain: A triage tool for cervical pap smears with atypical squamous cells of undetermined significance and low grade squamous intraepithelial lesion

ASC and LSIL comprise the majority of abnormal Pap smears. Currently, high‐risk human papillomavirus testing is utilized to triage women with ASC for colposcopy; however, no cost effective triage method is available for LSIL. p16 and Ki‐67 have each been shown to be good biomarkers for high grade cervical intraepithelial neoplasia (HG CIN).We evaluated the role of the CINtec® PLUS p16/Ki‐67 dual immunostain as a marker for underlying (U) or subsequent (S) HG CIN. One hundred and eighty eight cervical SurePath Pap smears with histological and/or cytological follow‐up were retrieved from our departmental files. The Pap stained slides were destained and then immunostained utilizing the CINtec® PLUS dual staining reagent kit. Results of the dual stain were correlated with follow‐up diagnoses. Sensitivity, specificity, and positive and negative predictive values of CINtec® PLUS for U or S HG CIN were compared with those of HR HPV testing and with p16 and Ki‐67 immunostaining alone. The sensitivity of CINtec® PLUS for U or S HG CIN was 91% in the ASC group and 100% in the LSIL group, while the corresponding specificities were 61 and 43%, respectively. The sensitivity and specificity of CINtec® PLUS for U or S HG CIN in both groups combined were 97 and 53%, respectively. CINtec® PLUS was more specific than HR HPV testing and Ki‐67 and p16 immunostains alone in detecting an U or S HG CIN. CINtec® PLUS is a helpful adjunct in identifying U or S HG CIN when applied to SurePath Pap smears with ASC or LSIL. Diagn. Cytopathol. 2013. © 2012 Wiley Periodicals, Inc.     

The use of p16INK4A immunocytochemistry in “Atypical squamous cells which cannot exclude HSIL” compared with “Atypical squamous cells of undetermined significance” in liquid‐based cervical smears

Even though p16^INK4a (p16) immunocytochemistry has proven a useful accessory tool verifying the identification of atypical squamous cells of undetermined significance (ASC‐US) categorized smears, the procedure still has limitations. To date few studies examining the usefulness of p16 immunocytochemistry in atypical squamous cells which cannot exclude HSIL (ASC‐H), compared with ASC‐US in liquid‐based cervical smears. Therefore, we examined the correlation of p16 immunocytochemical staining with follow‐up biopsy results on ASC‐H categorized smears and compared the data with those classified as ASC‐US on 105 liquid‐based cytology samples. We found no statistical significance in the p16 expression of ASC‐US smears and the presence of squamous intraepithelial lesions (SIL) in follow‐up biopsies (p = 0.546). However, p16 expression did significantly correlate with the presence of SIL (p = 0.002) in ASC‐H smears. There was a statistically significant relationship between p16 expression and presence of high grade squamous intraepithelial lesions (HSIL) or more on the follow‐up biopsies in both ASC‐US (p = 0.012) and ASC‐H (p < 0.001) categorized smears. In ASC‐US categorized smears, there was no statistical significance between p16 expression and the HR‐HPV viral load (p = 0.091). But there was a statistical significance between p16 expression and the HR‐HPV viral load (p < 0.001) in ASC‐H categorized smears. Our results indicate that p16 immunostaining is a much better useful marker for HR‐HPV infection and detection of SIL in ASC‐H categorized smears compared to those defined as ASC‐US. Diagn. Cytopathol. 2010. © 2009 Wiley‐Liss, Inc.     

Comparison of the clinical significance of the Papanicolaou test interpretations LSIL cannot rule out HSIL and ASC‐H

Despite the two‐tiered classification of dysplasia in The Bethesda System (TBS), rare cases fall into the category squamous intraepithelial lesion (SIL) of indeterminate grade. These Pap tests are often interpreted as “LSIL/ASC‐H” or “LSIL” with a comment indicating the presence of cells with features approaching HSIL. Patients with LSIL/ASC‐H have a significant risk of CIN 2 or worse (29–61.5%) on follow‐up cervical biopsies, similar to the risk of CIN 2 or worse in patients with ASC‐H Pap tests (24–68%). The purpose of this study was to compare patients with ASC‐H and LSIL/ASC‐H Pap tests. Women with LSIL/ASC‐H had a slightly lower incidence of CIN 2 or worse (PPV = 35.6%, 95% CI: 29.8–41.4%) on follow‐up cervical biopsy than the control ASC‐H group (PPV = 40.2%, 95% CI: 31.9–56.3%); this difference was not statistically significant. The difference in the distribution of the biopsy results between the two groups was statistically significant (P < 0.001). The current guidelines for the management of cervical cytologic abnormalities from the American Society for Colposcopy and Cervical Pathology (ASCCP) advocate similar treatment algorithms for both LSIL and ASC‐H. The main difference is the option of cytologic follow‐up or HPV testing for certain “special populations,” as an alternative to colposcopy, for LSIL Pap test results. Based on our results, we recommend (1)LSIL/ASC‐H to be added to TBS classification and (2) Pap test cases of LSIL/ASC‐H may need to be clinically followed in a manner similar to ASC‐H, i.e., colposcopy for all patients. Diagn. Cytopathol. 2010. © 2009 Wiley‐Liss, Inc.     

Triage of cervical cytological diagnoses of atypical squamous cells by DNA methylation of paired boxed gene 1 (PAX1)

Detection of cervical high‐grade squamous intraepithelial lesions (HSIL) in patients with equivocal cytological abnormalities, such as atypical squamous cells (ASC) of undetermined significance (ASCUS) or inability to exclude high‐grade squamous intraepithelial lesions (ASC‐H) is still a challenge. This study tested the efficacy of PAX1 methylation analysis in the triage of cervical ASCUS and ASC‐H and compared its performance with Hybrid Capture 2 (HC2) HPV test. A hospital‐based case–control study was conducted. Cervical scrapings from patients with ASCUS or ASC‐H were used for the quantitative methylation analysis of PAX1 methylation by MethyLight and HPV testing by HC2. Patients with ASC‐H or ASCUS with repeated abnormal smears underwent colposcopic biopsy and subsequent therapies. Diagnoses were made by histopathology at a follow‐up of 2 years. The efficacies of detecting high‐grade lesions were compared. Fifty‐eight cervical scrapings with cytological diagnosis of ASCUS (n = 41) and ASC‐H (n = 17) were analyzed. One of the 41 (2.4%) ASCUS patients and seven of 17 (41.2%) ASC‐H patients were confirmed to have HSIL. After dichotomy of the PMR, PAX1 methylation rates were significantly higher in ASC developing HSIL compared with those developing reactive atypia (87.5% vs. 12.5%, P < 0.001). Testing PAX1 methylation in cervical swabs of patients with ASC confers better sensitivity (87.5% vs. 62.5%) and specificity (98.0% vs. 86.0%) than HC2 HPV testing. We show for the first time that PAX1 hypermethylation analysis may be a better choice than HC2 in the triage of ASCUS and ASC‐H. Diagn. Cytopathol. 2013. © 2011 Wiley Periodicals, Inc.     

Clinical significance of HPV DNA cotesting in Korean women with ASCUS or ASC‐H

The purpose of this study was to evaluate the clinical significance of Human papillomavirus (HPV) DNA cotesting in Korean women with abnormal Papanicolaou (Pap) smear results based on colposcopic pathology. A total of 1012 women underwent liquid‐based Pap smears and hybrid capture II HPV DNA tests followed by colposcopy at the Korea University Hospital from January 2007 to May 2012. Of these women, 832 women were included in this retrospective study. The mean patient age was 45.4 ± 13.7 years (range:15–80). The distribution of Pap smear results was normal (4.7%), atypical squamous cells of uncertain significance (ASCUS) (42.1%), low‐grade squamous intraepithelial lesion (26.8%), ASC‐H (7.0%), and high‐grade squamous intraepithelial lesion (HSIL) (19.5%). In women with ASCUS, none of the 87 HPV‐negative had ≥cervical intraepithelial neoplasia (CIN2) (P < 0.001). In women with ASC‐H, only one out of 17 HPV‐negative vs. 14 out of 41 HPV‐positive had ≥CIN2 (P = 0.025). In patients with HSIL, 54.5% of HPV‐negative had ≥CIN2, as compared to 80.8% of HPV‐positive with ≥CIN2 (P = 0.039). Patients were further analyzed by age groups: <30 and ≥30 years. In HPV‐negative women, there was a significant difference in the ratio of ≥CIN2 (30.8% <30 vs. 4.5% ≥30, P = 0.005). When the HPV DNA test was negative in women ≥30, the risk of ≥CIN2 was significantly lower (P < 0.001). HPV DNA cotesting in women with ASCUS and ASC‐H furnish healthcare providers with informative data. There is a lower proportion of ≥CIN2 in HPV‐negative women and a higher proportion of ≥CIN2 in HPV‐positive. When HPV data were further evaluated by age group, the risk of ≥CIN2 was lower in HPV‐negative women, especially in women ≥30. Diagn. Cytopathol. 2014;42:1058–1062. © 2014 Wiley Periodicals, Inc.     

Prevalence of human papillomavirus genotypes in cytologic abnormalities from unvaccinated women living in north-western Spain

Cervical cancer and its precursors low-grade squamous intraepithelial lesions (LSIL) and high-grade squamous intraepithelial lesions (HSIL) are associated with infection by human papillomavirus (HPV), in particular HPV 16 and 18. The distribution of the HPV genotype varies with the severity of cervical disease, age and the geographic location of the patients. We report the results of a population study carried out in a region of north-western (NW) Spain aimed at determining the prevalence of single and multiple infections by 35 types of HPV using low-density microarrays for 113 cases with negative for intraepithelial lesions or malignancies; 588 with atypical squamous cells of undetermined significance (ASCUS)/LSIL; 183 with HSIL; and seven cases of squamous cell carcinomas. Of the 891 patients analysed, 50.2% had single infections and 49.8% had multiple HPV infections. In women aged below 30 years, there was a predominance of multiple infections (p = 0.027). ASCUS/LSIL was associated with multiple and HSIL with single infections (p = 0.025). We observed significant increases in the percentage of infections due to a high-risk (HR) type of HPV when the severity of the cytological lesion increased (p = 0.001). No relationship was found between greater aggressiveness in the cytological diagnosis and a higher number of HPV types involved in multiple infections. The five most frequent genotypes were HPV 16 (26.3%), 53 (18.2%), 51 (17.3%), 6 (14.8%) and 66 (13.1%). The prevalence of HPV 16, 33 and 58 increased significantly from ACUS/LSIL to HSIL and the prevalence of HPV 51, 53 and 66 decreased. HPV 16 was the only genotype that showed a significant increase in prevalence when the severity of the cytological disease increased in single infections (p = 0.0001). The implementation of bivalent prophylactic vaccination could potentially lead to prevention in 32% of the population included in the study - in at least a quarter of patients with ACUS/LSIL (26.7%), and in half of HSIL (50.2%).     

Impact of ASC‐H terminology on the detection of HSILs in medically underserved California women

In this study we evaluated CDP: EWC, a large public health screening program for low‐income women to determine whether the ASC‐H term, introduced in the 2001 revision to the Bethesda System, has facilitated the detection of the most serious precancerous cervical lesions such as CIN‐II–III/CIS, including accuracy of detection and minimizing diagnostic delays. Pap test and biopsy data from the period 2003–2006 were compared with those from 1995–1999, and included analysis of a subset of rarely and never‐screened for each study period. More ASC‐H Pap tests were followed by CIN‐II+ biopsies (20%) than ASCUS Pap tests (5%). Thirteen percent of ASC‐H showed CIN‐III/CIS on follow‐up biopsy. Following the introduction of ASC‐H, negative and LSIL Pap tests followed by CIN‐III/CIS biopsies decreased from 5.6% to 0.37% and 9.2% to 4.4%, respectively. Fewer CIN‐III/CIS cases were preceded by Pap tests with negative and ASCUS results in 2003–2005 as compared with 1995–1999. The number of days from screening to diagnosis for women aged 25–39 years with ASC‐H Pap test results (58 days) was less than for ASCUS results (78 days) for the current study period. In this high‐risk multi‐ethnic population, ASC‐H more effectively communicates equivocal findings likely to represent HSIL leading to more rapid follow‐up. The incidence of CIN‐II or CIN‐III/CIS following ASC‐H Pap tests is lower than generally appreciated for this population, including for rarely or never‐screened high‐risk women. Fewer CIN‐III/CIS lesions were detected following negative and ASCUS Pap results. Compliance with follow‐up is less than ideal for this population. Diagn. Cytopathol. 2009. © 2008 Wiley‐Liss, Inc.     

The role of human papillomavirus in cyto-histological practice: distribution and prevalenceof hig-risk strains (16, 18, 31, 33, and 35) in intraepithelial lesions and neoplasia of the uterine cervix

INTRODUCTION: Many studies have already shown the association of persistent infection of human high risk papillomavirus (HPV) with the development of pre-invasive and invasive cervical disease. MATERIALS AND METHODS: We evaluated the use of high risk HPV testing in a study of about 1908 women, aged 29-78, who attending, from 1996 to 1998, the Sant'Anna Hospital in Turin for routine, second level smears and histopathological diagnosis. We considered all cervical lesions: ASCUS, LSIL, HSIL, squamous and adeno invasive cancers. HPV testing was performed by polymerase chain reaction (PCR) using L1 consensus primers which can detect almost all infections (high and low risk types). The most important high risk HPV types (16, 18, 31, 33 and 35) were tested using specific primers. RESULTS: The prevalence of high risk HPV was: ASCUS 42.2%, LSIL 39%, HSIL 73.5%, squamous invasive cancers 98.3% and adeno 100%. In addition HPV 16 is the most represented type in all lesions: ASCUS 40%, LSIL 62%, HSIL 71.2% squamous invasive cancers 73.3% and adeno 50.6%. In addition we study the mean age of cervical cancer onset compared with the different high risk HPV types. We found that HPV 18 related cancer occurs in younger women (mean age 41 years; range 39-42). CONCLUSIONS: The addition of high risk HPV testing to cytology may improve early identification of women at risk for cervical cancer.     

Accuracy of a low priced liquid‐based method for cervical cytology in 632 women referred for colposcopy after a positive Pap smear

The aim of this quality controlling study was to determine the accuracy of liquid‐based cytology (LBC) with the Turbitec® cytocentrifuge technique. Cervical smears of 632 women, who were referred to our CIN outpatient department, after at least two smears with ASCUS or higher were evaluated and compared with the histological outcome. In 592 cases the smears revealed abnormalities of squamous epithelium, and in 40 cases the abnormalities of glandular epithelium. In the group of squamous epithelium abnormalities, the sensitivity for LSIL was 39.7% and the specificity was 89.2%; for the LSIL+ group, these values were 89.4% and 91.4%, respectively. For HSIL the sensitivity was 68.3% and the specificity 92.8%, for the HSIL+ group 82.3% and 92.3%, respectively. The ASCUS rate was low (2.4%). The Turbitec cytocentrifuge method was proved to be a very good LBC method for cervical smears. Because of a comparable accuracy together with a lower price, this LBC method outweighs commercial alternatives. Diagn. Cytopathol. 2009. © 2009 Wiley‐Liss, Inc.     

The significance of endocervical cells and metaplastic squamous cells in liquid‐based cervical cytology

We conducted a retrospective study to investigate whether the presence or absence of endocervical cells (EC) and metaplastic squamous cells (MSC) was associated with the detection of squamous intraepithelial lesions in liquid‐based cervical cytology. 90,376 cases of liquid‐based cervical cytology smears received in 2006 were included in the study. Low‐grade (LSIL) and high‐grade squamous intraepithelial lesions (HSIL) were classified according to the Bethesda system (2001). The rates of detecting LSIL and HSIL in smears with and without EC and/or MSC were determined. There were 1,540 LSIL and 396 HSIL. The ratio of HSIL/NILM (no intraepithelial lesion or malignancy) was 0.0022 in smears without EC or MSC, 0.0040 in smears with EC only, 0.0044 in smears with MSC only, and 0.0056 in smears with both EC and MSC present. Compared with smears without EC or MSC, this ratio was significantly higher (P < 0.05) when either EC or MSC was present. Compared with smears with EC only, the ratio was also significantly higher when both EC and MSC were present (P < 0.05). On the other hand, the presence or absence of EC had no effect on the detection rate of LSIL (0.0191 for both groups), while the presence of MSC was actually associated with lower detection rate of LSIL (0.0153, P < 0.05). The presence of endocervical and metaplastic cells was associated with higher detection rates of HSIL. MSC was associated with lower detection or LSIL. Diagn. Cytopathol. 2009. © 2009 Wiley‐Liss, Inc.     

Evaluation of p16 immunostaining to predict high‐grade cervical intraepithelial neoplasia in women with Pap results of atypical squamous cells of undetermined significance

p16 immunostaining has been examined to detect high‐grade cervical intraepithelial neoplasia grade (CIN2+) in Pap cytology specimens. However, the utility of p16 in predicting CIN2+ in Pap specimens with atypical squamous cells of undetermined significance (ASC‐US) or atypical squamous cells, cannot exclude high‐grade squamous intraepithelial neoplasm (ASC‐H), is controversial. In this study, we evaluated the utility of p16 immunostaining for predicting CIN2+ in 78 Pap specimens with ASC‐US/ASC‐H and compared the results in high‐risk HPV DNA and the follow‐up biopsies. p16 immunostaining was positive in 47% (37/78) of the Pap specimens. Of the 13 Pap specimens with follow‐up biopsy results of CIN2+, 7 (54%) were positive for p16. p16 positivity in the Pap specimens was not significantly associated with a CIN2+ biopsy result (P = 0.76). The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of p16 immunostaining for predicting CIN2+ were 54%, 52%, 19%, and 85%, respectively. High‐risk HPV DNA was detected in 40% (31/78) of the Pap specimens. The sensitivity, specificity, PPV, and NPV of HPV DNA for predicting CIN2+ were 100%, 72%, 42%, and 100%, respectively. High‐risk HPV genotypes were detected in six p16‐negative specimens with follow‐up biopsy results of CIN2+. Our findings suggest that the utility of p16 immunostaining for predicting CIN2+ in Pap specimens with ASC‐US/ASC‐H is limited. Scant abnormal cells in Pap specimens with ASC‐US/ASC‐H may have contributed to the low p16 sensitivity. Diagn. Cytopathol., 2011. © 2010 Wiley‐Liss, Inc.     

Detection and typing of human papillomavirus DNA by PCR using consensus primers in various cervical lesions of Korean women

The association between cervical cancers and human papillomavirus (HPV) is now well established. To estimate the extent of infection with common HPVs among Korean women, we have examined 224 cervical scrapes of various cervical lesions. Detection and typing of HPVs were done by polymerase chain reaction (PCR) using consensus primers followed by restriction enzyme digestion and PCR using type-specific primers. The prevalence of total HPV infection in patients with cervical intraepithelial neoplasia (CIN) and cervical cancer were significantly higher than those in healthy women and patients with atypical squamous cells of undetermined significance (ASCUS). HPV typing in 41 invasive carcinomas of the cervix revealed the prevalence of HPV 16 in 15 cases, followed by HPV 58, 18, 33, 31, 52 and 35. The distribution pattern of HPV types in CIN were not much different from carcinomas. HPV types except HPV 18 had a tendency to show higher prevalence in high-grade squamous intraepithelial lesion (HSIL) than low-grade squamous intraepithelial lesions (LSIL), however, HPV 18 was detected in LSIL but not in HSIL. HPV 18 tended to have the worse clinical stage, although it was not statistically significant. These findings suggest the importance of HPV typing other than HPV 16 and 18 and a different clinicopathologic significance of HPV 18.     

p16(INK4A) is a surrogate biomarker for a subset of human papilloma virus-associated dysplasias of the uterine cervix as determined on the Pap smear

Recently, p16(INK4A) has been identified as a biomarker for human papilloma virus (HPV)-induced dysplastic lesions of the cervix and it has been suggested that it may be a useful diagnostic aid for these lesions. This study therefore was performed to determine the utility of p16 expression in a series of Papanicolaou (Pap) smears collected in liquid medium and to determine its benefit, if any, over HPV testing. One hundred seven cases, including 23 negative cases, 34 with low-grade squamous intraepithelial lesion (LSIL), 16 with high-grade squamous intraepithelial lesion (HSIL), 29 with atypical squamous cells of uncertain significance (ASC-US), and 5 cases with ASC suspicious for HSIL (ASC-H), were evaluated for both p16 expression and HPV DNA. We observed p16 expression in only 36% of all cases with abnormal cytology (30/84) and in 40% of all cases associated with high-risk HPV. The highest rate of positivity (80%) and the highest levels of expression (more than three to five positive cells/10x field) were seen in HSIL. Similar results were observed with ASC-H cases. This suggests that in equivocal cases, p16 may be used for confirmation of the diagnosis. On the other hand, p16 positivity was noted in only 21% of LSIL and ASC-US cases. This raises the interesting possibility, given that only a minority of LSIL cases progress on to higher-grade lesions, that p16 might be useful for triaging these patients for closer follow-up and/or further evaluation. Additional studies are required for confirmation.     

Management of atypical squamous cells of undetermined significance or low‐grade squamous intraepithelial lesions of the uterine cervix with human papilloma virus infection among young women aged less than 25 years

Current ASCCP guidelines recommend repeat cytology 12 months after HPV‐positive results in women aged 21–24 years with either atypical squamous cells of undetermined significance (ASCUS) or a low‐grade squamous intraepithelial lesion (LSIL). The purpose of this study was to validate an algorithm in such women with ASCUS or LSIL. A multicenter cross‐sectional study was carried out at three academic hospitals involving 40,847 Korean women who underwent cervical cancer screening with cytology and HPV testing with or without subsequent colposcopic biopsies between January 2007 and December 2013. Among a total of 3,193 women with available histopathology data, 762 women with ASCUS and 758 with LSIL were HPV‐positive. Among HPV‐positive women with ASCUS, 38.5% of women aged 21–24 years had ≥CIN2, compared to 20.8% of women aged 30–65 years and 21.1% of the total women. Among HPV‐positive women with LSIL, 25.8% aged 21–24 years had ≥CIN2, compared to 21.2% of women aged 30–65 years and 21.9% of the total women. In HPV‐positive women with ASCUS/LSIL aged less than 25 years, the prevalence of ≥CIN2 lesions was 34.5%, which was significantly higher than that (21.0%) in women aged ≥25 years. The risk of ≥CIN2 lesions in HPV‐positive Korean women aged 21–24 years with ASCUS or LSIL was not lower than that in older women. Colposcopic examination should be considered for management of HPV‐positive young women with ASCUS or LSIL. Diagn. Cytopathol. 2016;44:959–963. © 2016 Wiley Periodicals, Inc.     

Detection of HPV16 and 18 by in situ hybridization in precancerous and cancerous lesions of cervix

Fifty cervical biopsies from women with preinvasive and invasive malignancies of uterine cervix and ten normal cervical biopsies were examined for the presence of human papilloma virus (HPV) 16 and 18 DNA sequences by in situ hybridization (ISH) method with biotinylated DNA probes. The overall positivity of HPV DNA was 48% (24/50). The positivity of HPV 16 DNA for low grade squamous intraepithelial lesion (LSIL), high grade squamous intraepithelial lesions (HSIL) and squamous cell carcinoma (SCC) were 33.33%, 45.45%, 42.30% respectively. The positivity for HPV 18 DNA for LSIL, HSIL and SCC were 0%, 18.18%, 30.76% respectively. Two cases of cervical adenocarcinomas showed positivity for HPV 18 DNA only.     

“Immunocytochemical expression of P53, PTEN,FAS (CD95), P16INK4A and HPV L1 major capsid proteins in ThinPrep cervical samples with squamous intraepithelial lesions”

The aim of this study was to further investigate the immunocytochemical expression of p53, PTEN, Fas, p16, and HPV L1 capsid proteins in cervical smears with low and high grade squamous intraepithelial lesions (LSIL and HSIL, respectively). A total of 92 ThinPrep cervical samples, comprising 11 cases of HSIL, 61 cases of LSIL, and 20 negative cases were studied by immunocytochemical methods. The results obtained in LSIL cases were correlated with the available follow up data. Abnormal p53, PTEN, or Fas expression was found in a subset of HSIL cases, while positive expression for p16 was significantly associated with the diagnosis of HSIL (P < 0.0001, P = 0.001, P < 0.0001, and P < 0.0001, respectively). Among cases positive for p16 expression, the staining pattern was weak in 88.9% of LSIL cases and strong in 80% of HSIL cases (P < 0.0001). The p16 negative/L1 positive and p16 positive/L1 negative staining patterns were significantly associated with the presence of LSIL and HSIL, respectively (P < 0.0001). None of these markers had a significant prognostic value in LSIL cases (P > 0.05). Our results suggest that loss of PTEN or Fas expression and p53 overexpression may be involved in the process of neoplastic transformation of the cervical epithelium. Furthermore, negative or weak immunocytochemical staining for p16 in a Pap smear may strongly argue against the presence of a high grade lesion, while the combined p16/L1 staining pattern may be useful as a diagnostic adjunct for differentiating between LSIL and HSIL. Diagn. Cytopathol. 2014;42:465–475. © 2013 Wiley Periodicals, Inc.     

Ki-67 immunocytochemistry in liquid based cervical cytology: useful as an adjunctive tool?

AIMS: To test the ability of Ki-67 to detect cytological lesions in a screening setting and its use as a surrogate marker of human papillomavirus (HPV) infection. METHODS: A study of liquid based cytology, HPV DNA testing by MY09/MY11 consensus polymerase chain reaction (PCR), type specific PCRs, and Ki-67 immunocytochemistry on a randomly selected series of 147 patients. RESULTS: Comparison of the number of Ki-67 immunoreactive cells/1000 cells in the different cytological groups showed that the HSIL group yielded a significantly higher mean count than did the other groups. The number of Ki-67 immunoreactive cells/1000 cells was significantly higher in HPV-16 positive samples than in samples containing infections with other high risk types. Receiver operating characteristic curves indicated a test accuracy (area under curve) of 0.68, 0.72, and 0.86 for atypical squamous cells of undetermined significance (ASCUS), low grade squamous intraepithelial lesions (LSIL), and high grade squamous intraepithelial lesions (HSIL), respectively. Thresholds for 95% sensitivity were 0.07, 0.08, and 0.15 Ki-67 immunopositive cells/1000 cells for ASCUS, LSIL and HSIL, respectively. The threshold for 95% specificity was 1.9 Ki-67 immunopositive cells/1000 cells. CONCLUSIONS: Ki-67 immunocytochemistry can be applied to liquid based cytology. The accuracy and diagnostic indices of the test are good when compared with those of other techniques. As part of a panel of screening procedures, it could be used as an adjunct to liquid based cytology to identify HSIL, and as a surrogate marker of HPV-16 infection.     

An evaluation of the diagnostic and prognostic significance of p16INK4a/p21WAF1/Cip1 immunostaining in squamous intraepithelial lesions of the uterine cervix using liquid‐based cytology specimens

Human papillomavirus (HPV) infection frequently causes squamous intraepithelial lesions (SIL) of the uterine cervix and consequently gives rise to squamous cell carcinoma. It is therefore important to identify cases that potentially develop higher grades of SIL at an early stage of the disease. In this study, we thus investigated whether immunocytochemistry for p21^WAF1/Cip1 and p16^INK4a could be applicable in the diagnosis and the prognostic prediction of SIL in combination with genomic analyses of HPV. The genomic analysis of high‐risk HPV (hrHPV), which was done by reversed dot blotting and by in situ hybridization, and immunocytochemistry were performed on liquid‐based cytological specimens. A cross‐sectional study comprising 145 cases of NILM, ASC‐US, LSIL, and HSIL indicated that the incidence of the positive cases for p16^INK4a and p21^WAF1/Cip1 and hrHPV increased with the grade of SIL. A double positive status for p16^INK4a and p21^WAF1/Cip1 was a significant discriminator between HSIL and LSIL/NILM, even when applied in conjunction with the genomic test for hrHPV (P = 0.006 by logistic regression analysis). However, a prospective study employing 61 NILM/ASC‐US cases, revealed that the p16^INK4a/p21^WAF1/Cip1 immunostaining was not a significant predictor for the progression of SIL, whereas the cytological diagnosis (NILM vs. ASC‐US) and the infection status of hrHPV conferred significant effects on the prognosis. Immunostaining of p16^INK4a and p21^WAF1/Cip1 provides additional information on the cytological diagnosis of SIL. A further analysis using a larger population is warranted to obtain a conclusive result regarding the prognostic significance of p16^INK4a/p21^WAF1/Cip1 immunocytochemistry in the diagnosis of SIL. Diagn. Cytopathol. 2014;42: 125–133. © 2013 Wiley Periodicals, Inc.     

Overexpression of p16INK4A in liquid-based specimens (SurePath) as marker of cervical dysplasia and neoplasia

Human papillomavirus (HPV) is recognized as a causal agent for cervical carcinomas. Assimilation of HPV oncogenes E6 and E7 into the host DNA promotes upregulation of cyclin dependent kinase inhibitor (CDKI) p16(INK4A), detectable by monoclonal antibody in the developing cervical cancer cells. The aim of this study was to 1) develop a protocol for p16(INK4A) immunocytochemical staining on SurePath preparations, and 2) determine its utility as an HPV marker on a spectrum of cervical reactive and neoplastic lesions. Seventy-two specimens consisting of 28 nonneoplastic/nondysplastic cases (NN), one reactive glandular cells (RGC), 27 low-grade squamous intraepithelial lesions (LSIL), 10 high-grade squamous intraepithelial lesions (HSIL), one squamous cell carcinoma (SCCA), four atypical glandular cells (AGUS), and two adenocarcinomas (ADCA) were reprepped by SurePath and antibody to p16(INK4A) applied at 1:100 dilution using the Dako Envision + System on the Dako Autostainer. Expression of p16(INK4A) within the nucleus principally and cytoplasm of at least 10-15 cells was considered positive. All initial Papanicolaou-stained discrepant cases (p16(INK4A) positivity of NN and RGC cases and lack of reactivity in LSIL, HSIL, and AGUS) were reviewed. Nine of ten (90%) HSIL, one (100%) SCCA, 21/27 (78%) LSIL, and some reactive and inflammatory specimens demonstrated the presence of p16(INK4A). Reevaluation of discrepant cases revealed that several were underinterpreted (four NN were LSIL, one RGC was AGUS) or overinterpreted (one LSIL was NN). Following reassessment, false-positive staining was present in only 1/25 (1.4%) NN. Six of 30 (20%) LSIL lacked p16(INK4A) positivity. One of 10 (10%) HSIL had no staining. Two of four AGUS did not react with p16(INK4A) antibody. Both SCCA (1) and ADCA (2) had positive expression. This study confirms the intimate relationship between p16(INK4A) and HPV cytopathic effect. The p16(INK4A) immunocytochemical stain can be applied to liquid-based cervical preparations. This technique offers a more objective approach to deciphering "gray areas" of gynecologic cytopathology.     

Telomerase and human papillomavirus as diagnostic adjuncts for cervical dysplasia and carcinoma

Telomerase and human papillomavirus (HPV) DNA were evaluated as potential markers of high-grade dysplasia in cervical cytological specimens. Cytology specimens were collected from patients at the time of colposcopic evaluation for management of a previous abnormal cytology test result. Telomerase activity was evaluated by the telomeric repeat amplification protocol (TRAP), and HPV DNA was detected by polymerase chain reaction with L1 consensus-sequence primers and filter hybridization genotyping. Telomerase was detected in 8 of 97 (8.2%) cases with normal cytology or benign cellular changes, in 7 of 98 (7.1%) cases of atypical squamous cells of undetermined significance (ASCUS), in 3 of 95 (3.2%) cases of low-grade squamous intraepithelial lesion (LSIL), and in 17 of 48 (35.4%) cases with high-grade squamous intraepithelial lesion (HSIL). High-risk HPVs were detected in 23 of 97 (23.7%) cases with normal/reactive cellular changes (RCC) cytology, in 28 of 98 (28.6%) cases of ASCUS, in 69 of 95 (72.6%) cases of LSIL, and in 35 of 48 (72.9%) cases of HSIL. Telomerase expression did not correlate with the detection of high-risk HPVs in any cytological diagnostic categories. Telomerase and HPV test results of cytological specimens were correlated with the histological diagnoses of concurrent cervical biopsy specimens. Telomerase showed a sensitivity of 29.9% and a specificity of 94.0% for biopsy-confirmed cervical intraepithelial neoplasia (CIN) II/III. In contrast, high-risk HPVs were detected in 70.1% of cases with underlying CIN II/III, with a specificity of 62.5%. A relatively high proportion of normal/RCC or ASCUS cases with telomerase-positive test results had underlying high-grade dysplasia on cervical biopsy. Thus, technical and practical limitations of the TRAP assay in cervical cytology specimens limit the practical application of telomerase as a diagnostic adjunct in cervical cytopathology.