人乳头状瘤病毒不同型别与宫颈病变的相关性研究

目的探讨人乳头状瘤病毒（ＨＰＶ）不同型别与宫颈病变性质的关系。方法应用ＰＣＲ技术和原位杂交方法对６１例宫颈上皮内瘤（ＣｅｒｖｉｃａｌｉｎｔｒａｅｐｉｔｈｅｌｉａｌＮｅｏｐｌａｓｉａＣＩＮ）和１２例宫颈鳞癌（ＳＣＣ）进行ＨＰＶ６Ｂ／１１、１６、１８ＤＮＡ检测。结果ＰＣＲ检测结果显示ＨＰＶ６、１１主要分布于低度鳞状上皮内病变（６１９％）和一部分ＣＩＮⅡ中（２０％），而在ＣＩＮⅢ和ＳＣＣ中检测不到；ＨＰＶ１６、１８的检出率随ＣＩＮ级别增高而增加，在ＳＣＣ中高达８３３％。原位杂交结果显示在低度鳞状上皮内病变中，地高辛（Ｄｉｇ）标记的ＨＰＶ６Ｂ／１１、１６、１８ＤＮＡ杂交物质在核中均呈细颗粒状，为“游离型”。上述杂交阳性信号形态亦出现于ＣＩＮⅡ的所有ＨＰＶ６Ｂ／１１及部分ＨＰＶ１６、１８型感染中，而ＣＩＮⅢ和宫颈鳞癌及部分ＣＩＮⅡ中，其杂交阳性信号均为非颗粒状的“整合型”。结论低度鳞状上皮内病变是以ＨＰＶ６、１１低危型为主的多型别病毒的繁殖性感染，ＣＩＮⅢ和宫颈鳞癌为ＨＰＶ１６、１８高危型病毒的整合型感染，而在ＣＩＮⅡ中存在着ＨＰＶ６，１１和ＨＰＶ１６，１８的繁殖性感染及ＨＰＶ１６，１８的整合型感染     

Implications of continued upregulation of p16(INK4a) through the evolution from high-grade squamous intraepithelial lesion to invasive squamous carcinoma of the cervix

On integration into the host cervical keratinocyte genome, human papillomavirus (HPV) E7 protein binds pRB,releasing E2F from normally incompetent pRB-E2F complexes and allowing propagation of G1-S transition by the E2F. p16(INK4a), a tumour suppressor protein, increases in reflex response to counter this. 29 histologically re-confirmed low-grade squamous intraepithelial lesions (LSIL), 27 high-grade squamous intraepithelial lesions (HSIL) and 30 invasive cervical squamous carcinoma (SCC) were immunohistochemically stained for p16(INK4a) expression using the CINtec Histology Kit (REF 9511, mtm laboratories AG, Heidelberg, Germany) to re-affirm the notion that integration of HPV occurs predominantly in SCC and possibly HSIL and less in LSIL and normal squamous epithelium (NSqE). Implicit was also the attempt to understand the role of E2F, as indicated by p16(INK4a), in evolution of SCC from HSIL. No ethnic predilection was noted for LSIL, HSIL or SCC. Patients with SCC were significantly older by about 14-years compared with HSIL (p < 0.05) while there was no significant age difference between HSIL and LSIL. p16(INK4a) expression was significantly increased (p < 0.05) in both HSIL (88.9%) and SCC (83.3%) compared with LSIL (3.4%) and NSqE (0%); the NSqE being normal squamous epithelium noted in 17 of the LSIL, 19 HSIL and 5 SCC. From these findings there is suggestion that fundamental upstream events viz HPV integration, E7 upregulation followed by E2F activation occurs at point of transformation to HSIL and continues unrelentingly for another one to two decades before hitherto unclear factors convert a non-invasive lesion into an overtly invasive malignant counterpart. Interestingly, the occurrence of HSIL and LSIL in almost the same age group could mean that alteration from episomal to integrated form of HPV may not incur a prolonged incubation period, unlike from HSIL to SCC.     

The predictive value of p16(INK4a) and hybrid capture 2 human papillomavirus testing for high-grade cervical intraepithelial neoplasia

We performed p16(INK4a) immunocytochemical analysis and Hybrid Capture 2 (HC2; Digene, Gaithersburg, MD) high-risk HPV testing on 210 abnormal SurePath (TriPath Imaging, Burlington, NC) Papanicolaou specimens diagnosed as low-grade squamous intraepithelial lesion (LSIL) or high grade squamous intraepithelial lesion (HSIL). The results were compared with 121 follow-up biopsy specimens. p16(INK4a) was positive in 57.9% of women with LSIL compared with 97.1% of women with HSIL. In contrast, HC2 testing was positive in 85.0% of women with LSIL and 86.4% of women with HSIL. The differences in the positive rates for16(INK4a) between LSIL and HSIL was significant (P < .001), whereas, for HC2, it was not (P = .264). In patients who had cervical biopsies following a cytologic diagnosis of LSIL, the positive predictive value (PPV) of p16(INK4a) for a biopsy of cervical intraepithelial neoplasia grade 2 or 3 (CIN2/3; 33.3%) was significantly higher than the PPV of HC2 results (21.2%) (P < .001). Using liquid-based cytology specimens, p16(INK4a) immunocytochemical analysis has a higher PPV than reflex HC2 HPV testing for identifying CIN2/3 among patients with LSIL and might be useful for selecting patients with LSIL for colposcopy.     

p16INK4a在宫颈细胞学鳞状上皮内瘤变中的意义

目的探讨p16^INK4a的表达在宫颈细胞学中鳞状上皮内瘤变中的意义及其与人乳头状瘤病毒（HPV）型别之间的关系。方法对88例经活检证实的液基细胞学标本[包括20例慢性宫颈炎、18例低度鳞状上皮内瘤变（LSIL）、34例高度鳞状上皮内瘤变（HSIL）及16例鳞状上皮细胞癌（SCC）],分别用免疫细胞化学（EnVision方法）检测其p16^INK4a的表达,并对所有标本用聚合酶链反应（PCR）方法检测HPV型别（包括HPV16、18、31、33、35、39、45、51、52、53、56、58、59、66、68、6、11、42、43及44型）。结果p16^INK4a在慢性宫颈炎组呈阴性,在LSIL、HSIL及SCC组的表达率分别为27．8％、100％及100％,LSIL、HSIL及SCC组p16^INK4a的表达均显著高于慢性宫颈炎组（P〈0．01）;p16^INK4a在高危型HPV感染组的表达率（96．4％）显著高于低危型HPV感染组（7．7％）,差异有统计学意义（t=4．32,P〈0．01）。结论p16^INK4a是一种敏感性较高,特异性较好的标记物,可以识别与高危型HPV有关的非典型鳞状上皮细胞。     

Immunohistochemical assessment of p16, COX-2 and EGFR in HPV-positive cervical squamous intraepithelial lesions

The protein capsid L1 of the human papilloma virus (HPV) - a key factor in the cervical carcinogenesis - is considered, together with p16, EGFR and COX-2, a characteristic marker for the evaluation of the malignancy progression and prognostic, in terms of tumoral aggressiveness. The purpose of the present study was to make a comparative assessment between the immunohistochemical pattern of p16, EGFR and COX-2 and immunochemical expression of L1 HPV capsid protein, in low grade and high-grade cervical squamous intraepithelial lesions, in order to determine the relationship of these tumoral markers with the infection status of HPV, and their practical applicability in patients diagnosis and follow-up. The study group included 50 women with cytological and histopathological confirmed LSIL (low grade SIL) and HSIL (high-grade SIL). The immunoexpression of L1 HPV protein was assessed on conventional cervico-vaginal smears and EGFR, COX-2 and p16 were immunohistochemically evaluated on the corresponding cervical biopsies. From all cervical smears, the HPV L1 capsid protein was expressed in 52% of LSIL and 23% of HSIL. From all cervical biopsies, p16 was positive in 64% of LSIL, 82% of CIN2 and 100% of CIN3, EGFR was overexpressed in 67% of HSIL (56% CIN2 and 43% CIN3) and 32% LSIL. For COX-2, the Allred score was higher in HSIL when compared to LSIL. Our data revealed 33 cases belonging to both LSIL and HSIL categories with the same Allred score. Immunochemical detection of L1 capsid protein, on cervico-vaginal smears, indicates an immune status induced by the HPV infection and may offer prognosis information, mainly in LSIL lesions. The assessment of p16, EGFR, and COX-2 allows to an integrative approach for the progression of squamous intraepithelial lesion, associated or not with the HPV infection.     

Combined detection of p16INK4a and IMP3 increase the concordance rate between cervical cytologic and histologic diagnosis

Currently, there are discrepancies in the interpretation between cervical liquid-based cytology (LBC) and histologic diagnoses. The aim of our study was to evaluate the utility of p16^INK4a (p16) and IMP3 staining of LBC specimens to increase the concordance rate. A total of 98 cell blocks with biopsy results, including 37 low-grade squamous intraepithelial lesions (LSIL), 36 high-grade squamous intraepithelial lesions (HSIL), and 25 squamous cell carcinomas (SCC), were selected for the immunocytochemical analysis of p16 and IMP3. The LBC diagnoses corresponded with histological diagnoses for 59.5% (22/37), 63.9% (23/36), and 88.0% (22/25) of LSIL, HSIL, and SCC lesions, respectively. We found a high frequency of p16 positivity in HSIL (72.2%) and SCC (100%), but not LSIL (29.7%). IMP3 was frequently expressed in SCC (84.0%), but rarely in LSIL (8.1%) and HSIL (25.0%). Cervical intraepithelial neoplasia 1 (CIN1) was negative for both p16 and IMP3, CIN2/3 tended to be positive for p16 and negative for IMP3, and SCC was positive for both p16 and IMP3. The combination of p16 and IMP3 immunostaining had a higher sensitivity and specificity for detecting CIN1 and CIN2/3 than cytology. For detecting SCC, p16/IMP3 had a higher sensitivity than cytology, but a lower specificity. IMP3 is a useful diagnostic immunomarker that can be used to identify SCC and the combination of p16/IMP3 expression was found to improve the discrepant results between cytologic and histologic diagnoses.     

Cell cycle and/or proliferation markers: what is the best method to discriminate cervical high-grade lesions?

The aim of this study on a series of biopsies diagnosed as normal, metaplastic, low-grade squamous intraepithelial lesions (LSILs), and high-grade squamous intraepithelial lesions (HSILs) was dual: to determine the chronology of cell cycle and proliferation abnormalities after human papillomavirus infection during the development of squamous intraepithelial lesions and to determine the best diagnostic indicator(s) linked to the appearance of an HSIL. Ninety-nine cervical biopsies, 18 normal, 9 with metaplastic changes, 29 LSIL, and 43 HSIL (23 cervical intraepithelial neoplasia 2 and 20 cervical intraepithelial neoplasia 3), were analyzed by image cytometry for DNA ploidy and p16INK4A determination, AgNOR counting, MIB-1, and ICBP90 immunostaining quantification. The human papillomavirus status had been previously determined on corresponding cytological smears with the Hybrid Capture II test. Suspect DNA profile and p16INK4A staining were the first significant events that preceded the increase of cell proliferation. Indeed, these markers were the best tests for the detection of a lesion, whatever its grade (positive predictive values of 90% and 100%, respectively). The presence of MIB-1- or ICBP90-positive cells in the upper two thirds of the epithelium was a very accurate feature to select HSIL (sensitivity, 100% for MIB-1) but with a low specificity. The sensitivity of a suspect DNA profile associated with a positive MIB-1 or ICPB90 immunostaining for the detection of an HSIL was, respectively, 92.8% and 92.7%; their specificities were 54.2% and 44%; their positive predictive values were 78% and 73%; their negative predictive values were 81.2% and 78.6%; and the global values were 78.8% and 74.3%. Thus, the most accurate test to distinguish an LSIL from an HSIL was the association of a suspect DNA profile and the presence of MIB-1- or ICBP90-positive cells in the upper two thirds of the epithelium.     

MALDI imaging as a specific diagnostic tool for routine cervical cytology specimens

Cervical squamous cell carcinoma (SCC) is among the most common malignancies in women worldwide. In developed countries routine cytology screening has dramatically reduced SCC mortality within the last three decades. High risk (HR) human papilloma virus (HPV) infection is the main causal factor in nearly 100% of invasive SCCs, in most cases of low grade squamous intraepithelial lesion (LSIL) and in nearly all cases of high grade squamous intraepithelial lesion (HSIL). Detection of HR-HPV DNA has been extensively evaluated for the triage of patients with low grade cytological abnormalities in order to identify those at greatest risk for underlying or developing HSIL or SCC. However, the vast majority of HR-HPV-positive precursor lesions will not progress to invasive cancer. A variety of other screening tools are available which aim to stratify clinically significant HPV infections and cytological alterations. Matrix assisted laser desorption/ionization (MALDI) imaging mass spectrometry is a promising technology to assist in this endeavor. It delivers accurate mass spectrometric information of the sample's proteins and enables the visualization of the spatial distribution of protein expression profiles in correlation with histological features. In this study, 18 samples with Pap IIID or higher (>LSIL) and 14 samples with Pap I-II (WNL) were analyzed by MALDI imaging mass spectrometry (IMS). A genetic algorithm was applied to classify spectra resulting in an overall cross validation, sensitivity for Pap IIID and Pap?I-II of 83.7%, 88.9% and 78.6%, respectively. As this IMS based approach allows for unbiased and automated classifiction of cytological samples it appears to be a promising tool for stratification of cervical Pap smears.     

Cytological physiognomies and genotype distribution of human papillomaviruses among HPV/HIV co-infected and HPV mono-infected women

BackgroundCo-infection of High Risk Human Papillomavirus (HR-HPV) and HIV is thought to favour initiation of intraepithelial squamous cell lesion and subsequent progression to cervical carcinoma.ObjectivesEvaluation of cytological physiognomies in relation to possible age influence and the genotype distribution of human papillomaviruses among HPV/HIV co-infected and HPV monoinfected women in Kisii, Kenya.MethodsThe case-control study enrolled 42 HPV/HIV co-infected and 42 HPV monoinfected women. Cervical swabs were collected in ThinPrep vials for HPV tying and cytological analysis. HPV subtypes were assayed by Xpert® HPV system (GXHPV-CE-10).ResultsMono-infected women aged 30–39 years had the highest proportion of low grade squamous intraepithelial lesion (LSIL) at 14 (16.67%) while the co-infected aged 50–59 years had the highest proportion of high grade squamous intraepithelial lesion (HSIL) at 9 (10.71%). HPV-16 genotype was the most predominant and it increased with age rise. Older coinfected and mono-infected women (>40 years) had HSIL and LSIL as the most predominant cytological grade respectively.ConclusionThe predominance of HPV-16 and HPV-18/45 genotypes in the study setting is a consideration that would benefit targeted prophylactic vaccination programs. HPV testing and cervical cancer screening for young and older women on a regular basis ought to be reinforced.     

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.     

Reflex high-risk human papilloma virus DNA test is useful in the triage of women with atypical squamous cells cannot exclude high-grade squamous intraepithelial lesion

This study is aimed to investigate the role of reflex high-risk human papilloma virus (HPV) DNA testing as an alternative triage method to colposcopy for women with atypical squamous cells cannot exclude high-grade squamous intraepithelial lesion (ASC-H) on Papanicolaou (Pap) tests. Reflex HPV DNA testing using Hybrid Capture II method was carried out on 88 women with ASC-H diagnosed by Thin Prep Pap test. Correlation with follow-up biopsies was available on 42 of these patients. The reflex HPV DNA test showed an overall positive rate of 67% and negative rate of 33% in 88 patients with ASC-H. Using age 30 as the cut off point, the positive rate had increased to 83.3% (35/42) in patients 30 yr or younger, while the positive rate for patients older than 30 yr had decreased to 52.2% (24/46). Follow-up colposcopic biopsy results were available in 35 of 59 HPV-positive women, which revealed 15 (43%) high-grade squamous intraepithelial lesions (HSIL), 12 low-grade squamous intraepithelial lesions (LSIL), and 8 negative for dysplasia. In 7 HPV-negative patients, the follow-up biopsies showed no evidence of HSIL or LSIL. Correlation between clinical risk factors and the HPV results demonstrated no significant differences in HPV positivity between the high-risk and low-risk patients. The high sensitivity (100%) and negative predictive rate (100%) in detecting HSIL in our study provide strong evidence that, instead of automatic referral to colposcopy, reflex HPV DNA testing may be used as an alternative triage method for women diagnosed with ASC-H on Thin Prep Pap test, especially for women older than 30 yr of age.     

Significance of Bcl-2 and Bax proteins in cervical carcinogenesis: an immunohistochemical study in squamous cell carcinoma and squamous intraepithelial lesions

Sixteen low grade (LSIL), 22 high grade (HSIL) squamous intraepithelial lesions, 28 invasive (13 stage I and 15 stage II-IV) squamous cell carcinoma (SCC) and 15 benign cervices were immunohistochemically studied for involvement of Bcl-2 and Bax proteins in cervical carcinogenesis. 4-microm sections of the cases were immunostained for Bcl-2 (Clone 124: Dako) and Bax (Dako) and staining intensity was rated as 1 (light), 2 (moderate) and 3 (strong) and percentage cellular staining as 0 (negative), 1 (1-25%), 2 (26-50%), 3 (51-75%) and 4 (>75%) with score derived by multiplying staining intensity and percentage positivity. The cut-off value, indicating upregulated expression, was computed as >2 for Bcl-2 and >8 for Bax. Bcl-2 was upregulated (p < 0.05) in HSIL and Bax in SCC when compared with benign cervical squamous epithelium. Bcl-2 expression was confined to the lower third of the epithelium in the benign cervices and LSIL. In HSIL, expression reached the middle and upper thirds. 4 (30.8%) HSIL with upregulated Bcl-2 demonstrated intensification of staining around the basement membrane. SCCs showed "diffuse" (evenly distributed) or "basal" (intensified staining around the periphery of the invading tumour nests) expression of Bcl-2. Of the 5 SCCs with upregulated Bcl-2, 1 of 2 (50%) stage I and 3 (100%) stage II-IV tumours exhibited the "basal" pattern. Benign cervical squamous epithelium, LSIL, HSIL and SCC showed a generally diffuse Bax expression. Thus, Bcl-2 and Bax appeared to be upregulated at different stages of cervical carcinogenesis, Bcl-2 in HSIL and Bax after invasion. Intensification of staining of Bcl-2 at the basement membrane in some HSIL and SCC is interesting and may augur for increased aggressiveness.     

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.     

Screening properties of human papillomavirus testing for predicting cervical intraepithelial neoplasia in atypical squamous cells of undetermined significance and low-grade squamous intraepithelial lesion smears: a prospective study

The aim of the present study was to determine the usefulness of human papillomavirus (HPV) testing for predicting cervical intraepithelial neoplasia (CIN) 1 and 2 to 3 on cervical biopsies in women who had atypical squamous cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesion (LSIL) on Papanicolaou tests. In this prospective cohort, 167 women with abnormal cytologic examination (ASCUS and LSIL) were evaluated by colposcopy-directed biopsy and endocervical curettage. Colposcopy was performed on all study participants to obtain cervical tissue for histologic examination for detection of underlying CIN in patients with an initial cytologic test result of ASCUS and LSIL. A sample for HPV DNA detection by polymerase chain reaction was obtained. The HPV type 16 was positive in 35.4% of the 167 women with abnormal cytologic examination result in our gynecologic outpatient's clinic. Histologic diagnosis of CIN 1 was found in 45 of 135 women with ASCUS and in 17 of 32 women with LSIL. According to the cytologic findings, the frequency of CIN grade 2 or 3 in patients classified as ASCUS and LSIL was 12.5% (17/135) and 18.7% (6/32), respectively. Of the ASCUS smears, 9.6% were positive for HPV type 16. The sensitivity of the HPV type 16 using polymerase chain reaction technique threshold in detecting CIN 1 and CIN 2 to 3 was 57% and 46% in ASCUS-LSIL cytologic examination, respectively. The positive predictive value of HPV type 16 ranged from 60% in patients with CIN 1 and 42% in CIN 2 to 3 in ASCUS-LSIL. By contrast, negative predictive value was 58% in patients with CIN 1 and 80% in CIN 2 to 3. The low positive predictive value of HPV testing with ASCUS smears suggests that HPV positivity could be not used for predicting the presence of CIN 2 to 3.     

Negative loop electrosurgical excision procedure (LEEP) following cervical biopsy diagnosis of high grade squamous intraepithelial lesion

Context: Loop Electrosurgical Excision Procedure (LEEP) is commonly performed after cervical biopsy diagnosis of high grade squamous intraepithelial lesion (HSIL/CIN2 or CIN 3). Histological and immunohistochemical assessments are made to differentiate reactive and metaplastic changes from dysplastic changes. A Human Papillomavirus (HPV) test is used for prognostic assessment after conization. Objective: We retrospectively reviewed cases where the cervical biopsy showed HSIL but the LEEP specimen was negative for high grade dysplasia. Our aim was to determine the cause of miscorrelation. Data: IRB approval was obtained and a search was made of all LEEP specimens received during 2018. We reviewed 25 of 137 LEEP specimens that did not correlate with the diagnosis of HSIL rendered on the cervical biopsy. These were from women between 25 to 54 years. All cases had positive high-risk HPV with 80% being non16/18 subtype. On review, 8/25 had HSIL with the remainder of cases falling short of HSIL diagnosis. Follow up cytology with HPV test after the LEEP procedure was negative in all but one case of LSIL with persistent non-16/18 HPV. Conclusion: The study highlights the diagnostic difficulties of distinguishing HSIL from immature squamous metaplasia. The practical implication is that in cases with non-16/18 high risk HPV which have thin epithelium and fall short of definite morphologic criteria of HSIL, presence of immature squamous metaplasia should be carefully evaluated. The specific role of CK7 and CK17 which highlight squamocolumnar junctional cells and metaplastic cells, respectively, needs to be explored in these cases.     

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.     

Utility of P16 expression and Ki‐67 proliferation index in ASCUS and ASC‐H pap tests

Current cervical screening uses a combination of cytology and high‐risk human papillomavirus (HR‐HPV) analysis in cases of atypical squamous cells of undetermined significance (ASCUS) and atypical squamous cells cannot exclude high‐grade intraepithelial lesion (ASC‐H). These diagnoses are subject to interobserver variability and HR‐HPV analysis can be limited by sampling inadequacy. This study correlates immunoexpression of P16 and Ki‐67 in residual cervicovaginal material against cytology category and HR‐HPV status. Eighteen pap tests were selected: 8 ASCUS, 4 ASC‐H, and 6 controls (2 LSIL and 4 HSIL). Digene Hybrid Capture II test was used to detect HR‐HPV. The cytospins were stained for P16/Ki‐67. Pap tests, P16, Ki‐67, HR‐HPV result and available biopsies were correlated. P16 expression correlated with HR‐HPV status in 15/17 cases. Discordant cases (1 ASCUS and 1 ASC‐H) were +P16/–HR‐HPV. Ki‐67 correlated with HR‐HPV in 8/15 cases. Discordant cases were +HR‐HPV/– Ki‐67 (HSIL, LSIL, and ASC‐H one each), and –HR‐HPV/+Ki‐67 (3 ASCUS, 1 LSIL, 1 ASC‐H). Two cases were + P16/+ Ki‐67/– HR‐HPV. None were ‐ P16/– Ki‐67/+ HR‐HPV. Histologic follow‐up in 13 cases varied from benign to CIN III. Two cases of +P16/ – Ki‐67/– HR‐HPV had benign cervical biopcies. Although a small sample size, our findings show a utility for adjunct P16/ Ki‐67 in addition to HR‐HPV testing in cases of squamous atypia when HR‐HPVs are non‐detected due to low DNA copies, or missed lesions in cervical biopsies. Diagn. Cytopathol. 2014;42:576–581. © 2013 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%).     

High prevalence of human papillomavirus type 58 in Mexican colposcopy patients.

BACKGROUND: Cervical cancer is the second most common cancer of the women worldwide. Infection with some genotypes of human papillomavirus is the most important risk factor associated to cervical cancer. OBJECTIVE: To determine the prevalence and genotypes of papillomavirus in biopsies of women with squamous intraepithelial lesion and cervical cancer. STUDY DESIGN: Two hundred sequential patients of colposcopy clinic were studied. HPV diagnosis was done by polymerase chain reaction using MY09/MY11 primers, for genotyping line blot hybridization was used. RESULTS: A total of 186 women were beta globin positive; 104 (55.9%) had histology diagnosis of low-grade squamous intraepitelial lesions (LSIL), 67 (36.0%) high-grade squamous intraepitelial lesions (HSIL) and 15 (8.1%) invasive cervical cancer (IC). The prevalence of HPV was 56.4% (104/185); HPV 58 was founded in 28.5% of all positive women, HPV 16 in 25.7%, HPV 18 in 13.3%, HPV 33 in 11.4% and 31 in 8.5%. In all grades of the lesions HPV 58 was the most frequently. CONCLUSIONS: The high prevalence of HPV 58 among Mexican women with HSIL and IC, has important implications in prophylaxis.     

Human papillomavirus in dysplasia and carcinoma of the cervix in Singapore

A prospective study was conducted in Singapore in 1985 where 107 women with abnormal cervical smears were studied for cervical neoplasia and its association with the human papillomaviruses (HPV), using HPV 11, 16 and 18 DNA as probes. Cervical biopsies were performed for histology as well as for DNA Southern Blot hybridization studies to detect the presence of HPV 11, 16 or 18 genome. The prevalence of the various types of papillomavirus DNA in cervical tissue samples from cervical carcinoma and dysplasias is presented. HPV types 16 and 18 were found in 5 of 8 (63%) cases of invasive squamous cell carcinoma (SCC), in 12 of 41 (29%) cases of squamous carcinoma-in-situ (CIN 3) and in 3 of 48 (6%) cases of lower grade dysplasia. None of our cases of SCC were associated with unknown HPV types detected using a mixture of HPV 16 or 18 DNA under conditions of low stringency. These unknown HPV types were present in approximately one quarter of our cases of dysplasia of all grades.