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.     

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.     

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%).     

Human papillomavirus-16 E6 variants in cervical squamous intraepithelial lesions from HIV-negative and HIV-positive women.

We studied 48 human papillomavirus (HPV)-16-positive squamous intraepithelial lesions (SILs) from HIV-negative patients (16 low-grade SILs [LSILs]; 32 high-grade SILs [HSILs]) and 13 HPV-16-positive SILs from HIV-positive patients with AIDS (1 LSIL; 12 HSILs). After HPV typing, the entire HPV-16 E6 coding region was amplified and sequenced in all samples. We detected 12 HPV-16 E6 prototypes and 4 variants among the LSILs in HIV-negative patients, and 15 HPV-16 E6 prototypes and 17 HPV-16 variants in the HSIL group. The most prevalent variant of SIL types was European 350G, present in 3 and 13 cases, respectively. In 3 HSILs and no LSILs we found mixed infection by an HPV-16 E6 prototype and a variant. Two variants (1 each in LSIL and HSIL) were of non-European lineage. The only LSIL in HIV-positive patients had an HPV-16 E6 prototype; in the HSILs, we found 8 HPV-16 E6-prototypes, 4 with mixed infection with HPV-31 and 4 variants, all European 350G. The higher proportion of HPV-16 E6 variants in HSIL than in LSIL in HIV-negative patients suggests a greater risk of progression. However, further studies are needed.     

Human papillomavirus infection among women with cytological abnormalities in Switzerland investigated by an automated linear array genotyping test

Limited data are available describing human papillomavirus (HPV) genotype distribution among females with cytological abnormalities in Switzerland. Cervical cell specimens obtained from 5,318 women were screened routinely by liquid-based Pap smear. All specimens with cellular abnormalities were analyzed subsequently for HPV DNA by the Linear Array HPV genotyping test. Cellular abnormalities were found in 202 (3.8%) specimens, of which 150 (74.3%) were positive for high-risk (HR) HPV. HR-HPV was detected in 20 (60.6%; 95% CI, 43.7-75.4%) of 33 specimens with atypical squamous cells of undetermined significance compared to 98 (72.1%; 95% CI, 64-78.9%) of 136 low-grade squamous intraepithelial lesions and 32 (97%; 95% CI, 83.4-99.9%) of 33 high-grade squamous intraepithelial lesions. The cumulative prevalence of HR-HPV other than HPV 16 and 18 was significantly higher than HPV 16 and/or 18 lesions with atypical squamous cells and low-grade lesions and was comparable in high-grade squamous intraepithelial lesions. The most common HR-HPV genotypes were HPV 16 (15.2%), HPV 31 (12.1%), HPV 58 (12.1%), HPV 51 (9.1%), and HPV 59 (9.1%) in women with atypical squamous cells, HPV 16 (25%), HPV 51 (16.9%), HPV 52 (11.8%), HPV 31 (9.6%), and HPV 56 (8.1%) in women with low-grade lesions (LSIL) and HPV 16 (57.6%), HPV 18 (18.2%), HPV 31 (15.2%), HPV 52 (12.1%), and HPV 58 (6.1%) in women with high-grade lesions (HSIL).     

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。     

Distribution of human papillomavirus genotypes in Paraguayan women according to the severity of the cervical lesion

The incidence of cervical cancer in Paraguay is among the highest in the world. This study aimed to determine the distribution of human papillomavirus (HPV) genotypes in Paraguayan women, according to the severity of the cervical lesion. This cross-sectional study included 207 women without a squamous intraepithelial lesion, 164 with low-grade squamous intraepithelial lesions, 74 with high-grade squamous intraepithelial lesions, and 41 with cervical cancer. Type-specific HPV was determined by the polymerase chain reaction with MY9/11 L1 and GP5+/GP6+ L1 primers, followed by restriction fragment length polymorphism and reverse line blotting hybridization, respectively. In total, 12 high-risk and 24 low-risk HPVs types were detected. HPV 16 was the most prevalent, followed by HPV 18 in cervical cancer (14.6%), HPV 31 in high-grade squamous intraepithelial lesions (14.9%), HPVs 58/42 in low-grade squamous intraepithelial lesions (9.1% each), and HPVs 31/58 (2.4% each) in women without squamous intraepithelial lesions. Among 285 positive samples, 24.2% harbored multiple HPV types, being this more prevalent in women with squamous intraepithelial lesions (30.8% in low-grade squamous intraepithelial lesions, 22.5% in high-grade squamous intraepithelial lesions, and 22.0% in cervical cancer) than in women without lesions (9.3%). The higher prevalence of HPV 16 and other high-risk HPVs in women both with and without cervical lesions may explain the high incidence of cervical cancer in Paraguay. This information may be of importance for local decision makers to improve prevention strategies. In addition, these results may be useful as baseline pre-vaccination data for a future virological surveillance in Paraguay.     

Easy and fast detection and genotyping of high-risk human papillomavirus by dedicated DNA microarrays

Persistent cervical high-risk human papillomavirus (HPV) infection is correlated with an increased risk of developing a high-grade cervical intraepithelial lesion. A two-step method was developed for detection and genotyping of high-risk HPV. DNA was firstly amplified by asymmetrical PCR in the presence of Cy3-labelled primers and dUTP. Labelled DNA was then genotyped using DNA microarray hybridization. The current study evaluated the technical efficacy of laboratory-designed HPV DNA microarrays for high-risk HPV genotyping on 57 malignant and non-malignant cervical smears. The approach was evaluated for a broad range of cytological samples: high-grade squamous intraepithelial lesions (HSIL), low-grade squamous intraepithelial lesions (LSIL) and atypical squamous cells of high-grade (ASC-H). High-risk HPV was also detected in six atypical squamous cells of undetermined significance (ASC-US) samples; among them only one cervical specimen was found uninfected, associated with no histological lesion. The HPV oligonucleotide DNA microarray genotyping detected 36 infections with a single high-risk HPV type and 5 multiple infections with several high-risk types. Taken together, these results demonstrate the sensitivity and specificity of the HPV DNA microarray approach. This approach could improve clinical management of patients with cervical cytological abnormalities.     

Qualification of ASCUS. A comparison of equivocal LSIL and equivocal HSIL cervical cytology in the ASCUS LSIL Triage Study

Cytologic detection of high-grade squamous intraepithelial lesions (HSILs) is critical to cervical cancer prevention. Therefore, identifying "equivocal HSIL" (ASCUS [atypical squamous cells of undetermined significance]-H) may be useful. Accordingly, we compared findings associated with "equivocal low-grade SIL" (ASCUS-L), ASCUS-H, and HSIL using data from the ASCUS LSIL (low-grade squamous intraepithelial lesion) Triage Study. The frequency of oncogenic human papillomavirus (HPV) DNA detection and underlying lesions cervical intraepithelial neoplasia (CIN) 2 or worse or CIN 3 or worse in women with ASCUS-H was intermediate between that of ASCUS-L and HSIL. Oncogenic HPV DNA was associated with 85.6% of ASCUS-H ThinPreps and 69.8% of ASCUS-H smears. Histopathologic lesions CIN 2 or worse were associated with 40.5% of ASCUS-H ThinPreps and 27.2% of ASCUS-H smears (mostly CIN 3). Nevertheless, numerically more lesions CIN 2 or worse were preceded by ASCUS-L than by ASCUS-H because ASCUS-L was more common. ASCUS-H is an uncommon interpretation that derives clinical usefulness from its high positive predictive value for lesions CIN 2 or worse.     

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.     

Atypical squamous cells of undetermined significance-favour reactive compared to atypical squamous cells of undetermined significance-favour dysplasia: association with cervical intraepithelial lesions and human papillomavirus infection.

BACKGROUND AND OBJECTIVES: The current study compared the cervical cytological sub-category "atypical squamous cells of undetermined significance-favour reactive (AFR)", recently recommended to be eliminated by the Bethesda system, to the sub-category "atypical squamous cells of undetermined significance-favour dysplasia (ASC-US)", in terms of prevalence of coexistent squamous intraepithelial lesions of either low-grade (LSIL) or high-grade (HSIL) and rate of human papillomavirus (HPV) infection. STUDY DESIGN: One hundred women with AFR and 100 with ASC-US were consecutively included in the study. All patients underwent colposcopy, followed by biopsy when necessary, and were screened for HPV infection by the combined use of Hybrid Capture II (DIGENE) and PCR with MY09/11 primers, the latter followed by direct sequencing of the amplifications products for HPV genotyping. RESULTS: LSIL were detected in 5.6% of AFR and 18.5% of ASC-US (p=0.00812), HSIL only in 4.3% of ASC-US. HPV infection was diagnosed in 11.2% of AFR and 38.0% of ASC-US (p=0.00003); high-risk HPV types (namely, HPV-16, -18, -31, -66, -67 and -70) were found in 6.7% of AFR and 22.8% of ASC-US (p=0.00239). Evidence of HPV infection in absence of SIL was proven in 7.1% of AFR and in 22.5% of ASC-US (p=0.00622). CONCLUSION: The association of AFR with SIL and high-risk HPV infection is low but not inexistent. Thus, to avoid the risk of leaving some high-risk AFR patients untreated or without follow-up, it could be proposed to keep AFR as a cytological category and to triage it by HPV testing, similarly to what has been already recommended for ASC-US.     

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.     

Cervical cancer precursors: cytohistologic correlation with the results of HPV testing

Persistence of high-risk types of human papillomavirus (HPV) is responsible for the development, maintenance and progression of squamous intraepithelial lesions (SILs). Cytohistologic correlation with the results of the HPV testing in 52 patients over a 3year period is presented. Two patients out of the 52 patients presented in this histologic follow up bore the diagnosis high-grade squamous intraepithelial lesion (HSIL) with the former cytology ASC-H. Low grade squamous intraepithelial lesions (LSIL) were found in eight patients, half of them diagnosed identically on cytology. Another four cases were formerly diagnosed cytologically as ASC-US. All women with the histologically confirmed dysplastic changes were HR HPV DNA positive. Our results indicate that significant histologic lesions may be discovered in patients exhibiting the high-risk HPV DNA positivity in the category of ASC-US (Atypical Squamous Cells of Undetermined Significance) and especially ASC-H (ASC cannot exclude high-grade squamous intraepithelial lesion). A combined screening test thus offers the possibility of greater protection and /or longer screening intervals.     

Localization of telomerase hTERT protein and hTR in benign mucosa, dysplasia, and squamous cell carcinoma of the cervix

Telomerase has been detected by telomerase repeat amplification protocol (TRAP) assay in cervical dysplasia and squamous cell carcinoma but not in most normal cervical tissues. In the present study, the cellular localization of the protein catalytic subunit of telomerase (hTERT) and the RNA component (hTR) were investigated by a sensitive immunohistochemical technique and by in situ hybridization, respectively. hTERT protein was detected in all diagnostic categories of cervical specimens. hTERT was localized predominantly to the lower suprabasal levels of normal squamous mucosa but was detected throughout virtually all levels of the lesional epithelium in low-grade squamous intraepithelial lesions (LSILs), high-grade squamous intraepithelial lesions (HSILs), and squamous cell carcinoma (SCC). Telomerase expression correlated with hTERT detection in SCC and HSIL but was not detected by TRAP assay in most samples of normal mucosa or LSIL. The distribution of hTR correlated with the localization of hTERT in HSIL and SCC but was restricted to the basal and suprabasal cell layers in normal mucosa and LSIL.     

The clinical significance of "squamous intraepithelial lesion of indeterminate grade" as a distinct cytologic category

The histologic and/or cytologic follow-up of 127 cases of cervical lesions termed "squamous intraepithelial lesion of indeterminate grade" (SIL) on Papanicolaou (Pap) smears by the 2001 Bethesda System was compared with 150 control cases of low-grade SIL (LSIL), high-grade SIL (HSIL), and atypical squamous cells, cannot exclude HSIL (ASC-H). A follow-up diagnosis of cervical intraepithelial neoplasia (CIN) 2 or higher was identified in 22.8% of SIL cases, which was 2.6 times higher than LSIL, 3 times lower than HSIL, and 1.5 times lower than ASC-H. A follow-up diagnosis of CIN 1 was identified in 31.5% of SIL cases, which was 2 times lower than the LSIL group, 1.5 times higher than the ASC-H cases, and 1.8 times higher than the HSIL group. We found that 22.0% of cases diagnosed as SIL were followed up by Pap smears rather than colposcopy and biopsy, compared with about 1% of LSIL and HSIL cases. Because SIL cases have a significant risk of harboring CIN 2 or greater, we recommend follow-up by colposcopy and biopsy.     

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.     

Anal cytology: Is there a role for reflex HPV DNA testing?

There is an increased incidence of anal squamous carcinoma and its precursor lesions (anal intraepithelial neoplasia [AIN]) among persons who engage in anal-receptive sex. Analogous to cervical cancer screening, anal Papanicplaou (Pap) smears currently are used to screen these high-risk populations. Human papilloma virus (HPV) has been implicated in anal carcinoma pathogenesis and this study was performed to assess the potential role of HPV DNA testing as an adjunct to anal cytology. We correlated cytological diagnoses and HPV DNA (Digene Hybrid Capture [HC II] assay) in anal specimens collected in SurePath liquid medium from 118 patients; 54.8% of cases diagnosed as atypical squamous cells of undetermined significance (ASC-US) and 87.8% diagnosed as low-grade squamous intraepithelial lesion (LSIL) or above tested positive for high- risk HPV DNA (B+). High-grade SIL (HSIL) was present in 31 of the 51 patients with follow-up. Although a cytological diagnosis of ASC-US or above was a reliable indicator for AIN, cytology frequently did not accurately predict the grade of SIL in subsequent biopsy. Our findings suggest that reflex HPV DNA testing would be helpful in triaging patients diagnosed with ASC-US. However, patients diagnosed with LSIL or above should go directly to ansocopic biopsy.     

Atypical immature metaplasia (AIM) of the cervix: is it related to high-grade squamous intraepithelial lesion (HSIL)?

Atypical immature metaplasia (AIM) is a poorly characterized cervical lesion with uncertain biological and clinical significance. AIM shares some, but not all, morphological features of squamous intraepithelial lesions (SILs). SILs are characterized by human papillomavirus (HPV) positivity and increased cellular proliferation, but these features have not been fully evaluated in AIM. Genomic DNA was extracted from 27 microdissected cervical biopsy specimens diagnosed as AIM. HPV DNA was detected by polymerase chain reaction (PCR), using two different sets of L1 gene consensus primers. HPV types were identified by sequence analysis of PCR products and comparison with published HPV sequences. The cell proliferation index was assessed by immunohistochemical staining for Ki-67 (MIB-1) antigen and expressed as the percentage of Ki-67-positive cells. Comparison groups included normal cervix (n = 10) and low-grade (LSILs, n = 19) and high-grade squamous intraepithelial lesions (HSILs, n = 11). Intermediate- or high-risk HPV DNA was detected in 67% (18 of 27) of AIM cases. Low-risk HPV DNA was not detected in any of the specimens. The Ki-67 index in AIM (mean, 33.0 +/- 20.3; median, 29) was comparable to that of LSILs (mean, 21.4 +/- 4.6; median, 21) and was significantly higher than that of normal cervix (mean, 11.0 +/- 2.1; median, 11) (P< .01) and lower than that of HSILs (mean, 60.4 +/- 13.2; median, 60) (P < .01). Of the cases with available follow-up, HPV-positive AIMs were significantly more likely to have a concurrent or subsequent diagnosis of typical HSIL (12 of 15, 80%) than HPV-negative AIMs (one of six, 45%) (P = .014). The wide range of Ki-67 indices and variable HPV status in AIM suggest that AIM represents a heterogeneous group of lesions including bona fide HSILs (high-risk HPV-positive, high Ki-67 index), antecedents (precursors?) of HSILs (high-risk HPV-positive, low to moderate Ki-67 index), and benign reactive conditions (HPV-negative, variable Ki-67 index). HPV testing may be useful in the assessment of atypical epithelial proliferations of the cervix for which a diagnosis of AIM is considered.     

Atypical glandular cells of undetermined significance (AGUS): cytopathologic features, histopathologic results, and human papillomavirus DNA detection.

We intensively reviewed 137 smears initially classified as atypical glandular cells of undetermined significance (AGUS) to refine cytological criteria for evaluating these cases, evaluate histological outcomes, and assess the value of human papillomavirus (HPV) DNA testing in management. Consenting, nonpregnant study participants were identified from a cohort of 46,009 women receiving routine Pap smear screening in a managed care setting. Colposcopy was performed on all women, and at least one histological sample was obtained from each. Review diagnoses were assigned to smears and biopsy specimens by two separate panels of pathologists. DNA testing for cancer-associated HPV types was performed on rinses of cytological samplers after a smear and thin-layer slide had been made. On review, 47 (34%) smears were reclassified as negative, 44 (32%) as AGUS, 30 (22%) as atypical squamous cells of undetermined significance (ASCUS), and 16 (12%) as squamous intraepithelial lesions (SIL). The 19 smears interpreted as high-grade intraepithelial lesions on review included 13 high-grade SIL (HSIL), two HSIL with AGUS, favor neoplastic (endocervical adenocarcinoma in situ [AIS]), and four AGUS, favor neoplastic (AIS). Review histological diagnoses were negative in 105 (77%), squamous or glandular atypia in four (3%), low-grade SIL (LSIL) in nine (7%), HSIL in 12 (9%), AIS in five (4%, including two with concurrent HSIL), and endometrial carcinoma in one (1%). HPV testing identified 11 (92%) of 12 women with histologically confirmed HSIL and all five with AIS (100%). A high-grade intraepithelial lesion or carcinoma is detected in approximately 14% of women with community-based diagnoses of AGUS who are referred for immediate evaluation. Use of refined cytological criteria and HPV DNA testing may permit improved management of women with AGUS.     

Clinicopathological Implications of Human Papilloma Virus (HPV) L1 Capsid Protein Immunoreactivity in HPV16-Positive Cervical Cytology

Background: The objective of this study was to investigate the expression of human papilloma virus (HPV) L1 capsid protein in abnormal cervical cytology with HPV16 infection and analyze its association with cervical histopathology in Korean women.Material and Methods: We performed immunocytochemistry for HPV L1 in 475 abnormal cervical cytology samples from patients with HPV16 infections using the Cytoactiv^® HPV L1 screening set. We investigated the expression of HPV L1 in cervical cytology samples and compared it with the results of histopathological examination of surgical specimens.Results: Of a total of 475 cases, 188 (39.6%) were immunocytochemically positive and 287 (60.4%) negative for HPV L1. The immunocytochemical expression rates of HPV L1 in atypical squamous cells of unknown significance (ASCUS), low-grade squamous intraepithelial lesions (LSIL), high-grade squamous intraepithelial lesions (HSIL), and cancer were 21.8%, 59.7%, 19.1%, and 0.0%, respectively. LSIL exhibited the highest rate of HPV L1 positivity. Of a total of 475 cases, the multiple-type HPV infection rate, including HPV16, in HPV L1-negative cytology samples was 27.5%, which was significantly higher than that in HPV L1-positive cytology samples (p = 0.037). The absence of HPV L1 expression in ASCUS and LSIL was significantly associated with high-grade (≥cervical intraepithelial neoplasia [CIN] 2) than low-grade (≤CIN1) histopathology diagnoses (p < 0.05), but was not significantly different between HPV16 single and multiple-type HPV infections (p > 0.05). On the other hand, among 188 HPV L1-positive cases, 30.6% of multiple-type HPV infections showed high-grade histopathology diagnoses (≥CIN3), significantly higher than the percentage of HPV16 single infections (8.6%) (p = 0.0004)Conclusions: Our study demonstrates that the expression of HPV L1 is low in advanced dysplasia. Furthermore, the absence of HPV L1 in HPV16-positive low-grade cytology (i.e., ASCUS and LSIL) is strongly associated with high-grade histopathology diagnoses. The multiplicity of HPV infections may have an important role in high-grade histopathology diagnoses (≥CIN3) in HPV L1-positive cases.