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.     

bcl-2 immunoreactivity, human papillomavirus DNA, and cervical intraepithelial neoplasia.

The aim of this study was to identify the role of bcl-2 protein expression in precancerous lesions of the cervix in patients from Johannesburg, South Africa and to correlate this expression with human papillomavirus (HPV) status. Archival cervical biopsy specimens (n = 107) of normal squamous epithelia (n = 18), pure HPV squamous epithelial lesions (n = 15), cervical intraepithelial neoplasia (CIN) I lesions (n = 17), CIN II lesions (n = 26), and CIN III lesions (n = 31) underwent bcl-2 immunohistochemical analysis with use of the streptavidin-biotin complex/horseradish peroxidase system and nonisotopic in situ hybridization for the detection of HPV DNA. Although 45 (61%) of the 74 CIN lesions demonstrated bcl-2 protein expression in the epithelia, most seemed to be in a patchy basal cell distribution, with a 1+ to 2+ intensity. Furthermore, comparison of bcl-2 immunoreaction between the low and high grades of the CIN lesions did not reveal significant differences. In addition, there was no apparent link between the presence of HPV DNA and bcl-2 expression in the CIN lesions. In contrast to previous studies that showed an increase in bcl-2 immunostaining intensity with increasing severity of CIN, only 4 (5.4%) of our 74 CIN specimens satisfied this pattern. Hence, we suggest that bcl-2 protein expression might not play a significant role in the majority of CIN lesions in this population group and that it might not correlate with HPV status.     

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     

Comparison of the hybrid capture tube test and PCR for detection of human papillomavirus DNA in cervical specimens.

The strong association of human papillomavirus (HPV) and cervical cancer makes it important to study HPV detection methods that may play a role in cervical cancer screening. We compared two DNA methods that are commonly used for HPV research in the United States: the MY09/MY11 L1 consensus primer PCR-based test and the first-generation Hybrid Capture tube method (HCT). Laboratory assays by each method were performed with 596 cervicovaginal specimens collected from participants in a large cohort study conducted in Portland, Oreg. Included were 499 specimens from women whose cytology was normal and 97 specimens from women with squamous intraepithelial lesions (SILs). The overall HPV DNA positivity for known types was 22.5% by PCR compared to 13.6% by HCT. When the analysis was restricted to the 14 HPV types detectable by both methods, the sensitivity of HCT, with PCR used as the standard for HPV status, was higher for specimens from women with concurrent SILs (81.0%) than for specimens from women with normal cytology (46.7%). Among specimens testing positive by both methods, 97.2% of the time the two methods agreed on whether specimens were positive for cancer-associated HPV types. Both of these HPV test methods provide information that supplements the information provided by the Pap smear. The PCR method has higher analytic sensitivity than HCT in detecting HPV, but HCT may be helpful in identifying women with concurrent SILs.     

P16INK4A和人乳头状瘤病毒检测对宫颈上皮高度病变的预测意义

目的 探讨在液基细胞学(TCT)未明确意义的不典型鳞状上皮细胞(ASCUS)中,P16INK4A免疫细胞化学染色和高危型人乳头状瘤(HR-HPV)检测对宫颈上皮内高度病变(HSIL)的预测意义.方法 应用免疫细胞化学方法检测P16INK4A在89例液基细胞学不典型鳞状上皮细胞(ASC-US 55例和ASC-H 34例)中的表达,用第二代杂交捕获法(HC-2)检测每一患者的13种HR-HPV DNA,并同组织病理诊断结果比较.结果 在89例ASCUS中,组织病理证实:HSIL(CIN2/3)22例,宫颈癌2例.占26.97%(24/89).P16INK4A免疫细胞化学染色阳性26例中,组织病理证实:CIN2/3 20例,宫颈癌2例,CIN1和宫颈炎4例;HC-2HR-HPV DNA检测阳性64例,组织病理证实:CIN2/3 21例,宫颈癌2例,CIN1和宫颈炎41例;差异有统计学意义(P<0.01).P16INK4A免疫细胞化学染色对宫颈鳞状上皮内高度病变(HSIL)的阳性预测值(PPV)和特异性(Sp)分别为84.62%和93.85%,明显高于HC-2 HR-HPV DNA检测的PPV 35.94%和Sp 36.92%.结论 在TCT-ASCUS中,P16INK4A特异免疫染色比HG2 HR-HPVDNA检测能够更可靠地鉴别宫颈良、恶性疾病,二者联合检测将会为病人提供一种更有效、更合理的处理方案.     

Distribution of human papillomavirus genotypes in cervical intraepithelial neoplasia and invasive cervical cancer in Canada

Infection with high‐risk human papillomavirus (HPV) causes cervical intraepithelial neoplasia (CIN) and invasive cervical cancer (ICC). The distribution of HPV types in cervical diseases has been previously described in small studies for Canadian women. The prevalence of 36 HPV genotypes in 873 women with CIN and 252 women with ICC was assessed on cervical exfoliated cells analyzed with the Linear Array (Roche Molecular System). HPV16 was the most common genotype in CIN and ICC. The seven most frequent genotypes in order of decreasing frequency were HPV16, 51, 52, 31, 39, 18, and 56 in women with CIN1, HPV16, 52, 31, 18, 51, 39, and 33 in women with CIN2, HPV16, 31, 18, 52, 39, 33, and 58 in women with CIN3, and HPV16, 18, 45, 33, 31, 39, and 53 in women with ICC. HPV18 was detected more frequently in adenocarcinoma than squamous cell carcinoma (P = 0.013). Adjustment for multiple type infections resulted in a lower percentage attribution in CIN of HPV types other than 16 or 18. The proportion of samples containing at least one oncogenic type was greater in CIN2 (98.4%) or CIN3 (100%) than in CIN1 (80.1%; P < 0.001 for each comparison). Multiple type infections were demonstrated in 51 (20.2%) of 252 ICC in contrast to 146 (61.3%) of 238 women with CIN3 (P < 0.001). Adjusting for multiple HPV types, HPV16 accounted for 52.1% and HPV18 for 18.1% of ICCs, for a total of 70.2%. Current HPV vaccines should protect against HPV types responsible for 70% of ICCs in Canadian women. J. Med. Virol. 83:1034–1041, 2011. © 2011 Wiley‐Liss, Inc.     

Comparison between the Hybrid Capture II test and a PCR-based human papillomavirus detection method for diagnosis and posttreatment follow-up of cervical intraepithelial neoplasia

Human papillomavirus (HPV) infection is the major cause of cervical cancer and its precursor, cervical intraepithelial neoplasia (CIN), and HPV testing has therefore been proposed for improved triaging and follow-up of women treated for CIN. We compared two common HPV DNA detection tests (Hybrid Capture II [HCII] and PCR-enzyme immunosorbent assay (EIA) using the primers GP5+/GP6+ followed by HPV typing with reverse dot blot hybridization) for sensitivity and specificity for detection of CIN and of CIN recurrence after treatment. Two hundred and thirty-nine women referred to the Department of Obstetrics and Gynaecology in V?ster?s, Sweden, were enrolled because of atypical Pap smears; 177 of these were later treated for dysplasia by conization or loop diathermy. Samples for HPV DNA testing were taken before and 4 to 6 months after treatment. There was substantial agreement between the HCII and PCR-EIA (kappa, 0.70 before treatment and 0.72 after treatment). The sensitivity for histopathologically confirmed CIN III was 100.0% for PCR-EIA and 95.6% for HCII. For patients with CIN II or worse (CIN II+), the sensitivities were 92.9% (PCR-EIA) and 91.8% (HCII). The specificities for CIN II+ in the pretreatment setting were 30.4% for PCR-EIA and 24.1% for HCII. After treatment, the sensitivities for CIN III in cytology were 100.0% by both methods, and for CIN II+, sensitivities were 80.0% by both methods. The specificities for CIN II+ in the posttreatment setting were 83.5% for PCR and 85.4% for HCII. In conclusion, the sensitivities of both PCR-EIA and HCII are high and almost equal, suggesting that both methods are suitable as tools for detection and posttreatment follow-up of CIN II-III.     

Analysis of human papillomavirus type-16 variants in Italian women with cervical intraepithelial neoplasia and cervical cancer

Human papillomavirus type 16 (HPV-16) classes (E, AA, As, Af1, Af2) and their variants have different geographic distribution and different degrees of association with cervical lesions. This study was designed to examine HPV-16 variants among Italian women and their prevalence in case patients (affected by invasive cervical carcinoma or cervical intraepithelial neoplasia grade 2-3 and cervical intraepithelial neoplasia grade 1), versus control subjects with normal cervical epithelium (controls). A total of 90 HPV-16 positive cervical samples from women of Italian Caucasian descent have been tested, including 36 invasive cervical carcinomas, 21 with cervical intraepithelial neoplasias grade 2-3, 17 with cervical intraepithelial neoplasia grade 1 and 16 controls. HPV-16 was detected with an E6/E7 gene-specific polymerase chain reaction, and variant HPV-16 classes and subclasses were identified by direct nucleotide sequencing of the region coding for the E6 and the E7 oncoproteins, the MY09/11-amplified highly conserved L1 region, and the long control region (LCR). Among the 90 HPV-16 samples, nine viral variants have been identified belonging to the European (Ep-T350 and E-G350) and non-European (AA and Af-1) branches. The E-G350 is the prevalent variant in all analyzed different disease stages being present in 55.5% of ICC, 52.4% of cervical intraepithelial neoplasias 2-3, 47.1% of cervical intraepithelial neoplasia grade 1, and 50.0% of control samples. The non-European variants AA and Af1, rarely detected in control samples, represent 33.3% of all HPV-16 infections in invasive cervical carcinoma (with a peak of 19.4% and 13.9%, respectively), showing a statistically significant increase in frequency in more advanced lesions (chi(2) trend = 7.2; P < 0.05). The prevalence of HPV-16 Ep-T350, however, is higher in controls (43.7%) and in of cervical intraepithelial neoplasia grade 1 (41.2%) than in cervical intraepithelial neoplasia grade 2-3 (28.6%) and in invasive cervical carcinoma (11.1%) cases strongly suggesting lack of progression for pre-neoplastic lesions associated with such variant. The increased frequency of non-European variants in invasive lesions suggests that they are more oncogenic than European variants. This could have implications for future diagnostic and therapeutic strategies.     

High‐risk human papillomavirus load and biomarkers in cervical intraepithelial neoplasia and cancer

The purpose of this study was to examine the implication of high‐risk human papillomavirus (HPV) load in cervical intraepithelial neoplasia (CIN) and cancer, and to detect biomarkers in cervical disease. We conducted high‐risk HPV DNA load and cervical cytology tests in 343 women, cervical tissue biopsy in 143 women, and immunohistochemistry for p16^INK4A, cyclin D1, p53, cyclooxygenase‐2, Ki‐67, GLUT1, hPygopus2, and beta‐catenin. As a result, HPV load [relative light units (RLU) value] was correlated with the histological severity of cervical disease (p < 0.05). In the ‘atypical squamous cells of undetermined significance’ cytology group, 2.385 of HPV load seemed to be the cut‐off value at which ‘benign’ or CIN I can be differentiated from ‘CIN II or more severe’ (AUC = 0.712), but not statistically significant. The relative risk (odds ratio) of p16^INK4A and GLUT1 overexpression increased gradually according to the histological severity of cervical disease. The p16^INK4A showed statistically significant odds ratios in CIN II, CIN III, and cancer; GLUT1, in CIN II and CIN III; hPygopus2, in CIN III; and beta‐catenin, in CIN III and cancer. Conclusively, HPV load, p16^INK4A, and GLUT1 can be instrumental in predicting the severity of HPV‐related cervical disease. The beta‐catenin/hPygopus2 signaling may be involved in proceeding to CIN III.     

New biomarkers of human papillomavirus infection in acute cervical intraepithelial neoplasia

Acute human papillomavirus (HPV) infection of the cervix (cervical intraepithelial neoplasia, CIN) is marked by high copy episomal viral DNA and L1/L2 capsid protein expression (productive infection) in the cells towards the surface that facilitate sexual viral transmission. Viral DNA is low copy and not associated with viral capsid protein expression in the less differentiated lower part of the CIN (nonproductive infection). The purpose of this study was to examine the host response in these two areas. Serial section and co-localization analyses demonstrated that in 29/33 (88%) of cases the NF-κB pathway was activated and localized to the suprabasal nonproductively infected cells in the CIN lesions. There was a concomitant increased expression of importin-β, exportin-5, Mcl1, p16, Ki67 and cFLIP in 32/33 (96%) of CIN lesions that likewise localized primarily to the nonproductively infected cells. Only Ki67 and exportin-5 were expressed, though much less so, in the adjacent, normal squamous epithelia. The viral proteins E1^E4 and L1 were localized to productively infected cells whereas E6/E7 protein/RNA was rarely present in early CIN. It is concluded that the host viral response to acute cervical HPV infection includes strong increased expression of proteins besides p16 and Ki67. These include importin-β, exportin-5, Mcl1, and cFLIP in cells with low copy and relatively quiescent viral DNA that, in turn, may serve as new biomarkers of this disease.     

Type‐specific human papillomavirus prevalence in cervical intraepithelial neoplasia and cancer in Iran

In Iran, human papillomavirus (HPV) vaccination is not currently included in the national vaccination program and there are no comprehensive approaches to cervical screening program. Regional data on distribution of HPV types in women is important to predict the impact of current HPV vaccines. Although several studies on distribution of HPV types in cervical precancer and cancer have been conducted in Iran, in most of them HPV positive samples were subjected to specific‐primer genotyping (mainly 16 and 18), and leaving the other HPV genotypes almost undetermined. Therefore, the present study aimed to investigate the distribution of HPV types in cervical neoplasia from West and Northwest of Iran. A total of 112 women with atypia, cervical intraepithelial neoplasia, and invasive cervical cancer were included. A PCR assay was performed in all samples to detect the presence of the HPV genome using the GP5+/6+ L1 consensus primer set. All HPV positive samples were subjected for sequencing. In overall, HPV prevalence was 20% in atypica, 44.5% in cervical intraepithelial neoplasia I, 92.3% in cervical intraepithelial neoplasia II‐III, and 98.2% in invasive cervical cancer. The most frequent HPV type was HPV 16 (79.2%), which was followed by HPV types 18, 6, and 33 at the frequencies of 6.5%, 5.1%, and 2.7%, respectively. The least HPV types were found to be 31, 45, 53, 58, and 66. In conclusion, this study shows that the current HPV vaccines could have great impact to reduce the burden of cervical cancer in Iran.     

Prognostic value of human papillomavirus types 16 and 18 DNA physical status in cervical intraepithelial neoplasia

The aim of this work was to assess the value of the physical status of human papillomavirus (HPV) DNA as a disease marker for cervical cancer development in a set of 248 DNA samples previously genotyped as HPV 16 or 18, by calculating the E2/E6 ratio through real-time PCR. There was a significant difference in integration status according to disease grade for both genotypes (p <0.001). Furthermore, especially for HPV 18, determining the DNA physical status could be a useful biomarker in predicting cervical cancer risk development, with a lower E2/E6 ratio clinically associated with the development of a precancerous lesion.     

HPVDNA永生化人宫颈上皮细胞的生长方式与宫颈上皮内肿瘤的比较

目的：研究人乳头瘤病毒（HPV）DNA永生化人宫颈上皮细胞在器官培养的生长特点及将其与体内宫颈上皮内肿瘤（CIN）进行比较。方法：先用HPV16和18型DNA转染人宫颈上皮细胞,建立永生化的人宫颈上皮细胞株,再用胶原筏培养方法分析永生化人宫颈上皮细胞在器官培养的生长特点,将其与体内CIN的形态进行了比较。结果：人宫颈上皮细胞经HPV16和18DNA转染后,可以使其变成一种永生化细胞。细胞生物学研究显示永生化细胞是一种前恶性细胞。胶原筏培养显示水生化人宫颈上皮细胞的生长行为类似体内CIN。结论：HPV感染主要影响宫颈癌的发生的早期阶段。     

Comparison of prevalence of human papillomavirus antigen in biopsies from women with cervical intraepithelial neoplasia.

Human papillomavirus antigen was found in 39 (16%) of 253 colposcopic biopsies from a group of women at high risk for cervical cancer who had been examined in the early 1970s. Immunohistochemical evidence of papillomavirus infection was found in 20 (30%) of 67 cervical intraepithelial neoplasia lesions infected with wart virus from these patients. When these results were compared with results of a similar study carried out in the early 1980s there was no significant difference in the prevalence of human papillomavirus antigen in cervical intraepithelial neoplasia lesions infected with wart virus from women who had been examined over a decade apart.     

宫颈瘤变程度与其相对应的HPV基因型分布规律的研究

目的研究不同级别宫颈上皮内瘤变（cervical intraepithelial neoplasia,CIN）与人乳头瘤病毒（Human papil-lomavirus,HPV）基因型分布之间的关系。方法收集321例经组织病理学诊断确定为CIN2及以上（CIN2＋）病人的宫颈分泌物标本,其中CIN2 67例,CIN3/ACIS 247例,浸润性宫颈癌7例。采用深圳亚能生物技术有限公司的人乳头瘤病毒（HPV）基因分型检测试剂盒对所选标本进行HPV检测及基因分型。结果在所有321例标本中,HPV阳性300例,阳性率93.5%,两型及两型以上感染138例,占比43.0%。HPV16最常见,检出率为41.1%（132/321）,其次为HPV31,33,52,18和51。HPV16和33的单型感染在CIN3＋中比CIN2更常见,具有统计学差异（P〈0.05）。在包含有HPV16或HPV33的多型感染中,含有HPV16或HPV33的多型感染在CIN3＋中比CIN2更常见,具有统计学意义（P〈0.05）。结论 HPV16和HPV33比其他型别HPV具有更强的潜在致癌性。     

Genotyping of human papillomavirus in cervical intraepithelial neoplasia in a high-risk population

Infection with the human papillomavirus (HPV) is responsible for 99.7% of cervical cancers, the second most prevalent neoplasia in women worldwide and the fifth leading cause of death by cancer in this population. In Chile, the incidence rate is 14.4 cases per 100,000 women per year and it is considered a significant public health problem. The natural history of cervical cancer begins gradually from low-grade and high-grade squamous intraepithelial lesions to an invasive disease. In this study the frequency of HPV types was determined by HPV genotyping with reverse line blot hybridization in 200 cytobrushes of women with preneoplastic lesions in a high-risk population. HPV DNA was found in 89% of the lesions (83.3% of low-grade squamous intraepithelial lesions and 93.6% of high-grade squamous intraepithelial lesions). Multiple HPV infections were found in 14.4% and 15.5% of low- and high-grade lesions, respectively. HPV 16 was the most frequent genotype in single infections, followed by HPV 18. These results show that most of the preneoplastic lesions of the cervix (60%) were associated with HPV 16 and/or HPV 18, supporting the implementation of an HPV vaccination program in this high-risk population.     

Hybrid capture II, a new sensitive test for human papillomavirus detection. Comparison with hybrid capture I and PCR results in cervical lesions

AIM: To test a new assay for the detection of human papillomavirus (HPV) DNA, hybrid capture II (HC II), compared with the previous commercialized hybrid capture I (HC I) and polymerase chain reaction (PCR) results on cervical scrapes from fresh cone excision biopsy samples. METHODS: The three methods were used on cervical scrapes from 42 fresh cone excision biopsy samples. There were nine metaplastic and inflammatory lesions, five low grade lesions, and 28 high grade lesions. PCR was performed using the general primers GP5+/GP6+. The viral load of high risk HPV DNA was estimated by the ratio of relative light units to positive control values in the samples. RESULTS: The sensitivity of HC I for the detection of high grade lesions was 71.4%, while it was 92.8% for HC II and 96.4% for the PCR. Considering only the absence of detectable cervical in situ neoplasia, the specificity was 88.9% for HC I, 66.7% for HC II, and 66.7% for PCR. With HC II, for a ratio of cervical sample to normal control of > 200, the sensitivity for the detection of high grade lesion was only 34.6% with a specificity of 66.7%. CONCLUSIONS: HPV detection with the HC II assay is more sensitive than the previous HC I and represents a more convenient and easier test than PCR for routine use. Nevertheless the viral load estimated with this test cannot be a reliable predictive indicator of high grade lesions.