宫颈病变中HPV16感染状况、微卫星不稳定性及错配修复基因表达的研究

目的通过检测HPV16、微卫星不稳定性（microsatellitein stability,MSI）和错配修复基因（hMLH1、hMSH2）的表达,探讨三者在宫颈病变进展中的作用及意义。方法将宫颈病变组织分为宫颈炎、宫颈上皮内瘤变（cervicalintraepithelialneoplasia,CIN）CIN1、CIN2～CIN3、宫颈癌4组。用PCR方法检测HPV16感染状况,用聚合酶链-单链构象多态性分析方法检测宫颈组织中MSI的表达,采用免疫组织化学SP法来检测宫颈组织中的hMLH1和hMSH2的表达,并分析三者的相关性。结果CIN120例中25.0%（5/20）检测到HPV16,CIN2～CIN331中54.8%（17/31）检测到HPV16,二者比较χ2=4.413,P=0.034;宫颈癌中各期HPV16感染比较,差异均无统计学意义。选取的三个微卫星位点D3S2832E、RH91127和SHGC-56838均在宫颈炎中未检测到MSI,在CIN和宫颈癌中均检测到MSI的存在。但总体的表达率较低,最高只有46.7%。错配修复基因的低表达检测结果和MSI的检测结果一致,唯一不同的是前者的总体表达率较高,最高的低表达率为80%;宫颈病变中MSI与错配修复基因（本文即hMLH1、hMSH2）蛋白低表达及HPV16为正相关。结论HPV16的感染导致错配修复基因的低表达,进一步引起微卫星不稳定性,可能是宫颈癌发生、发展的重要机制,HPV16、微卫星不稳定性和错配修复基因有望作为宫颈癌高危人群的检测指标。     

子宫颈癌微卫星不稳定性和HPV感染的研究

目的　探讨微卫星不稳定性 (microsatelliteinstability ,MI)及人乳头瘤病毒 (HPV)感染与宫颈癌的相关性。方法　 2 2例宫颈浸润性鳞癌石蜡标本 ,选取 3、 5、 6号染色体上的 3个微卫星位点D3S1 2 89、D5S4 0 6、D6S2 77进行MI分析 ;选用HPV 1 6 / 1 8型特异引物进行HPV检测 ;应用免疫组化法检测Ki6 7蛋白的表达。结果　在宫颈癌标本中 3个位点均未发现MI和杂合性缺失 (LOH)的改变 ;HPV1 6 / 1 8检出率为 77 3% ,Ki6 7指数明显高于对照组。结论　 3个微卫星位点未见MI和LOH的改变 ,与国外报道不同 ,可能与种族差异有关。HPV分型及Ki6 7蛋白表达的检测有助于宫颈病变的评价     

人乳头状瘤病毒16型E6、E7基因诱导的人子宫颈永生化上皮细胞系的建立及其生物学特性的鉴定

目的 建立人宫颈上皮的永生化细胞系,并进行生物学特性进行鉴定。方法 用含人乳头状瘤病毒(HPV)16型E6、E7基因的腺病毒伴随病毒感染人胎儿宫颈上皮细胞,培养、传代。采用激光共聚焦免疫荧光和PCR技术检测HPV16型E6、E7基因的表达情况;取20代细胞,采用软琼脂培养、染色体核型分析和Scid小鼠皮下接种等方法检测细胞系的生物学特性;采用电子显微镜观察细胞的形态。结果 20代细胞的表型仍保留原代上皮细胞的特征,表现为单层生长和锚锭依赖性生长。激光共聚焦免疫荧光和PCR技术检测显示,细胞内有HPV16型E6、E7基因的表达,其基因片断长度为829bp。20代细胞进行软琼脂培养不形成克隆;scid小鼠皮下接种未成瘤;染色体核型分析：勾二倍体和多倍体,11号染色体可能为HPV16型E6、E7基因整合的部位。电子显微镜观察可见张力原纤维,证实细胞为鳞状上皮来源。结论 成功建立了HPV16型E6、E7基因诱导的宫颈上皮永生化细胞系,且其生物学特性稳定,可进一步用以宫颈癌病因和发病相关机制的研究。     

Role of human papillomavirus testing in reducing the number of surgical treatments for precancerous cervical lesions

PURPOSE OF INVESTIGATION: To determine whether the addition of the Hybrid Capture II (HC II) test (Digene Corp., Gaithersburg, MD, USA) to cytological, colposcopical and histological results could reduce the number of surgical treatment procedures for precancerous cervical lesions. METHODS: Surgical treatment of precancerous cervical lesions was performed in 181 women. Priorly, the women were tested for high-risk human papillomavirus (HPV). Sensitivity, specificity, positive and negative predictive value were calculated to assess the performance characteristics of HC II in the detection of cervical intraepithelial neoplasia grade 2 or worse (CIN 2+) and grade 3 or worse (CIN 3+). RESULTS: Eighty (44.2%) women had a histological result < CIN 2; 117 (64.6%) women had < CIN 3. Fifty-three (29.3%) women with < CIN 2 tested HPV negative; 69 (38.1%) women with < CIN 3 tested HPV negative (p < 0.05). The sensitivity of HC II for detecting CIN 2+ and CIN 3+ was 76.2% and 87.5%, respectively. CONCLUSION: A high proportion of women were overtreated probably due to cytological and histological overestimations. HPV testing would reduce the number of unnecessary surgical treatments and should be used as an additional screening tool.     

Cervical response to vaccination against HPV16 E7 in case of severe dysplasia

OBJECTIVE: To evaluate the tolerance to vaccination against human papillomavirus (HPV)16 E7 (in SB adjuvant ASO2B) and its histological and immunohistological effects on HPV16 associated high-grade cervical dysplasias associated with HPV16. STUDY DESIGN: Five patients with histologically demonstrated severe cervical dysplasia (CIN3) HPV16 positive were injected three times before conization was performed 2 months after the first injection. We studied cytological, histological, proliferative pattern and immune profile before and after vaccination. The slides were compared with those obtained from non-injected patients. RESULTS: The injections were well tolerated and the specimens displayed a limited regression of the lesions. Nevertheless, massive CD4 and CD8 T cell lymphocytic infiltration was noticed after vaccination. DISCUSSION: We conclude that the vaccination we used provides an obvious immune histological reaction in the HPV infected cervix and that the 2 months delay before the final step (conization) is done is probably too short.     

Persistence of human papillomavirus as a predictor for treatment failure after loop electrosurgical excision procedure

We aimed to investigate whether postconization human papillomavirus (HPV) DNA testing can predict treatment failure and improve the accuracy of conventional follow-up in women with high-grade cervical intraepithelial neoplasia (CIN). Between March 2001 and October 2005, 120 patients with confirmed CIN 2 or 3 were treated with loop electrosurgical excision procedure (LEEP) and were enrolled. Six patients were lost to the follow-up. Postconization follow-up was performed at every 3-6 months during the first year and then annually. Specimens were tested for the presence of HPV, using the Hybrid Capture 2 (Digene Co, Gaithersburg, MD) and HPV DNA chip (Mygene Co, Seoul, Korea) test. Persistent HPV infection was defined as persistently (two times or more) positive HPV tests with the same HPV subtype(s) at initial diagnosis. Twenty-two (19.3%) patients showed treatment failure after conization. The only significant risk factor for redevelopment of CIN after conization was persistence of the same HPV subtype (P < 0.0001). And women with recurrent or residual CIN had higher HPV load during the 6-month follow-up postconization. In conclusion, the persistence of the same HPV subtype after LEEP conization was an important predictor of treatment failure. The follow-up protocol after conization of CIN should include both cervical cytology and HPV test, and HPV DNA chip test is needed to detect a persistent HPV infection.     

Expanded cytological referral criteria for colposcopy in cervical screening: comparison with human papillomavirus testing

OBJECTIVE: The goal of this study was to investigate whether expanded cytologic referral criteria for colposcopy or the addition of human papillomavirus (HPV) testing on cervical screening could improve the rates of detection of cervical intraepithelial neoplasia (CIN). METHODS: HPV testing by semiquantitative polymerase chain reaction/ELISA was performed in 1000 women who were self-referred for routine Pap smear. They underwent colposcopy following an abnormal smear result or a positive HPV test. As abnormal smear results were considered reports of low- or high-grade squamous intraepithelial lesion, atypical squamous cells of undetermined significance, and even HPV-associated reactive cellular changes (mild koilocytosis, mild dyskeratocytosis, hyperchromatic nuclei, bimultinucleation, and cleared cytoplasm). Loop excision of the transformation zone was performed in women with cytology and colposcopy indicative of CIN, as well as in women with normal cytology but positive HPV test and colposcopic impression of CIN. RESULTS: The Pap test was abnormal in 89% of the cases of CIN 1 (34/38) and 96% of CIN 2/3 (27/28) diagnosed in our population. HPV testing picked up four additional cases of CIN 1 (11%) and one case of CIN 2/3 (4%). Overall the HPV test detected 95% of the cases of CIN 1 (36/38) and 89% of the cases of CIN 2/3 (25/28). CONCLUSION: HPV testing does not appear to add significantly to cytology in terms of positive predictive value or detection rate, if extended cytologic indications for colposcopy are used.     

Interest of Human Papillomavirus DNA quantification and genotyping in paired cervical and urine samples to detect cervical lesions

Background

Cervical cancer is caused by persistent infection with high-risk human papillomavirus (HR-HPV). Conventional human papillomavirus (HPV) testing requires cervical sampling. However, vaginal and urine self-sampling methods are more acceptable for patients and result in increased participation when they are available in screening programs. In this context, we have developed a non-invasive screening method via the detection of HPV DNA in urine samples.

Purpose

To compare HPV viral loads and genotypes in paired cervical and urine samples, and to assess correlation between virological and cytological results in women seeking gynecological consultation.

Methods

Paired urine and cervical specimens were collected and analyzed from 230 of 245 women participating in the previously described prospective PapU study. HPV DNA detection and quantification were performed using a real-time PCR method with short fragment PCR primers. Genotyping was carried out using the INNO-LiPA HPV genotyping assay.

Results

The prevalence of HPV in the 230 paired urine and cervical smear samples was 42 and 49 %, respectively. Overall agreement for HPV positivity and negativity between the paired samples was 90 % (κ = 0.80). High HPV viral load in both cervical and urine samples was associated with cytological abnormalities. HPV-positive women were mostly infected with HR-HPV types. The agreement between high- and low-risk HPV (LR-HPV) detection in both samples was 97 % (κ = 0.95 for HR-HPV and κ = 0.97 for LR-HPV).

Conclusions

High concordance rates for HPV-DNA quantification and high/low-risk HPV genotyping in paired urine/cervical samples suggest that urinary HPV DNA testing could be useful for cervical lesion screening.     

Interobserver agreement in the evaluation of digitized cervical images

OBJECTIVE: To estimate the agreement among multiple expert colposcopists evaluating high-resolution digitized cervigrams taken from patients with a variety of human papillomavirus (HPV) infection states and previous cervigram interpretations. METHODS: Twenty expert colposcopists evaluated 939 digitized images of the uterine cervix obtained after the application of 5% acetic acid during the ASCUS-LSIL Triage Study. Twenty images selected to represent a broad range were graded by all the colposcopists. The remaining 919 pictures were distributed by stratified random sampling, such that each image was evaluated by two colposcopists, and each expert evaluated 112 images with similar distributions of cervigram diagnoses and HPV DNA test results. We evaluated interrater agreement among the pairs of colposcopists and confirmed the conclusions using the 20 images they all graded. RESULTS: Pairs of colposcopists agreed on the diagnosis for only 56.8% of images. Similar agreement was seen regarding number of visible lesions (of low-grade or greater). This variability in ratings remained when the images were stratified by final histologic diagnosis or HPV status. The results were confirmed by the presence of large variability in ratings (ranging in some cases from normal to cancer) for the 20 images graded by all colposcopists. CONCLUSION: Colposcopic diagnosis using static images is poorly reproducible and might reflect similar problems in clinical practice. Researchers should question the use of colposcopic images as a reference standard for teaching and evaluating the presence or severity of disease.     

HPV16 and increased risk of recurrence after treatment for CIN

OBJECTIVE: Addition of high-risk human papillomavirus (hrHPV) testing to post-treatment monitoring policies of women treated for high-grade cervical intraepithelial neoplasia (CIN) may improve the effectiveness of detecting recurrent/residual disease. Recent studies have shown that HPV type 16 confers an increased risk of high-grade CIN and cervical cancer. This study aimed to find out whether the post-treatment CIN3 rate is increased in HPV16-positive women treated for CIN3. METHODS: We included 229 hrHPV-positive women treated for CIN3. HPV typing was performed by GP5+/6+-PCR followed by reverse line blotting on a cervical scrape taken before treatment. HPV typing data were related to the occurrence of post-treatment CIN3 within a median follow-up time of 20.1 months (range 3-85.4 months) following treatment. RESULTS: Twenty nine of the 151 (19%) HPV16-positive women versus 6 of the 78 (8%) women with other hrHPV types had recurrent/residual CIN3. Post-treatment CIN3 rate was significantly increased in women with HPV16 compared to those harboring other hrHPV types (p=0.03). None of the other hrHPV types were associated with higher post-treatment CIN3 rates. CONCLUSION: Women treated for HPV16 containing CIN3 should be monitored more intensively because of their increased risk of post-treatment CIN3. Thus, the HPV genotype should be considered in post-treatment monitoring policies.     

Cost-effectiveness analysis of liquid-based cytology and human papillomavirus testing in cervical cancer screening

OBJECTIVE: To compare the outcomes of several cervix cancer screening strategies in a military population using a model that considers both direct and indirect costs of health care. METHODS: A Markov model of the natural history of cervical cancer was used to simulate an age-stratified cohort of 100,000 active duty women in the U.S. Army. Total costs and incremental cost-effectiveness ratios were estimated for different modalities of screening: liquid-based cytology with testing for human papillomavirus (HPV) irrespective of cytologic results compared with liquid-based cytology with HPV detection for cytologic results of atypical cells of undetermined significance (reflex HPV). The costs and outcomes of these screening methods were evaluated separately as well as in combination (liquid-based cytology and reflex HPV before age 30 years and DNA and Pap test every 3 years thereafter). Each of these screening methods was evaluated at 1-, 2-, and 3-year intervals. RESULTS: A screening strategy of liquid-based cytology and reflex HPV every 2 or 3 years is the least costly strategy among active duty women irrespective of age, especially when accounting for time costs associated with screening, diagnosis, and treatment of cervix cancer. A strategy of liquid-based cytology and HPV testing irrespective of cytology results is the most effective strategy; however, it is also the most costly of the strategies tested, even when performed in patients older than 30 years of age. CONCLUSION: In the U.S. Army, cervix cancer screening performed with liquid-based cytology and reflex HPV testing of atypical squamous cells of undetermined significance performed every 2 years is cost-effective, especially when indirect costs are considered.     

基于CT数据人离体胎盘血管网数字化三维模型重建研究

目的 探讨利用CT原始数据进行人离体胎盘血管网数字化三维模型重建的方法及应用。方法   选取2012年5月南方医科大学南方医院妇产科人正常妊娠足月离体胎盘20例,胎盘动脉灌注后进行增强CT扫描并获取DICOM原始数据集,随后行胎盘静脉灌注,胎盘标本再次行增强CT扫描,得到DICOM原始数据集。将两次数据导入Mimics 10.01软件分别重建得到胎盘动脉血管网及胎盘动静脉血管网的数字化三维模型。对CT扫描后的胎盘标本进行强酸腐蚀,得到胎盘血管铸型。对比同一胎盘标本的血管网数字化三维模型及血管铸型,明确数字化三维模型反映胎盘血管形态结构及走行的真实性和准确性。结果   基于CT原始数据,利用Mimics 10.01软件成功构建出胎盘血管网数字化三维模型。重建得到的模型立体感强,三维效果逼真,具有较佳的视觉效果,与胎盘血管铸型一致性高,可以清晰地再现胎盘动静脉各级血管分支的解剖形态结构,且可进行任意缩放、任意角度旋转观察。结论   基于CT原始数据构建的胎盘血管网数字化三维模型可全面、真实地展示胎盘血管,是一种胎盘血管研究的新方法;该模型的建立为后续研究提供了一定的三维重建的技术基础。     

Risk factors for the progression or persistence of untreated mild dysplasia of the uterine cervix

To identify the factors that may predict the progression or persistence of untreated mild dysplasia of the uterine cervix, we performed a retrospective review of 118 patients with histologically verified mild dysplasia who underwent colposcopic biopsies between January 1999 and December 2003. Regression to normal occurred in 70.3%, progression to moderate dysplasia or worse occurred in 11.0%, and persistence of mild dysplasia occurred in 18.7%. In regression/progression analysis, progression of untreated mild dysplasia was 34.5% (10/29) in patients with high viral loads (> or =100 relative light units/positive control [RLU/PC]) and 4.5% (3/67) in those with low viral loads (1 to <100 RLU/PC) and negative human papillomavirus (HPV) tests (P < 0.001). Women with high viral loads had a 13-fold greater chance of progression of untreated mild dysplasia than those with low viral loads and negative HPV tests (CI: 2.494-95.297; P = 0.0022). Those associated with both positive smear and positive HPV test (12/45 = 26.7%) were at a greater risk of progression of untreated mild dysplasia as compared with those with positive smear and negative HPV (0/17 = 0.0%) or those with negative smear and positive HPV test (1/18 = 5.6%). Those with high viral loads and both with positive smear and positive HPV test should be followed closely because of their increased risk of progression of untreated mild dysplasia.     

Impact of human papillomavirus coinfections on the risk of high-grade squamous intraepithelial lesion and cervical cancer

Objective

The molecular and epidemiologic effect of human papillomavirus (HPV) coinfections in the risk of developing cervical cancer is yet unclear. The aim of this study was to determine the frequency HPV coinfections at different stages of cervical lesions in the development of cervical cancer and the impact of HPV specific type interactions on high-grade squamous intraepithelial lesions (HSIL) and invasive cervical cancer (ICC) risk.

Methods

HPV testing was performed in 931 cervical samples diagnosed as: negative for intraepithelial lesion or malignancy (NILM); low-grade squamous intraepithelial lesion (LSIL); HSIL; and ICC. For HPV detection and typing two sets of primers from the L1 region were used in the polymerase chain reaction method (PCR) (MY09/MY11/HMB01 and L1C1/L1C2.1/L1C2.2) and HPV type was determined by PCR product sequence. To look for multiple HPV infections, the E6 nested multiplex PCR method was performed in all DNA samples. Odds ratios were calculated as indexes of the strength of the association between the sample category (LSIL/NILM or ICC/HSIL) and the presence of a given viral combination.

Results

In HPV positive samples, coinfections are as common in ICC/HSIL as in LSIL/NILM (47.12% and 40.17%, respectively). There is an increased risk to ICC/HSIL when multiple high-risk HPV types are present. The coinfection of HPV68 with HPV16 increases the risk of ICC/HSIL (OR = 14.54, P = 0.012, after multivariate adjustment), related to the presence of HPV16 or HPV68 alone.

Conclusions

These results sustain that specific HPV coinfections confer an increased risk to develop ICC/HSIL.     

Cytologic correlates of cervical papillomavirus infection

A fundamental question in Papanicolaou smear screening is the specificity of cytologic criteria for the recognition of genital human papillomavirus (HPV) infection. To address this problem, we conducted a two-phase study of routinely screened women to determine the efficiency with which cytologic findings identified the presence of HPV DNA, focusing on the criteria for identifying smears as "atypical." In phase 1, 25 of 290 (8.6%) smears were designated atypical, but only 3 (12%) of the samples contained HPV nucleic acids. Four of five (80%) smears designated as diagnostic of HPV/cervical HPV infection were associated with HPV nucleic acids. By applying more stringent criteria for the diagnosis of atypical in phase 2, only 3 of 166 (1.8%) were identified as atypical. Of these, two (67%) contained HPV nucleic acids. The criteria that most efficiently correlated with HPV nucleic acids included prominent nuclear enlargement with either multiple nuclei or nuclear hyperchromatism. On review of the 19 HPV-positive and 20 control HPV-negative smears originally diagnosed as cytologically negative, the above criteria identified an additional 3 cytologically atypical/positive smears versus none (0 of 20) in the control group. This study supports the concept that cytologic abnormalities suggesting "subtle" HPV infection may be extremely difficult to distinguish from non-HPV-related changes, and that criteria used to imply "suggestive but not diagnostic for HPV infection" should be continually reevaluated. The potential role of HPV DNA analysis in Papanicolaou smear interpretation is discussed.     

A repetitive DNA sequence that characterizes human papillomavirus integration site into the human genome is present in vulvar vestibulitis

OBJECTIVE: To determine the DNA sequence of polymerase chain reaction (PCR) products obtained from surgical specimens of patients with severe vulvar vestibulitis, in order to identify and type the human papillomavirus (HPV)-DNA associated with vulvar vestibulitis. STUDY DESIGN: Fifty three women, referred for dyspareunia and diagnosed as having severe vestibulitis, underwent perineoplasty operation consisting of surgical removal of the sensitive vestibule. PCR analysis using L1 HPV primer was performed, and DNA sequencing of the samples that were found to contain HPV-DNA was undertaken, using the dideoxy chain termination method. RESULTS: Using PCR, HPV-DNA was detected in 31 of 53 tissue specimens (58%). DNA sequencing of 12 HPV-positive PCR products revealed extensive homology to human Alu consensus sequence, albeit not to any known HPV sequence. CONCLUSIONS: The presence of interspersed, repetitive-DNA sequence Alu, which is known to be the preferred site for HPV integration into human genome, in the PCR product reinforces previous observations, suggesting that HPV may have a role in the pathogenesis of vulvar vestibulitis. It further implies a possible integration of the HPV into human DNA in these cases.     

Adding HPV16 testing to abnormal cervical smear detection is useful for predicting CIN3: a prospective study

BACKGROUND: The aim of this prospective study was to estimate whether adding human papillomavirus 16 (HPV16) testing to abnormal cervical smears is useful in the prediction of cervical intraepithelial neoplasia 3 (CIN3). METHODS: Between October 1994 and May 1996, a total of 207 patients at the Akita University Hospital had abnormal smears. Of these patients, 153 patients with CIN1,2 or atypical squamous cells of undetermined significance (ASCUS) were enrolled in this study and followed until June 2001. At the initial visit, a cervical swab was collected for cytology and for HPV16 testing using nested polymerase chain reaction (PCR). When the HPV16 test was positive, HPV16 testing was performed every 3 to 6 months. We compared the prevalence of progression to CIN3 between the HPV16-positive group (n = 16) and the HPV16-negative group (n = 137). We also investigated the risk of progression to CIN3 associated with persistent HPV16 infection. RESULTS: At the end of the study period, four patients (25%) in the HPV16-positive group developed CIN3, and all of these patients were found to have persistent HPV16 infection during this period. Only three patients (2.2%) in the HPV16-negative group developed CIN3. CONCLUSIONS: The prevalence of progression to CIN3 in the HPV16-positive group was significantly higher than that in the negative group (p = 0.0023). The odds ratio of progression to CIN3 was 14.9 [95% confidence interval (CI) 2.98-74.4]. In particular, the risk of progression to CIN3 increased with persistent HPV16 infection. Adding HPV16 testing when abnormal cervical smears are detected promises to be useful for predicting CIN3.     

Detection and genotyping of human papillomaviruses and their role in the development of ovarian carcinomas

Purpose

The objective of this study was to investigate the presence of human papillomavirus (HPV) infection in ovarian carcinoma samples from Serbian women and correlate them with clinicopathological characteristics of disease and patients?? characteristics.

Methods

Fifty-four ovarian carcinoma patients were included in the study. DNA was isolated by salting out method from tumor tissue obtained after surgical treatment. Presence of HPV infection was detected through polymerase chain reaction amplification of a 150-bp fragment of L1 viral gene by GP5+/GP6+ primers. HPV genotyping was performed by DNA sequencing.

Results

HPV DNA was present in 4/54 (7.4?%) ovarian carcinomas. All HPV-positive tumors contained high-risk HPV16 type. HPV infection was more common in advanced opposite to localized disease. The median age of diagnosis of disease varied from 57?years for patients with HPV infection to 59?years for patients without HPV infection.

Conclusions

Our results indicate that HPV infection may play a limited role in ovarian carcinogenesis.     

Immunocytochemistry (p16INK4a) as an additional examination for cytological specimens Class III D (Munich Classification II)

Pap smears classified as III D (munich classification II) describes cytological abnormalities according to low and/or moderate squamous epithelial dysplasia (CIN 1 and/or CIN 2). This cytological diagnosis-group is in routine procedure marked by an uncertain predictive statement: the positive predictive value is low, but relatively often occurs a higher degree dysplasia (CIN 3) in follow up examinations. Therefore un-certainties arise both for gynecologists and their patients with a view to the ensuring control- and therapy management respectively. In order to clarify the question, whether an additional immunocytochemical examination using p16 (INK4a) can improve the prognostic statement for patients with cytological diagnosis of group III D, the cytological diagnosis, the HPV test results and the results of the CINtec (TM) p16 (INK4a)-Immunocytochemistry in 357 patients were being drawn up parallel or documented. In the case of 115 patients with actual cytological diagnosis group III D a validation could be performed after histological (56) and cytological (59) follow up respectively. In 63.5 % of these III D-patients p16 was graded immunocytochemically positive, in 73.9 % high risk HPV (HC II) was positive. In relation to the entire follow up cytological diagnosis, high risk HPV und p16 detection have a positive predictive value of 53, 65.5 and 82.4 % at the time of evaluation. The negative predictive values are 80.6 % for high risk HPV and 100 % for p16 respectively. In spite of some methodical restrictions and an uncertain predictive statement concerning the graduation of CIN during the histological clarification in the follow up, the use of p16 (INK4a)-Immunocytochemistry can distinctly improve the prognostic statement in routine procedure for patients with cytological diagnosis of group III D.