Assessment of precancerous lesions of the uterine cervix for evidence of human papillomavirus infection: a histological and immunohistochemical study

Cervical biopsies obtained by colposcopic direction from 358 women were histologically examined for squamous dysplasia (cervical intra-epithelial neoplasia; CIN) and human papillomavirus (HPV) infection. Of the 358 biopsies, 136 were stained by an immunoperoxidase method using an antiserum against genus-specific (common) antigen of bovine papillomavirus. HPV antigens were detected in 40% of biopsies showing definite histological evidence of HPV effect, and in 7.9% and 2.6% of those with possible or no HPV effect, respectively. HPV effect was commonly seen in association with CIN. The frequency of histological evidence of HPV effect and positive immunoperoxidase staining decreased with increasing grades of CIN. HPV antigen was found in 57% of areas of HPV change with minor atypia, 34% of zones of CIN I and in only 8% of zones of CIN II. No antigenic staining or definite histological evidence of HPV effect was observed within areas of CIN III. Antigen was generally confined to the nuclei of superficial koilocytes, cells with lesser degrees of perinuclear clearing and parakeratotic cells. These results how a strong association between HPV infection and precancerous lesions of the uterine cervix and are consistent with the hypothesis that production of the HPV structural antigen requires a high degree of squamous cell maturation. The immunoperoxidase findings and the histopathological observations support the view that HPV change and dysplasia are part of a morphological continuum in which the cytopathic effect of HPV is expressed mainly in lower grades of dysplasia.     

p53 immunoreactivity in cervical intraepithelial neoplasia and non-neoplastic cervical squamous epithelium.

AIMS--To determine the pattern of p53 immunoreactivity in cervical squamous epithelium and to investigate the relation between p53 immunostaining and human papillomavirus (HPV) infection. METHODS--Immunocytochemistry for p53 was performed in 65 specimens of formalin fixed, paraffin wax embedded cervical tissue using a polyclonal antibody against recombinant p53. Microwave oven heating was used for antigen retrieval. Eight normal biopsy specimens, eight cases with histological features of HPV infection, and 49 cases of cervical intraepithelial neoplasia (CIN) were examined. Thirty one cases of CIN were examined. Thirty one cases of CIN were examined for evidence of HPV infection using in situ hybridisation with probes directed against wide spectrum HPV, HPV 16 and HPV 18. RESULTS--p53 immunoreactivity was seen in seven of eight (87%) of specimens with histological features of HPV infection, five of eight (62%) normal specimens, 13 of 22 (59%) CIN III, three of 14 (21%) CIN II and five of 13 (38%) CIN I specimens. The numbers of positive nuclei were small in cases of CIN and the location of positive nuclei within the epithelium paralleled the degree of dysplasia. Eleven of 15 (73%) CIN specimens which were immunoreactive for p53 yielded a positive signal for HPV by in situ hybridisation. A positive signal for HPV was also seen in 10 of 16 (63%) of CIN specimens in which p53 staining was absent. CONCLUSIONS--p53 immunoreactivity can be demonstrated in a small proportion of cells in the cervical squamous epithelium in a significant proportion of cases of CIN. This immunoreactivity seems to be independent of the presence of HPV, as assessed by in situ hybridisation. p53 immunoreactivity also occurs in non-neoplastic cervical squamous epithelium with a pattern of distribution within the epithelium which differs from that seen in CIN. Antigen retrieval by microwave oven heating enhances p53 immunostaining and may result in visualisation of cellular p53 in the absence of mutation.     

Immunohistochemical identification of human papillomavirus infection in tissues from cervical precancerous and early cancerous lesions among Indian women

Paraffin sections of 50 cervical condyloma biopsies from patients with precancerous and early cancerous lesions of the uterine cervix were screened for human papillomavirus (HPV) antigen by peroxidase-antiperoxidase (PAP) staining using genus specific anti-bovine papillomavirus serum (rabbit). The intra-nuclear HPV positivity was observed in 20 percent (10/50) of biopsies studied. Further, proportionately larger number of cases with moderate dysplasia (CIN II) associated with koilocytotic changes were found to be positive for HPV antigen (40%). No HPV positivity was detected from inflammation and mild dysplasia (CIN I) cases. The study strongly supports the association of HPV infection in the process of neoplastic transformation of the uterine cervix.     

人乳头状瘤病毒分型检测在宫颈细胞学诊断为ASCUS分层处理中的意义

目的 探讨人乳头状瘤病毒(human papillomavirus,HPV)分型检测在宫颈细胞学诊断为不典型鳞状细胞意义不明确(atypical squamous cell of undetermined significance.ASCUS)分层处理中的意义.方法 对184例宫颈细胞学诊断为ASCUS的患者,分别进行HPV检测和阴道镜下宫颈组织活检.结果 184例宫颈细胞学诊断为ASCUS的患者中,经组织病理学证实炎症112例(60.87%),CIN Ⅰ级33例(17.93%),CIN Ⅱ级17例(9.24%),CIN Ⅲ级8例(4.35%),宫颈鳞癌4例(2.17%),宫颈湿疣10例(5.43%).其中124例经检测呈高危型HPV(high-risk types HPV,HR-HPV)阳性,阳性率为67.39%(124/184),随后经病理学证实炎症66例(53.23%),CIN Ⅰ级22例(17.74%),CIN Ⅱ级16例(12.90%),CIN Ⅲ级8例(6.45%),宫颈鳞癌4例(3.23%),宫颈湿疣8例(6.45%).HPV阳性组CIN以上病变检出率明显高于HPV阴性组(P<0.003).结论对宫颈细胞学诊断为ASCUS的患者,建议作HPV检测,若HR-HPV阳性,则需进一步阴道镜下宫颈活检;若HPV阴性,酌情处理.     

HPV testing in patients with low grade cervical cytological abnormalities: a follow up study.

AIM: To assess the diagnostic performance of human papillomavirus (HPV) analysis in predicting cervical intraepithelial neoplasia (CIN) grades 2 and 3 in patients with persistent low grade cervical cytological abnormalities. METHODS: Cervical smears from 167 women referred for colposcopy with persistent borderline, wart virus or mildly dyskaryotic changes on cervical screening were analysed by Papanicolaou staining, non-isotopic in situ hybridisation and generic and type specific polymerase chain reaction (PCR) amplification of HPV sequences. Follow up was by cytological and, where appropriate, histological analysis. RESULTS: CIN grade 2 or 3 was identified in 46 patients after a median follow up of 27 months. HPV positivity by both techniques was associated with high grade CIN and with age less than 30 years (median age 33 years). Non-isotopic in situ hybridisation was more predictive but less sensitive than either generic or type specific PCR, but prediction was greater using either molecular technique in women over 30 years of age. CONCLUSIONS: Although the degree of prediction found is of only limited clinical value, the strong association of HPV positivity with both high grade CIN and patient age suggests that further studies of HPV testing in this patient group are warranted.     

人乳头状瘤病毒感染与宫颈癌前病变的关系

目的 探讨人乳头状瘤毒（ＨＰＶ）感染与宫颈湿疣、癌前病变的相关性。方法 对１７９例宫颈细胞涂片异常患者行宫颈多点活检,病理形这观察,其中１２８例同时采用ＰＣＲ方法检测ＨＰＶ－ＤＮＡ,１０例行原位杂交分析。结果（１）形态学观察：宫颈湿疣多为扁平型（９７．３％）;除表现经典的诊断性控空细胞（３９．７％）外,国可见异型性明显的非典型性控空细胞（６０．３％）;宫颈湿疣常伴宫颈上皮内肿瘤（ＣＩＮ,４２．５％     

Detection of human papillomavirus DNA in gynaecological swabs by filter in situ hybridization.

Gynaecological smears from the endo- and ectocervix of women with and without cytological and colposcopic abnormalities of the epithelium were investigated for human papillomavirus (HPV) types 6, 11, 16, and 18 by filter in situ hybridization (FISH). The data were compared with cytological, colposcopic, and histological findings. Of the 266 gynaecological smears, HPV DNA was detected in 84 (32%); of 101 cytologically and colposcopically HPV negative cases, HPV DNA was found in 10%. Of 56 women, cytologically and colposcopically positive for HPV infection, HPV DNA was detected in 68%. The sensitivity of the method was controlled by comparing the results of FISH with those of Southern-blot analysis of five cervical tumour biopsies. The data presented demonstrate the necessity of FISH for identification of the HPV type that might be of prognostic value in cervical pathology. Cytological and colposcopic positivity is a reliable sign in about 70% of the cases where HPV infection was proved by FISH.     

Human papillomavirus infection of the uterine cervix: histological appearances in 28 cases identified by immunohistochemical techniques

Twenty eight biopsy specimens of the cervix showed positive immunohistochemical staining when treated with an antiserum raised against an internal capsid antigen of human papillomavirus (HPV). Histological examination of adjoining sections from the same blocks showed a much wider range of abnormalities than those already described in association with HPV infection. The picture was usually diagnostic. It rested chiefly on identifying the koilocyte--the cell with the perinuclear halo that carries the viral antigen in its nucleus--but abnormal keratinisation was also a feature. The accompanying epithelial findings ranged from normal to CIN III (cervical intraepithelial neoplasia). The latter was of an unusual but distinct appearance, in which cytoplasmic maturation was preserved to some degree but in which gross nuclear atypia was seen in all layers of the epithelium.     

Human papillomavirus genotyping and p16INK4a expression in cervical intraepithelial neoplasia of adolescents.

Adolescents have high rates of human papillomavirus (HPV) infection, and persistent high-risk HPV infection can lead to the development of cervical cancer. The cyclin-dependent kinase inhibitor, p16(INK4a) is overexpressed in cervical intraepithelial neoplasia (CIN), probably due to a persistent and integrated HPV infection. This study investigated p16(INK4a) expression, grades of CIN, and high-risk HPV infection in adolescent cervical biopsies. Biopsies were immunohistochemically stained for p16(INK4a). The presence of wide-spectrum, low-risk, or high-risk HPV was determined by amplifying DNA extracted from the cervical biopsies. Biopsies were classified as cervicitis, 15 cases; CIN 1, 48 cases; CIN 2, 46 cases, and CIN 3, 52 cases. The distribution of p16(INK4a) staining was graded as patchy, diffuse basal, and diffuse full thickness. Pearson's chi(2) tests analyzed the relationships between p16(INK4a) staining, HPV infection, and CIN. Biopsies of cervicitis were negative for HPV and for p16(INK4a) expression. High-risk HPV 16, 18, and 31 increased from 18% in CIN 1 to 66% in CIN 2/3 (P<0.001). In CIN 1, p16(INK4a) was positive in 44% of biopsies with 35% showing patchy, 7% diffuse basal, and one case (2%) showing diffuse full thickness staining. In CIN 2/3, p16(INK4a) was positive in 97% of biopsies with 23% showing patchy, 21% diffuse basal, and 53% diffuse full thickness staining. The difference in the proportions of biopsies showing patchy p16(INK4a) staining in CIN 1 and diffuse full thickness staining in CIN 2/3 was significant (P<0.001). In CIN 1, 61% of high-risk HPV-positive biopsies were p16(INK4a) negative, while all high-risk HPV-positive CIN 2/3 biopsies were p16(INK4a) positive. Diffuse, full thickness p16(INK4a) expression discriminated low-grade from high-grade CIN and appears to be a marker of persistent high-risk HPV infection.     

Human papillomavirus infection, centrosome aberration, and genetic stability in cervical lesions.

DNA replication and centrosome duplication have to be strictly synchronized to guarantee genomic stability. p53, pRb, cyclin E, and cyclin A are reported to be involved in the synchronizing process. We investigated the relationship between papillomavirus infection, centrosome aberration and aneuploidy during genesis of cervical carcinoma. The number of centrosomes found in cells from normal cervical epithelium (n = 5), condyloma acuminata (n = 5), cervical intraepithelial neoplasia (CIN) I, II, and III (n = 14) and invasive cervical carcinoma (n = 5) was analyzed by gamma tubulin immunofluorescence staining. The nuclear DNA content was investigated by image cytometry and human papillomavirus (HPV) infection was determined by polymerase chain reaction. Normal epithelia and condyloma acuminata showed cells with one or two centrosomes, whereas CIN lesions showed cells with an increasing number of centrosomes. This abnormality was found to be lowest in CIN I lesions, increased with advancing grade of CIN and was highest in lesions of invasive carcinomas. In parallel, an increasing number of cells with aberrant DNA content was seen. All carcinomas and all except one of the CIN III lesions showed aneuploidy. Three CIN II cases were aneuploid and two cases with CIN I were tetraploid. Normal epithelia and condyloma acuminata showed diploidy. All invasive carcinomas and lesions with CIN were positive for high-risk HPV types 16, 18, or 31, except one invasive carcinoma and one CIN II lesion positive for universal primers only. Three condyloma acuminata were HPV 16-positive and one HPV 6-positive. The results suggest that high-risk HPV infection is correlated to a progressive numerical disturbance of centrosome replication followed by progressive chromosomal aberrations in CIN lesions and invasive carcinomas.     

Detection of human papillomavirus DNA sequences by in situ DNA-DNA hybridisation in cervical intraepithelial neoplasia and invasive carcinoma: a retrospective study.

Human papillomavirus (HPV) infection of cervical intraepithelial neoplasia (CIN) and invasive cervical carcinoma was investigated using in situ DNA-DNA hybridisation on histological sections of formalin fixed, paraffin embedded tissue to assess the technique's sensitivity and to assess retrospectively the association between HPV16 and invasive cervical carcinoma. HPV DNA was detected in 16 of 33 biopsy specimens of CIN. Cells containing viral DNA were more numerous than those positive for viral structural proteins. HPV DNA was also present in less differentiated cells deeper in the epithelium. The detection rate in CIN was lower than that reported for other hybridisation techniques such as Southern blotting. In a retrospective study of biopsy specimens of invasive squamous carcinoma of the cervix HPV16 DNA, the virus most commonly associated with cervical malignant disease, was found in 20 of 25 cases, including those dating from as far back as 1932. The level of sensitivity was similar to that reported for other hybridisation techniques. DNA positive cells were focally distributed in the invasive tumours, and most tumour cells were negative for viral DNA, a result consistent with the low copy number found in malignant cells. It is concluded that HPV16 is not a new virus but that its prevalence is a result of changes in sexual behaviour and that in situ hybridisation is useful in the localisation of HPV DNA replication in CIN and invasive carcinoma.     

p16INK4A as a marker for cervical dyskaryosis: CIN and cGIN in cervical biopsies and ThinPrep smears

AIM: To examine the potential of p16(INK4A) as a biomarker for dysplastic squamous and glandular cells of the cervix in tissue sections and ThinPrep smears. METHODS: Immunocytochemical analysis of p16(INK4A) expression was performed on 22 normal cervical tissue samples, five cervical glandular intraepithelial neoplasia (cGIN), 38 cervical intraepithelial neoplasia 1 (CIN1), 33 CIN2, 46 CIN3, and 10 invasive cancer cases (eight squamous and two adenocarcinomas). All samples were formalin fixed and paraffin wax embedded, and immunohistochemical analysis was carried out using a mouse monoclonal anti-p16(INK4A) antibody after antigen unmasking. The staining intensity was assessed using a 0 to 3 scoring system. In addition, the expression status of p16(INK4A) was examined in 12 normal ThinPrep smears, one smear exhibiting cGIN, and a total of 20 smears exhibiting mild, moderate, and severe dyskaryosis. Human papillomavirus (HPV) detection was carried out using a modified SYBR green assay system. Fluorogenic polymerase chain reaction (PCR) and solution phase PCR were used for specific HPV typing. RESULTS: p16(INK4A) immunoreactivity was absent in all normal cervical tissues examined. Dysplastic squamous and glandular cells were positive for p16(INK4A) expression in all cases included in this study, except for one CIN3 case. p16(INK4A) expression was mainly nuclear in CIN1 cases, and both nuclear and cytoplasmic in CIN2, CIN3, cGIN, and invasive cases. All cases positive for HPV expressed the p16(INK4A) protein, although not all cases found positive for p16(INK4A) were HPV positive. In general, the p16(INK4A) staining intensity was lower in cases negative for HPV or those containing a low risk HPV type. CONCLUSION: This pattern of overexpression demonstrates the potential use of p16(INK4A) as a diagnostic marker for cervical squamous and also glandular neoplastic lesions. In addition, the technique can be used to identify individual dyskaryotic cells in ThinPrep smears. Thus, p16(INK4A) is a useful marker of cervical dyskaryosis.     

Detection of human papillomavirus DNA by molecular hybridization in tube: interest in cervical neoplasia]

The presence of human papillomavirus (HPV) DNA in 79 cervical specimens obtained from 70 patients was studied by using a molecular hybridization technique performed in tube. The results were compared to those of the cytological and histological studies. The molecular hybridization technique in tube (Hybrid Capture I) detects two groups of HPV types. One group is highly associated with the development of cancer (types 16, 18, 31, 33, 35, 45, 51, 52, 56) whereas the second group (types 6, 11, 42, 43, 44) is not. Among 42 patients with cervical lesions before any treatment, high risk DNA of HPV was found in 50% of those with low grade cytology and 90% with high grade cytology. In total, 32 out of the 42 patients (76%) who presented histological lesions, were actually infected by HPV. Samples were obtained before and after treatment from 9 patients. Seven out of 9 presented high grade cervical intraepithelial neoplasia (CIN) and 2 other patients had low grade CIN. HPV DNA was not detected in any of the patients after treatment. Detection of HPV DNA by molecular hybridization in tube is simple, sensitive, standardized, inexpensive and is well adapted to screening programs. It can be used in complement of the cytological diagnosis, in the surveillance of equivocal cytological abnormalities, and in the follow-up of treated patients.     

Cytopathic effects of human papillomavirus infection and the severity of cervical intraepithelial neoplasia : A frequency study

Cytopathic effects related to the human papillomavirus (HPV) infection are more frequently found in cervical intraepithelial neoplasia (CIN) 1; however, there are indications that at least half the histological diagnoses of CIN2 and CIN3 include koilocytosis areas. The objective of this study was to evaluate the frequency of the cytological criteria suggestive of HPV infection in the cervical smears of women with a histological diagnosis of CIN. One hundred and sixty-two women with abnormal cervical smears and a diagnosis of CIN confirmed by histopathology were selected, including 46 cases of CIN 1, 42 of CIN 2 and 74 cases of CIN 3. Koilocytosis was found in 63% of the smears from women with a histopathological diagnosis of CIN 1. This sign was observed in 26.2% and 25.7% of smears of women with a diagnosis of CIN 2 and CIN 3, respectively. Cytomegaly also was frequent in cervical smears of women with histopathological diagnosis of CIN 1 (71.8%). On the other hand, spindle cells and atypical metaplasia were more frequent in women with CIN 2 and CIN 3. Atypical parakeratosis showed similar frequency in all grades of CIN diagnosis. Koilocytois and cytomegaly were inversely correlated with the diagnosis of CIN2 or CIN 3, with OR values respectively of 0.30 (95%CI 0.13-0.68) and 0.26 (95%CI 0.11-0.58). The others signs analyzed did not show any significant association. Koilocitosis and cytomegaly can provides good reassurance that a patient with atypical cervical smear have CIN 1.     

子宫颈癌及癌前病变HPV16、Ki-67的表达及其相关性

目的对子宫颈癌及癌前病变中HPV感染和增殖细胞核抗原Ki-67表达情况进行研究。方法对45例子宫颈浸润性鳞状细胞癌、5例子宫颈腺癌、35例子宫颈上皮内瘤变（CIN）和5例正常宫颈组织采用免疫组化EnVision法进行HPV16和Ki-67表达的检测。结果HPV16阳性率为78．9％（71／90）,其中HPV16阳性率在子宫颈癌组织中为76．0％（38／50）,在CIN病变中为94．3％（33／35）,在正常子宫颈黏膜上皮组织中为阴性（0／5）。Ki-67阳性细胞在正常子宫颈、CIN和子宫颈癌组织中表达逐级增加,显示Ki-67表达程度与组织学类型有关。HPV和Ki-67在CIN和癌组织中的表达呈显著相关（P〈0．005）。结论瑞安地区子宫颈癌及癌前病变组织中存在HPV感染,HPV感染可能在子宫颈癌的发生、发展中起着重要作用。联合应用Ki-67与HPV可作为筛选子宫颈癌高危个体有价值的生物学标记。     

Assessment of the effectiveness of HPV16/18 infection referred for colposcopy in cervical cancer screening in Northwest of China

To evaluate the effectiveness of Human papillomavirus16/18 infection referral to colposcopy in cervical cancer screening for women aged 25 years and older in Chinese northwest region Shaan'xi province. A total of 2224 women were diagnosed with primary high‐risk HPV infection by HPV‐DNA genotyping technology during August 2014 to August 2015. A total of 1916 cases referred for colposcopy with histological evidence were enrolled, including 1124 women with HPV16/18 genotype and 792 with other High‐risk human papillomavirus genotypes. A total of 1916 women aged 25 years and older with HR‐HPV positive were referred to colposcopy. The distribution of HPV16, HPV18, and other HR‐HPVs infection were 49.22%, 9.45%, and 41.33%, respectively. 71.56% had normal cervical histology, 7.05% had Cervical Intraepithelial Neoplasia1, 8.82% had CIN2, 7.25% had CIN3, and 5.32% had cervical cancer. The percentage of positivity of HPV16 and HPV18 was highly associated with the relative risk of cervical lesion. The sensitivity and specificity of HPV16/18 for detection of CIN2+ (CIN3+) was 82.68% (92.12%) and 47.87% (46.15%), respectively. The positive predictive value and negative predictive value of HPV16/18 for detection of CIN2+ (CIN3+) was 30.16% (19.75%) and 91.03% (97.60%), respectively. HPV16 and HVP18 are the most common genotypes in high grade cervical lesions in Shaan'xi province. Meanwhile, these two types play predominant roles in the progression of high grade cervical lesion. Primary HPV16/18 detection has high sensitivity and negative predictive value in cervical cancer screening and the strategy for women with HPV16 and HPV18 infection referral to colposcopy is efficient and feasible in northwestern region of China.     

Papillomavirus and c-myc antigen expression in normal and neoplastic cervical epithelium.

Cervical punch biopsy specimens or brushings were collected from 33 patients with cervical human papilloma virus (HPV) infection, cervical intraepithelial neoplasia (CIN), or invasive cervical carcinoma, and from eight control patients with recent normal cervical cytology. Prostatic chippings obtained from six men with benign prostatic hypertrophy were used as further controls. Biopsy specimens and brushings were assayed by flow cytometry for c-myc oncogene antigen and papillomavirus antigen expression and rate of cell division (by measuring DNA content). Results obtained from analysis of specimens and brushings were similar in terms of c-myc antigen and total DNA content, but when the percentages of nuclei from biopsy and brush specimens staining positively with antibody to papilloma viral antigens were compared, brush specimens gave consistently higher percentages than biopsy specimens. More specimens from normal epithelium were c-myc antigen positive (five of eight, (63%) than specimens from CIN II or III (two of 10, 20%), or invasive carcinoma (0%). No association was found between c-myc antigen expression and cell division. HPV antigen positive specimens were found to contain more dividing cells than negative specimens.     

p16(INK4a) as a complementary marker of high-grade intraepithelial lesions of the uterine cervix. I: Experience with squamous lesions in 189 consecutive cervical biopsies

AIM: To test the usefulness of p16(INK4a) immunostaining for improving the diagnostic accuracy of cervical punch biopsies referred to a routine laboratory setting during the investigation of women with abnormal Papanicolaou smears. METHODS: A total of 188 consecutive and unselected colposcopically directed cervical biopsies and a single contemporaneous cervical polyp were accessioned prospectively over a 3-month period, step-serially sectioned and examined by H&E and immunostained for p16(INK4a). The clinical context, results of concurrent Papanicolaou smears/ThinPrep slides and Digene hybrid capture tests for high-risk human papillomavirus (HPV) subtypes, as well as follow-up cervical smears/ThinPrep, biopsies and loop excisions of transformation zones or cone biopsies were all correlated with the morphological and immunohistochemical findings. RESULTS: Seventy-seven biopsies (40.7%) displayed a high-grade squamous intraepithelial lesion (HGSIL; cervical intraepithelial neoplasia [CIN] 2-3), 27 (14.3%) showed a low grade squamous intraepithelial lesion (HPV +/- CIN1) and 85 (45%) showed a range of non-dysplastic (inflammatory or reactive) changes. Diffuse strong parabasal immunostaining for p16(INK4a), suggestive of integrated high-risk HPV DNA into the host genome, was observed in 81 biopsies (42.9%, including the cervical polyp) and correlated (>90%) with HGSIL in the H&E sections. Only one case revealed irreconcilable discordance between the histological features and this strong parabasal immunostaining pattern. Focal and weaker midzonal or superficial p16(INK4a) immunostaining, suggestive of episomal HPV infection, was noted in 19 biopsies (10%) and these biopsies exhibited a range of histological changes but predominantly low grade squamous intraepithelial lesion (LGSIL). No staining of the squamous epithelium was seen in 89 biopsies (47.1%). Again, only one case revealed irreconcilable discordance between the histological features and this negative immunostaining pattern. On review of all cases where discordant results were noted between the H&E appearances and expected p16(INK4a) immunostaining, we found 26 cases (13.7%) in which this discordance prompted justifiable modification of the original diagnosis. CONCLUSIONS: Thus, within a routine diagnostic laboratory, p16(INK4a) immunostaining appears to be a very useful adjunctive test in the examination of colposcopically directed cervical biopsies, in the diagnostic cascade of women investigated for abnormal Papanicolaou smears. It is possible, as further data accumulate concerning the importance of integration of high-risk HPV DNA into the host cell genome and the reliability with which this can be identified by p16(INK4a) immunostaining, that this will become the diagnostic 'lesion of interest', replacing the subjective histological grading of cervical dysplasia, in the management of such patients; i.e., the discriminatory watershed between continued surveillance and active intervention.     

p16INK4a- und Ki-67-Immunzytochemie als Zusatzmethode für die Gruppe IIID der gynäkologischen Zytologie

Especially the cytological diagnoses of mild and moderate dysplasia are often followed by unnecessary stigmatization of patients and uncertainty in further clinical follow-up and therapy. Data from 222 patients including additional investigations by high-risk human papillomavirus (HPV) testing and combined immunocytochemistry for p16^INK4a and Ki-67 were documented, including cytological and histological follow-up. Overall for cytology, high risk HPV testing and dual staining the following characteristics concerning the presence of cervical intraepithelial neoplasia (CIN) 2+ were calculated (in %): sensitivity 100, 95.8 and 92.4, specificity –, 23.3 and 72.8, positive predictive value 53.6, 59.1 and 79.7, negative predictive value –, 82.8 and 89.3, respectively. There was a statistically significant advantage for higher specificity and positive predictive value for dual staining, especially for cytological diagnosis of low grade dysplasia. An objective individual risk stratification of patients with cytology of mild or moderate dysplasia is not available but the uncertainty in the management of these patients will be clearly reduced.     

Human papillomavirus DNA status after loop excision for cervical intraepithelial neoplasia grade III - A prospective study

The aims of the study were to investigate the relationship between human papillomavirus (HPV) DNA status and recurrence of cervical intraepithelial neoplasia (CIN) after loop excision (LEEP/LLETZ). Women (n=161) who underwent loop excision for CIN III and who were followed up prospectively for at least 4 years were the study cohort. Cervical smear cytology and testing for HPV DNA was performed at 3, 6 and 12 months prospectively and thereafter at intervals of 6-12 months, using the PCR method with a consensus primer targeting the L1 region. There has been no recurrence in 141 (81.6%) out of 161 subjects, while squamous intra-epithelial lesions (SIL) of low or high grade on cytology and CIN grade I-III on histology have been detected in 20 subjects. Prior to loop excision, HPV DNA was detected in 17 subjects who developed recurrence (9 had type 16, 2 each had types 18 and 52, and 1 each had types 31, 51, 58, and unknown). Within 3 months postoperatively, 12 (70.7%) subjects became negative for HPV, but 2 remained positive for the same type (1 each had types 16, 18), along with high-grade SIL on cytology, and CIN III on histology within 6 months, so repeat loop excision was performed. On the other hand, cytological findings were normalized in all transiently infected subjects within 18-36 months. Our results suggest that loop excision has improved HPV infection in many cases of CIN III and the persistent infection with a high-risk type of HPV is a predictor of the recurrence of CIN grade III.