The increased detection of cervical intraepithelial neoplasia when using a second biopsy at colposcopy.

OBJECTIVE: It has been suggested that colposcopy can miss a significant percentage of high-grade cervical intraepithelial neoplasia (CIN2+). Improved disease ascertainment was evaluated by taking multiple lesion-directed biopsies. METHODS: In a cross-sectional multicenter study in the Netherlands and Spain, 610 women referred to colposcopy following abnormal cervical cytology results were included. Multiple directed biopsies were collected from lesions and ranked according to impression. A non-directed biopsy of normal-appearing tissue was added if fewer than four biopsies were collected. We evaluated the additional CIN2+ yield for one and two directed biopsies. Colposcopic images were reviewed for quality control. RESULTS: In women with at least two lesion-directed biopsies the yield for CIN2+ increased from 51.7% (95%CI; 45.7-57.7) for one directed biopsy to 60.4% (95%CI; 54.4-66.2, p<0.001) for two biopsies. The highest CIN2+ yield was observed in women who were HPV16-positive, had high-grade squamous intraepithelial lesion (HSIL) cytology, and high-grade colposcopy impression. The yield increased from 83.1% (95%CI; 71.5-90.5) with one directed biopsy to 93.2% (95%CI; 83.8-97.3) with two directed biopsies. Only 4.5% additional CIN2+ were detected in biopsies not targeting abnormal areas on the cervix. CONCLUSIONS: A second lesion-directed biopsy is associated with a significant increase in CIN2+ detection. Performing a second lesion-directed biopsy and using a low threshold for abnormality of any acetowhitening should become the standard clinical practice of colposcopy.

The impact of human papillomavirus genotype on colposcopic appearance: a cross-sectional analysis.

OBJECTIVE: To study colposcopic performance in diagnosing high-grade cervical intraepithelial neoplasia or cervical cancer (CIN2+ and CIN3+) using colposcopic characteristics and high-risk human papillomavirus (hrHPV) genotyping. DESIGN: Cross-sectional multicentre study. SETTING: Two colposcopy clinics in The Netherlands and Spain. POPULATION: Six hundred and ten women aged 17 years and older referred for colposcopy because of abnormal cytology. METHODS: A cervical smear was obtained. Colposcopists identified the worst lesion, graded their impression and scored the colposcopic characteristics of the lesions. Up to four biopsies were collected, including one biopsy from visually normal tissue. MAIN OUTCOME MEASURES: CIN2+ and CIN3+, positive for HPV16 or other high-risk HPV types (non-16 hrHPV-positive). RESULTS: The mean age in HPV16-positive CIN2+ women was 35.1 years compared with 39.1 years in women with other hrHPV types (P = 0.002). Sensitivity for colposcopy to detect CIN2+ was 87.9% (95%CI 83.2-91.5), using colposcopic cut-off of 'any abnormality'. The remaining CIN2+ were found by a biopsy from visually normal tissue or endocervical curettage (ECC). Detection of CIN2+ by lesion-targeted biopsies was not different between HPV16-positive women [119/135; 88.1% (95%CI 81.2-92.9)] and non-16 hrHPV-positive women [100/115; 87.0% (95%CI 79.1-92.3); P = 0.776]. In multivariate analysis, 'acetowhitening' [odds ratio (OR) 1.91, 95%CI 1.56-3.17], 'time of appearance' (OR 1.95, 95%CI 1.21-3.15) and 'lesion >25% of visible cervix' (OR 2.25, 95%CI 1.44-3.51) were associated with CIN2+. CONCLUSIONS: In this population following European screening practice, HPV16-related CIN2+ lesions were detected at younger age and showed similar colposcopic impression as non-16 hrHPV high-grade lesions. There was no relationship between any of the colposcopic characteristics and HPV16 status.

Development and clinical evaluation of a highly sensitive PCR-reverse hybridization line probe assay for detection and identification of anogenital human papillomavirus.

Human papillomavirus (HPV) can be detected by amplification of viral DNA. A novel PCR primer set generating a short PCR fragment (SPF PCR) was used for amplification of a fragment of only 65 bp from the L1 region and permitted ultrasensitive detection of a broad spectrum of HPV genotypes. The intra- and intertypic sequence variations of the 22-bp interprimer region of this amplimer were studied. Among 238 HPV sequences from GenBank and clinical specimens, HPV genotypes were correctly identified based on the 22-bp sequence in 232 cases (97.2%). Genotype-specific probes for HPV genotypes 6, 11, 16, 18, 31, 33 to 35, 39, 40, 42 to 45, 51 to 54, 56, 58, 59, 66, 68, 70, and 74 were selected, and a reverse hybridization line probe assay (LiPA) (the INNO-LiPA HPV prototype research assay) was developed. This LiPA permits the use of amplimers generated by the SPF as well as the MY 09/11 primers. The assay was evaluated with a total of 1, 354 clinical specimens, comprising cervical scrapes (classifications ranging from normal cytology to severe dyskaryosis) and formalin-fixed, paraffin-embedded cervical carcinoma samples. LiPA results were highly concordant with sequence analysis of the SPF amplimer, genotype-specific PCR, and sequence analysis of amplimers generated by MY 09/11 primers. The sensitivity of the SPF primers was higher than that of the GP5(+)/6(+) primers over a broad range of HPV types, especially when multiple HPV genotypes were present. In conclusion, the SPF LiPA method allows extremely sensitive detection of HPV DNA as well as reliable identification of HPV genotypes in both cervical smears and paraffin-embedded materials.

Detection and typing of human papillomavirus in cervical carcinomas in Russian women: a prognostic study.

BACKGROUND: The correlation between human papillomavirus (HPV) infection and tumor prognosis in 159 Russian women with cervical carcinoma was investigated. The presence of various HPV types was correlated with the histologic parameters of the carcinomas and with their immunoreactivity with antibodies to p53, Ki-67-Ag, and bcl-2. METHODS: Formalin fixed, paraffin embedded tissue specimens representing 159 cases of International Federation of Gynecology and Obstetrics Stage I and II were used. HPV DNA was detected by polymerase chain reaction (PCR) using a general primer set that targets the L1 region and synthesizes a product of only 65 base pairs. The HPV types were determined by direct sequencing and compared with known HPV types. RESULTS: All 159 carcinomas were positive for HPV. HPV 16 (64.8%) was most frequently found, followed by HPV 18 (10.7%) and HPV 45 (8.2%). In 6 patients (3.8%), HPV types could not been further classified, and these cases were therefore categorized as HPV X. Although a trend was noted toward poorer prognosis for women with carcinomas harboring HPV types 16, 18, and 45 than for patients with carcinomas harboring HPV types 31, 33, 35, 52, 56, 58, and 68, the differences were not statistically significant. The prevalence of adenocarcinoma and adenosquamous carcinoma was higher among HPV 18 positive patients than among patients with the other known HPV types (P=0.0002). CONCLUSIONS: The rate of HPV positivity in these 159 cervical carcinomas was 100%. These findings challenge the assumption that HPV negative cervical carcinomas exist. This high rate might be attributed to the use of a new broad-spectrum HPV PCR test. HPV typing in cervical carcinoma was not significantly related to clinical outcome. HPV 18 was significantly more frequently found in adenocarcinoma and adenosquamous carcinoma. The possibility of classifying HPV 45 as an oncogenic high risk type should be considered.

Relationship between human papillomavirus type 16 in the cervix and intraepithelial neoplasia.

OBJECTIVE: To evaluate a temporal relationship between the presence of cervical human papilloma virus (HPV) type 16 and the risk of developing cervical intraepithelial neoplasia (CIN). METHODS: Fifty-four women with HPV 16 polymerase chain reaction (PCR)-positive tests were selected from the gynecologic outpatient clinic of the Reinier de Graaf Hospital, Delft, The Netherlands. At least three successive PCR tests were performed in each woman at intervals of 6 months. The PCR HPV 16 assay was performed in conjunction with cervical smear, and colposcopy and biopsy, if indicated. Women with at least three consecutive positive PCR tests were defined as having persistent HPV 16 infections. Women with one positive test followed by two negative tests were defined as having transient infections. Subdivided into two groups, 25 women had persistent infections and 29 had transient infections. RESULTS: In significantly more women in the persistent group compared with the transient group, CIN developed (11 of 25 versus six of 29, P = .036). Lesions in women with persistent HPV 16 infection were more severe (six of 11 were CIN III versus one of six P = .041). CONCLUSION: Persistent infection with HPV 16 is associated with a higher risk of developing CIN, which is often high-grade.

Novel short-fragment PCR assay for highly sensitive broad-spectrum detection of anogenital human papillomaviruses.

A novel set of polymerase chain reaction (PCR) primers, designated SPF1 and SPF2 and located in the L1 region, was developed for universal detection of human papillomavirus (HPV). A short PCR fragment (SPF) of only 65 pb was synthesized. SPF amplimers were detected in a microtiter-based hybridization system, using a mixture of oligonucleotide probes. The SPF system allowed detection of at least 43 different HPV genotypes. The clinical performance of the novel SPF system was assessed in three different patient groups. 1) Analysis of 534 cervical scrapes, obtained from treated patients, showed that the detection rate in 447 (83.7%) scrapes with normal cytology was significantly higher using the SPF system as compared with the universal primer set GP5+/6+ (P < 0.001). 2) The SPF assay detected HPV DNA in 299 (98.4%) of 304 scrapes with cytological dyskaryosis. 3) The SPF system detected HPV DNA in 100% of 184 formalin-fixed, paraffin-embedded cervical carcinoma specimens. In conclusion, the novel SPF system permitted universal and highly sensitive detection of HPV DNA in diverse clinical materials and may improve the molecular diagnosis and epidemiology of this important virus.

Progressing imbalance between proliferation and apoptosis with increasing severity of cervical intraepithelial neoplasia.

The equilibrium between cell proliferation and protection against apoptosis was studied immunohistochemically using monoclonal antibodies against Ki-67-Ag and bcl-2, respectively, in consecutive sections from normal and metaplastic cervical epithelia and cervical intraepithelial neoplasia (CIN) lesions and cervical carcinomas. A high percentage of Ki-67-Ag positive cells was seen in the parabasal cells of normal ectocervical and mature squamous metaplastic epithelium, although the basal cells were virtually negative. In preneoplastic lesions, however, the basal cells showed high proliferative activity and an increasing frequency of Ki-67-Ag positive cells was observed in the higher epithelial layers with increasing severity of CIN. In squamous cell carcinomas, variable numbers of Ki-67-Ag positive cells were observed and in adenocarcinomas expression increased with the degree of anaplasia. bcl-2 expression was observed only in the basal cells of normal endo- and ectocervix including reserve cells. With increasing severity of CIN, staining intensity and number of bcl-2 positive cells gradually increased. Five of eight squamous cell carcinomas were variably positive. All five adenocarcinomas showed extensive bcl-2 expression. Increased expression of both Ki-67-Ag and bcl-2 with increasing severity of CIN indicates an increasing imbalance between cell proliferation and protection from apoptosis. It is therefore proposed that an increasing proliferative fraction combined with a higher number of cells protected from apoptotic cell death contributes to progression of CIN. This phenotype may identify premalignant lesions with the potential to transform to cervical cancer.

Glutathione S-transferase pi is expressed in (pre) neoplastic lesions of the human uterine cervix irrespective of their degree of severity.

BACKGROUND: Glutathione S-transferase pi (GST pi) is involved in a variety of cell detoxification processes. In the uterine cervix its presence has been associated with high grade cervical intraepithelial neoplasia (CIN), but the reports are conflicting. For this reason we immunohistochemically investigated glutathione S-transferase pi expression in a well documented sequence leading to cervical cancer. MATERIALS AND METHODS: The series of tissue samples that were examined comprised normal, metaplastic, dysplastic (CIN 1, 11 and 111) and malignant cervix. GST pi expression was examined in 15 cases of uterine cervix lined with normal epithelia, in 11 cases of CIN 1, 9 cases of CIN 11, 10 cases of CIN 111, 6-cases of squamous cell cervical carcinomas and 5 cases of adenocarcinoma of the cervix. RESULTS: Both nuclear and cytoplasmic staining reactions were noted. In normal ectocervical epithelia a moderately strong nuclear and cytoplasmic staining reaction was noted, while in immature squamous metaplasia staining was more intense. Only 50% of the endocervical cells were immunostained while almost 100% of the reserve cells stained weakly, mainly restricted to the cytoplasm. Irrespective of severity, CIN lesions showed a moderate staining intensity in both cytoplasm and nuclei. Cervical carcinoma, irrespective of their type, showed significantly less staining activity. CONCLUSIONS: GST pi occurs in normal cervical epithelium and in all stages of premalignant cervix, suggesting an important role in the detoxification process in all these stages. Its ubiquitous presence indicates, in contrast to the earlier reports, that the enzyme does not play a crucial role in the initiation of the carcinogenic cascade. However, the absence of this detoxificating enzyme in the nucleus of the majority of cervical carcinomas may indicate that xenobiotic compounds are not catabolized and may therefore exert their mutagenic activity, resulting in tumor progression.

BCL-2 immunoreactivity increases with severity of CIN: a study of normal cervical epithelia, CIN, and cervical carcinoma.

The presence of the BCL-2 protein, a marker for inhibition of programmed cell death, was studied in a series of routinely processed cervical tissues, consisting of normal endocervical (n = 40) and ectocervical epithelium (n = 27), squamous metaplastic epithelium (n = 30), CIN (cervical intraepithelial neoplasia) lesions (n = 32), and cervical carcinomas (n = 13). BCL-2 was strongly expressed in the basal cell compartment of normal ectocervical squamous epithelium and in nearly all reserve cells, while in endocervical columnar cells it was moderately expressed. In immature squamous metaplastic epithelium, BCL-2 expression varied. Half of the cases showed only basal cell staining, while the other half showed staining also in suprabasal layers. BCL-2 could be detected in all premalignant lesions, showing a striking increase in the number of positive cells with increasing severity of CIN, in combination with a mild increase in staining intensity. All adenocarcinomas were positive (n = 5), while five of eight squamous cell carcinomas expressed BCL-2. Based on these results, it is hypothesized that both the larger number of cells staining with BCL-2 in higher grades of CIN and the increase in staining intensity imply an increasing protection of these neoplastic conditions against programmed cell death. This protection facilitates not only continuing proliferation, but also the induction of genetic instability in dysplastic epithelial cells; it may thus reflect the greater capacity of the more severe CIN lesions to evolve into cervical carcinoma.

Unexpected borderline malignant and malignant smooth muscle cell tumors of the uterine corpus in women treated with LH-RH analogues.

In this report, two cases of uterine smooth muscle cell tumors, one of uncertain malignant potential and one clearly malignant, are described in women treated for prolonged periods with luteinizing hormone-releasing hormone (LH-RH) analogues. Due to lengthy monitoring of LH-RH therapy, surgical intervention and histologic classification of these tumors was late in the course of disease, resulting in a delay in definite treatment. The risk to these women is discussed. The effects of LH-RH analogue therapy on fibroids is reviewed. Suggestions are put forward for monitoring LH-RH analogue therapy.

p53 and human papilloma virus type 16 in cervical intraepithelial neoplasia and carcinoma.

A series of cervical biopsies and excision specimens representing normal cervical epithelium, metaplastic epithelium, cervical intraepithelial neoplasia and cervical carcinoma were examined for the presence of p53 in relation to the human papillomavirus type 16 (HPV 16). The results show that p53 accumulation in premalignant cervical lesions is almost identical to the low levels detected in normal endo- and ectocervical epithelia, reserve cells, immature and mature squamous metaplastic epithelium. p53 levels were low and seem to be independent of the grade in cervical intraepithelial neoplasia (CIN). However, carcinomas of the cervix contained high levels of immunohistochemically detectable p53. Apparently, a p53 mutation is not an early factor in cervical carcinogenesis. Furthermore, our studies demonstrate that alterations in p53 levels and the presence of HPV 16 are not mutually exclusive markers of cervical tumorigenesis. This contrasts with several reports in the literature and underlines the observation that p53 expression is probably an inadequate prognosticator for estimating progression or regression of CIN lesions.