Elle K.J. Risse

Relation of human papilloma virus status to cervical lesions and consequences for cervical-cancer screening: a prospective study

BACKGROUND A relation has been established between infection with high-risk types of human papillomavirus and development of cervical cancer. We investigated a role for testing for human papillomavirus as part of cervical-cancer screening. METHODS We monitored by cytology, colposcopy, and testing for high-risk human papillomavirus 353 women referred to gynaecologists with mild to moderate and severe dyskaryosis. The median follow-up time was 33 months. At the last visit we took biopsy samples. Our primary endpoint was clinical progression, defined as cervical intraepithelial neoplasia (CIN) 3, covering three or more cervical quadrants on colposcopy, or a cervical-smear result of suspected cervical cancer. FINDINGS 33 women reached clinical progression. All had persistent infection with high-risk human papillomavirus. The cumulative 6-year incidence of clinical progression among these women was 40% (95% CI 21-59). In women with end histology CIN 3, 98 (95%) of 103 had persistent infection with high-risk human papillomavirus from baseline. Among women with mild to moderate dyskaryosis at baseline, a second test for human papillomavirus at 6 months predicted end histology CIN 3 better than a second cervical smear. INTERPRETATION Persistent infection with high-risk human papillomavirus is necessary for development and maintenance of CIN 3. All women with severe dyskaryosis should be referred to gynaecologists, whereas women with mild to moderate dyskaryosis should be referred only after a second positive test for high-risk human papillomavirus at 6 months.

Addition of high-risk HPV testing improves the current guidelines on follow-up after treatment for cervical intraepithelial neoplasia

We assessed a possible role for high-risk human papillomavirus (HPV) testing in the policy after treatment for cervical intraepithelial neoplasia (CIN) 2 or 3 (moderate to severe dysplasia). According to the Dutch guidelines follow-up after treatment consists of cervical cytology at 6, 12 and 24 months. Colposcopy is only performed in case of abnormal cervical cytology. In this observational study 184 women treated for CIN 2 or 3 were prospectively monitored by cervical cytology and high-risk HPV testing 3, 6, 9, 12 and 24 months after treatment. Post-treatment CIN 2/3 was present in 29 women (15.8%). A positive high-risk HPV test 6 months after treatment was more predictive for post-treatment CIN 2/3 than abnormal cervical cytology (sensitivity 90% and 62% respectively, with similar specificity). At 6 months the negative predictive value of a high-risk HPV negative, normal smear, was 99%. Largely overlapping, partly different groups of women with post-treatment CIN 2/3 were identified by HPV testing and cervical cytology. Based on these results we advocate to include high-risk HPV testing in monitoring women initially treated for CIN 2/3. In case of a high-risk HPV positive test or abnormal cervical cytology, colposcopy is indicated. All women should be tested at 6 and 24 months after treatment and only referred to the population-based cervical cancer screening programme when the tests are negative on both visits.

The presence of high-risk HPV combined with specific p53 and p16INK4a expression patterns points to high-risk HPV as the main causative agent for adenocarcinoma in situ and adenocarcinoma of the cervix.

Adenocarcinoma in situ (ACIS) and adenocarcinoma (AdCA) of the cervix are frequently missed in population‐based screening programmes. Adding high‐risk HPV (hrHPV) testing to cervical cancer screening might improve the detection rate of ACIS and AdCA. Since the exact proportion of AdCAs of the cervix that can be attributed to hrHPV infection is still a matter of debate, a comprehensive study was performed of hrHPV presence in ACIS and AdCA of the cervix. Archival formalin‐fixed specimens of indisputable ACIS (n = 65) and AdCA (n = 77) of the cervix were tested for hrHPV DNA by GP5+/6+ PCR‐enzyme immunoassay (EIA) and type‐specific E7 PCR for 14 hrHPV types. Further immunostaining for p16INK4A and p53 was performed to assess alternative pathways of carcinogenesis potentially unrelated to HPV. hrHPV DNA was found in all (100%) ACISs and 72 (94%) cervical AdCAs, whereas none of 20 endometrial AdCAs scored hrHPV‐positive. HPV 18 was most prevalent and found as single or multiple infection in 68% of ACISs and 55% of cervical AdCAs. Diffuse immunostaining for p16INK4a, a potential marker of hrHPV E7 function, was significantly more frequent in hrHPV‐positive cervical AdCAs (19/20; 95%) than in those without hrHPV (1/5; 20%; p < 0.001). Immunostaining for p53, pointing to stabilized wild‐type or mutant p53 protein, was significantly more frequent in hrHPV cervical AdCAs negative for hrHPV (p = 0.01). No difference in p16INK4a and p53 immunostaining was found between hrHPV‐negative cervical AdCAs and endometrial AdCAs. Hence, only a minority of cervical AdCAs displayed absence of HPV DNA and immunostaining profiles suggestive of an aetiology independent of HPV. Since all ACISs and nearly all cervical AdCAs were hrHPV‐positive, the incorporation of hrHPV testing in cervical cancer screening programmes is likely to decrease markedly the incidence of cervical AdCA. Copyright

Multiple suspicious lesions detected by autofluorescence bronchoscopy predict malignant development in the bronchial mucosa in high risk patients

Autofluorescence bronchoscopy (AFB) has been shown to be sensitive to detect preneoplastic lesions in central airways. Apart from bronchial mucosa thickness, tissue autofluorescence is also related to the biochemical properties of the target cells. Genetic studies have shown molecular abnormalities to be present in histologically normal mucosal specimens. Forty-six high-risk individuals, free of micro-invasive cancer at the initiation of the study, were included in this analysis and have been subjected to repeat bronchoscopic examinations every 4-6 months. They had previous curatively treated lung cancer (n=18), ENT tumor (n=11) or were at risk to acquire lung cancer primaries (n=17). Baseline AFB is scored for each suspicious lesion, thus the total score represents the number of AFB suspicious lesions present in each individual at risk. Baseline AFB score was correlated to outcome, i.e. the development of squamous-cell cancer (SCC) in each individual. So far, 11/46 (24%) of the individuals acquired SCC. Follow up has been 12-80 months. All five individuals with >/=3 lesions (100%, 12-36 months), five of the ten (50%, 12-48 months) individuals who had two lesions and one among the 12 (8%, 36 months) individuals with one suspicious AFB lesion, developed SCC. Up till now (12-80 months), the remaining 19 individuals without any suspicious AFB lesion have not acquired SCC. The average AFB score for the group of individuals which developed SCC was significantly different (P<0.001) from the remaining individuals who did not acquire SCC (2.64+/-1.1 vs. 0.6+/-0.7 S.D.). The number of suspicious lesions at baseline AFB is a good predictor for the development of SCC in the individuals at risk in our study population. This finding is compatible with field carcinogenesis and warrants a more upfront use of AFB in a lung cancer screening to sift the different risk-cohorts in a population mainly at risk for developing metachronous lung cancer.

The contribution of immunocytochemistry in diagnostic cytology. Comparison and evaluation with immunohistology

This study reports on the use and evaluation of immunocytochemistry in diagnostic cytology by correlation with (immuno)histology. The cytologic material was collected during a period of 3 years. Sixty‐four patients were selected and studied retrospectively. The reactivity of a number of polyclonal and monoclonal antibodies was investigated and applied to a diverse group of tumors. Marker expression on routine cytologic and histologic material was compared and the use of immunocytochemistry in diagnosis was evaluated. Immunocytochemistry proved to be an easy and valuable technique for the identification and classification of tumor cells, and there was a good concordance with immunohistology. A discrepancy in marker expression was found in 10% of the slides that were investigated. Immunocytochemistry led to a misdiagnosis in only four cases; however, in these cases cytology alone would not have led to the correct diagnosis. The causes for the discrepancies are discussed. It is estimated that immunocytochemistry contributed to the diagnosis of approximately 50% of the cases, supplying either additional or essential information. A wide variety of markers can be applied reliably on cytologic preparations. The use of panels of antibodies is mandatory.

Elevated hTERT mRNA levels: A potential determinant of bronchial squamous cell carcinoma (in situ)

Expression levels of hTERT mRNA were investigated by RT‐PCR in tissue specimens of patients with (Group A) and without (Group B) clinically overt bronchial carcinoma, respectively. Bronchial carcinoma (n = 9) and distant normal (n = 9) specimens were analyzed in Group A. The chance of carcinoma seemed to increase with increasing hTERT mRNA levels (OR = 6.04, 95% CI = 1.02–37). Group B was comprised of 21 patients who underwent autofluorescence bronchoscopy. After analysis of 66 bronchial biopsies the chance of prevalent carcinoma in situ or carcinoma increased with increasing hTERT mRNA levels (OR = 6.19, 95% CI = 1.55–25). Variables like age, gender, smoking history, history of cancer within the airways or the degree of lymphocyte infiltrate in the specimens did not modify this relation. In 7 Group B patients in whom bronchial cancer was diagnosed during follow‐up, biopsies taken before cancer diagnosis from both the area of the newly developed tumor and distantly from this area had been analyzed for hTERT expression. The median hTERT mRNA level in the biopsies from the area of future cancer was significantly higher than in biopsies taken from distant sites (p < 0.03). These data indicate that elevated hTERT mRNA is associated with an increased relative risk of prevalent and incident bronchial squamous cell carcinoma (in situ).

Detection and Staging of Preinvasive Lesions and Occult Lung Cancer in the Central Airways with 18F-Fluorodeoxyglucose Positron Emission Tomography: A Pilot Study

Purpose: To evaluate the role of 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) in radiologically occult preinvasive lesions and lung cancer in the central airways. Experimental Design: Twenty-two patients with 24 preinvasive lesions and early squamous cell cancer (SCC) being occult on high-resolution computed tomography were studied. All lesions were diagnosed based on histology sampled using autofluorescence bronchoscopy. FDG-PET findings were correlated with WHO histologic classification. FDG-PET was considered true-positive when the final diagnosis was SCC and true-negative when the lesions were classified as severe dysplasia or less. Results: FDG-PET was true-positive in 8 of 11 and true-negative in 11 of 13 cases corresponding with a sensitivity of 73% [95% confidence interval (CI), 0.43-0.91] and specificity of 85% (95% CI, 0.57-0.97). Positive and negative predictive values were 80% (95% CI, 0.48-0.96) and 79% (95% CI, 0.52-0.93), respectively. Conclusions: Our very preliminary data suggest that FDG-PET might be useful for the evaluation of early central airway lesions, being positive in most SCC and negative in cases of severe dysplasia. Validation in a larger multicenter study is needed.

Telomerase activity in high-grade cervical lesions is associated with allelic imbalance at 6Q14–22

Our study attempts to establish the relationship between telomerase activity and allelic imbalance (AI) on chromosomes 3p and 6 in high‐risk HPV‐containing cervical lesions. These chromosomes were implicated previously in telomerase regulation in HPV containing immortalized cells and cervical cancer cells. Allelotyping and telomerase analysis were carried out on 28 high‐grade cervical lesions (CIN III: n = 20; cervical carcinomas: n = 8), using 23 microsatellite markers on 3p, 6p and 6q. Clear telomerase activity was found in 17 of 28 lesions (61%). Allelic imbalance frequency at 6q14–22 was significantly higher in lesions with detectable telomerase activity, compared to lesions without telomerase activity (p = 0.02). No association was found between telomerase activity and AI at any of the remaining regions studied on 3p and chromosome 6. In addition, in telomerase positive passages of the HPV 16 immortalized cell line FK16A, shown recently to be responsive to chromosome 6 mediated telomerase repression, AI was found in the overlapping region of 6q14–27. These data suggest that 6q14–22 may contain 1 or more genes involved in telomerase deregulation and immortalization during cervical carcinogenesis.

Comparison of Light Microscopic Grading and Morphometric Features in Cytological Breast Cancer Specimens

A study was set up to investigate correlations between different light microscopic grades and morphometric features in cytological breast cancer specimens and to evaluate the discriminative power of morphometry for the three grades. 76 slides (smears and imprints) were graded independently in two different laboratories, and the 54 unequivocally graded slides were used as a training set for computing classification rules. In all slides, 100 nuclei and their nucleoli were measured on a graphic tablet at a final magnification of 2800x. A total of 43 morphometric features was evaluated. Univariate analysis showed significantly different values for most of the morphometric features for the three grades. Discriminant analysis revealed that a combination of the mean nuclear area and the number of mitoses per slide provided optimal discrimination between grades one and two (87.5% correct classification). One more feature (mean nuclear shape factor) was needed to obtain optimal discrimination between grades two and three (83.3% correct classifications). 16 of the 22 (72.7%) equivocally graded slides could be classified with high probability using these classification rules. We conclude that morphometry has discriminative power for the three grades in cytologic breast cancer specimens and may therefore be a useful instrument for classification of difficult cases.

Suprabasal p53 immunostaining in premalignant endobronchial lesions in combination with histology is associated with bronchial cancer.

Endobronchial carcinoma develops through a continuum of morphologically recognizable pre-neoplastic changes. At present, no marker has been identified that can reliably predict the biological behavior of these lesions. Endobronchial lesions (n=39) sampled from patients (n=20) without clinically overt lung cancer, were analyzed by immunohistochemistry (IHC) for abnormal expression regarding the p53 protein, i.e. suprabasal p53 expression. Clear suprabasal p53 immunostaining was found in two (12%) of the hyperplastic or squamous metaplastic lesions, in one (10%) of the mildly or moderately dysplastic lesions and in nine (75%) of the severely dysplastic or carcinoma in situ (CIS) lesions. Suprabasal p53 immunostaining was found significantly more frequent in severe dysplasia or CIS (P<0.01). Of 17 patients follow-up data were available. After a median follow up of 7 months (range 2-37 months), six patients presented with bronchial carcinoma within the same lobe or bronchial spur where biopsies had been taken. Four of these patients revealed suprabasal p53 immunostaining in the biopsies obtained from the sites of future cancer. In three patients biopsies were obtained from future cancer sites as well as from distant sites in the ipsilateral lung. Suprabasal p53 immunostaining was found exclusively at future cancer sites of these patients (P=0.02). Suprabasal p53 immunostaining in addition to histology improved the specificity and the positive predictive value for bronchial carcinoma development in the same lobe or bronchial spur, compared with histology alone. These results suggest that suprabasal p53 immunostaining is associated with bronchial cancer and might have additive value to predict the biological behavior of pre-neoplastic endobronchial lesions in the population at risk of bronchial cancer.