Exploiting the residual of cervical thin layer brush samples through cytohistology in cases with invasive carcinoma with application of antibodies.

OBJECTIVE: To exploit cervical thin layer brush samples through cytohistology in cases with invasive carcinoma with application of antibodies. STUDY DESIGN: Fourteen cases from women with carcinoma diagnosed in 2006 were selected out of 29 invasive carcinomas. From these 14 cases liquid-based cervical cytology material was available to prepare cytohistology. Eight women had squamous cell carcinoma, 4 endocervical adenocarcinoma, 1 endometrial adenocarcinoma and 1 ovarian adenocarcinoma. The residual material from the thin layer sample, collected by brushes by general practitioners, was used to prepare paraffin sections. These were stained with the Papanicolaou method and for the biomarkers Ki-67 and p16 and, if desired, for differentiation markers, including carcinoembryonic antigen, vimentin, cytokeratin 7 and cytokeratin 20 to establish the immunoprofile of the carcinoma. RESULTS: The morphologic details in the cancer nuclei in the paraffin sections were excellent, while in all cases the thin layer cytology slide contained thick epithelial fragments with blurred nuclei. In 5 of the 6 adenocarcinomas, the glandular architecture diagnostic of adenocarcinoma was visible in the cytohistology, which was highlighted in the biomarker stainings, particularly so in the Ki-67 sections. With the exception of endometrial adenocarcinoma, all p16(INK4a) stainings were positive, as they were in the ovarian adenocarcinoma case. CONCLUSION: Cytohistology is an adjunct to routine cervical cytologic examination of thin layer samples, allowing an unequivocal and refined diagnosis.

Biomarker p16(INK4a) on paraffin sections prepared from cervical brush samples highlights nuclear changes resulting in unquestionable cytohistodiagnosis.

OBJECTIVE: To optimize the morphology of pl6-positive atypical and (pre)neoplastic cells in paraffin cytoblock sections with the aim to minimize equivocal diagnoses on cytology samples. STUDY DESIGN: Patients with negative cytology results or results within normal limits (WNL) (n=38), atypical squamous cells of undetermined significance (ASCUS) (n=25) and high grade squamous intraepithelial lesion (HSIL) (n=16) were selected. The residual material of the cervical brush samples was processed to cytoblock paraffin sections and stained for pl1. The cytohistodiagnosis of the pl1-stained paraffin sections was based on the cytologic and histologic morphology. RESULTS: Of the 38 cytologically negative cases, only 4 contained afe w faintly positive pl61cells. Of the 25AS CUS cases, I 1as cytohistologically upgraded to low grade squamous intraepithelial lesion (LSIL). All 16 HSIL cases contained cells with outspoken diffuse positive immunostaining, highlighting chromatin clumping in the (pre)malignant cells. In the paraffin sections the tissue fragments showed architecture consistent with that of HSIL. The nuclear to cytoplasmic ratio in the HSIL was severely disturbed. CONCLUSION: Cervical brush samples allow an unequivocal cytohistodiagnosis based on the (pre)malignant nuclear changes highlighted by the p16 staining of the paraffin sections.

Membrane-type matrix metalloproteinases and vascularization in human endometrium during the menstrual cycle.

Endometrial angiogenesis is essential for a vascularized receptive endometrium. Previously, we described that membrane type-3 metalloproteinase (MT3-MMP) is associated with endometrial angiogenesis in vitro. The association of MT-MMPs with endometrial angiogenesis in vivo is unknown. Therefore, this study analysed the presence of MT-MMPs in human endometrium and their correlation with neovascularization. RNA/protein expressions of the six MT-MMPs were determined in cultured endometrial cells. Vascularization parameters and MT-MMP expressions in vivo were evaluated by immunohistochemistry in serial endometrium sections. MT1-, MT2-, MT3- and MT4-MMP antigens were expressed in cultured endometrial endothelial cells. MT2-, MT3- and MT4-MMP were expressed by endothelium during the proliferative and secretory phase. Strikingly, these phases showed elevated vascularization, elevated total vascular surface in proliferative phases, elevated number of vessels in proliferative/late secretory phases and increased luminal surface in the proliferative phases. All MT-MMP antigens were expressed in various endometrial cell types in vivo, with decreased levels during the early secretory phase. In conclusion, all MT-MMPs are expressed in endometrium in a cycle-dependent pattern. The vascular expression of MT2-, MT3- and MT4-MMP correlated with angiogenic episodes of the cycle. Since MT2- and MT3-MMP are known to regulate tube formation, these findings support earlier in vitro data on the role of MT3-MMP in endometrial angiogenesis. Additionally, MT2-MMP appears to be associated with endometrial neovascularization also.

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.

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.

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.

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.

Penile lesions and human papillomavirus in male sexual partners of women with cervical intraepithelial neoplasia.

BACKGROUND: Genital human papillomavirus infection (HPV) is causally associated with cervical carcinomas and premalignant lesions. Limited information is available about the prevalence of HPV and penile lesions in male sexual partners of women with cervical intraepithelial neoplasia (CIN). OBJECTIVE: The aim of this study was to identify the presence of penile lesions and HPV in penile scrapings from male sexual partners of women with CIN. METHODS: One hundred seventy-five male sexual partners of women with CIN were screened by peniscopy after acetowhite staining and HPV testing on penile scrapings. RESULTS: Penile lesions were seen in 68% of the male sexual partners. More than one lesion type was diagnosed in 15%. Flat lesions, papular lesions, and condylomata acuminata were seen in 83%, 29%, and 4%, respectively. HPV was detected in 59% of the penile scrapings, containing mainly oncogenic HPV types. When penile lesions were present at peniscopy, 67% of penile scrapings were positive for HPV, whereas 37% were HPV-positive when no lesions were visible. CONCLUSIONS: Penile lesions are frequently found in sexual partners of women with CIN. Most of these lesions are subclinical (ie, only visible after acetowhite staining) and are often associated with the presence of high-risk HPV, indicating that male sexual partners of women with CIN might constitute a reservoir for high-risk HPV.