Immunocytochemical analysis of cisplatin-induced platinum-DNA adducts with double-fluorescence video microscopy.

To detect low-level DNA platination, a sensitive immunocyto- and histochemical technique was developed using a polyclonal antibody. The antibody GPt, derived after immunization of rabbits with highly platinated DNA and purified with affinity chromatography, detected the main platinum (Pt)-containing intrastrand and interstrand adducts. Double-fluorescence microscopy image analysis was used to quantify Pt-DNA adducts with Hoechst 33258 fluorescence to locate the nuclei and with fluorescein isothiocyanate fluorescence to measure the immunosignal. A two- to five-fold dose-dependent difference in the level of cisplatin (CDDP)-induced Pt-DNA adducts between a CDDP-sensitive and -resistant human tumour cell line was detected. Large differences in Pt-DNA adduct levels after in vitro CDDP incubation between human buccal cells, lymphocytes and biopsies of different tumour types were observed. Pt-DNA adduct levels were fivefold higher in human testicular tumours than in colon tumours, representing CDDP-sensitive and -resistant tumours, respectively, in the clinic. These data suggest the possibility of predictive testing by measuring Pt-DNA adduct levels. Pt-DNA adducts in patients after treatment with CDDP were shown in normal buccal cells and in imprints of fresh tumour biopsies as well as in paraffin-embedded tumour cells. The analysis of Pt-DNA adducts at a single-cell level in small samples of normal and tumour cells during and/or after treatment is feasible with GPt and will hopefully enable more selective treatment of patients

Epidemiological evidence of cervical intraepithelial neoplasia without the presence of human papillomavirus.

The aim of this paper was to provide epidemiological evidence to support the notion that cervical intraepithelial neoplasia (CIN) without human papillomavirus (HPV) is a true entity. If a diagnosis of HPV-negative cervical neoplasia is erroneous, one would not expect there to be any differences in risk factors between HPV-positive and HPV-negative patients. Patients at a gynaecological outpatient clinic of a university hospital [a total of 265 consecutive women with dyskaryotic cervical smears who were subsequently diagnosed with CIN I (n=37), CIN II (n=48) or CIN III (n=180)] completed a structured questionnaire regarding smoking habits and sexual history. Analysis of an endocervical swab for Chlamydia trachomatis, analysis of a cervical scrape for HPV, and morphological examination of cervical biopsy specimens were also performed. HPV was found in 205 (77.4%) out of the 265 women. Univariate analysis showed that current age (P=0.02), current smoking behaviour (P=0.002) and the number of sexual partners (P=0.02) were significantly associated with the presence of HPV. Age at first sexual intercourse, a past history of venereal disease or genital warts, and current infection with Chlamydia trachomatis were not associated with the presence of HPV. Using multivariate logistic regression analysis, the number of sexual partners and current smoking behaviour showed an independent significant association with HPV. HPV-negative and HPV-positive CIN patients differ with respect to the risk factors for HPV. These findings suggest that HPV-negative CIN is a separate true entity

What is the added value of digital image analysis of HER2 immunohistochemistry in breast cancer in clinical practice? A study with multiple platforms

Aims We aimed to compare digital image analysis (DIA) of human epidermal growth factor receptor 2 (HER2) immunohistochemistry (IHC) in breast cancer by two platforms: (i) to validate DIA against standard diagnostics; and (ii) to evaluate the added value of DIA in clinical practice. Methods and results HER2 IHC and in-situ hybridisation (ISH) were performed on 152 consecutive invasive breast carcinomas. IHC scores were determined with DIA using two independent platforms. Manual scoring was performed by two independent observers. HER2 status was considered positive in 3+ and ISH-positive 2+ cases. HER2 status using DIA was compared to HER2 status with standard diagnostics (manual scoring with ISH in 2+ cases). Interplatform agreement of IHC scores was 'moderate' (linear weighted kappa = 0.58), agreement between manual scoring and platform A was 'moderate' (kappa = 0.60) and between manual scoring and platform B 'almost perfect' (kappa = 0.85). Compared to manual scoring, DIA resulted in a reduction of 2+ cases from 17.1 to 1.3% with platform A and from 17.1 to 15.8% with platform B. However, compared to standard diagnostics, there were three false-negative cases with DIA using platform A [81.3% sensitivity, 100% specificity, 100% positive predictive value (PPV), 97.8% negative predictive value (NPV)]. Sensitivity, specificity, PPV and NPV were 100% with DIA using platform B. Conclusions DIA of HER2 IHC is a valid tool in determining HER2 status in breast carcinoma. Algorithms in different platforms can behave differently, and optimal calibration is essential. In clinical practice, DIA offers an objective alternative to manual scoring, but a reduction in 2+ cases could result in loss of sensitivity

Feasibility of endometrial sampling by vaginal tampons in women with Lynch syndrome

Background: Endometrial sampling for the surveillance of women with Lynch syndrome is an invasive and painful procedure. The aim of this study was to evaluate the feasibility of a less invasive procedure of collecting vital cells by vaginal tampons. Methods: This was a prospective feasibility study of women scheduled to undergo annual gynecological surveillance, including endometrial sampling. We included consecutive asymptomatic women with Lynch syndrome or first-degree relatives and asked them to insert a vaginal tampon 2-4 h before attending their outpatient appointment. Feasibility was evaluated by the following metrics: Patient acceptance, pain intensity of each procedure (assessed by visual analog scale; range 0-10), and the presence of vital cells obtained by tampon-based or endometrial sampling methods. Two pathologists independently evaluated all samples. Results: In total, 25 of 32 approached women completed the tampon-based procedure, with 23 of these subsequently undergoing invasive endometrial sampling. The median visual analog scale scores for tampon use and invasive endometrial sampling were 0 (range, 0-10) and 5.5 (range, 1-10) (p < 0.001). None of the tampon samples analyzed by cytology showed endometrial cells, but they did contain vital squamous cells and granulocytes. By contrast, 18 (78%) of the invasive endometrial samples contained enough endometrial tissue for analysis. No endometrial abnormalities were found by endometrial sampling. Conclusions: Tampon-based endometrial surveillance was a well-accepted and non-painful procedure, and although tampons contained vital cells, they did not provide endometrial cells. However, this study was limited to asymptomatic women with Lynch syndrome (no endometrial pathology), indicating that research is needed to evaluate whether the tampon method has any utility for endometrial surveillance in women with Lynch syndrome

Glutathione S-transferase activity and isoenzyme composition in benign ovarian tumours, untreated malignant ovarian tumours, and malignant ovarian tumours after platinum/cyclophosphamide chemotherapy.

Glutathione S-transferase (GST) isoenzyme composition, isoenzyme quantities and enzymatic activity were investigated in benign (n = 4) ovarian tumours and malignant ovarian tumours, before (n = 20) and after (n = 16) chemotherapy. Enzymatic activity of GST in cytosols was measured by determining 1-chloro-2,4-dinitrobenzene conjugation with glutathione, cytosolic GST subunits were determined by wide pore reversed phase HPLC, using a S-hexylglutathione-agarose affinity column, and isoelectric focussing. Both GST activity and GST pi amount were not related to histopathologic type, differentiation grade, or tumour volume index in untreated malignant tumours. GST isoenzyme patterns were identical in benign tumours and malignant tumours before and after platinum/cyclophosphamide chemotherapy, while GST pi was the predominant transferase. Mean GST activity and GST pi amount were decreased (P < 0.05) in malignant ovarian tumours after platinum/cyclophosphamide chemotherapy compared to untreated ovarian malignant tumours. No relation was found in untreated ovarian tumours between GST pi amount and response to platinum/cyclophosphamide chemotherapy. Thus, within the limitations of the current study no arguments were found for a role of GST in in vivo drug resistance of malignant ovarian tumours to platinum/cyclophosphamide chemotherapy

L1CAM expression in uterine carcinosarcoma is limited to the epithelial component and may be involved in epithelial-mesenchymal transition

Uterine carcinosarcoma (UCS) has been proposed as a model for epithelial-mesenchymal transition (EMT), a process characterized by a functional change facilitating migration and metastasis in many types of cancer. L1CAM is an adhesion molecule that has been involved in EMT as a marker for mesenchymal phenotype. We examined expression of L1CAM in UCS in a cohort of 90 cases from four different centers. Slides were immunohistochemically stained for L1CAM and scored in four categories (0%, 50%). A score of more than 10% was considered positive for L1CAM. The median age at presentation was 68.6years, and half of the patients (53.3%) presented with FIGO stage 1 disease. Membranous L1CAM expression was positive in the epithelial component in 65.4% of cases. Remarkably, expression was negative in the mesenchymal component. In cases where both components were intermingled, expression limited to the epithelial component was confirmed by a double stain for L1CAM and keratin. Expression of L1CAM did not relate to overall or disease-free survival. Our findings suggest L1CAM is either not a marker for the mesenchymal phenotype in EMT, or UCS is not a good model for EMT

L1CAM expression in uterine carcinosarcoma is limited to the epithelial component and may be involved in epithelial–mesenchymal transition

Uterine carcinosarcoma (UCS) has been proposed as a model for epithelial–mesenchymal transition (EMT), a process characterized by a functional change facilitating migration and metastasis in many types of cancer. L1CAMis an adhesion molecule that has been involved in EMT as a marker for mesenchymal phenotype.We examined expression of L1CAM in UCS in a cohort of 90 cases from four different centers. Slides were immunohistochemically stained for L1CAMand scored in four categories (0%, 50%). A score of more than 10% was considered positive for L1CAM. The median age at presentation was 68.6 years, and half of the patients (53.3%) presented with FIGO stage 1 disease. Membranous L1CAM expression was positive in the epithelial component in 65.4% of cases. Remarkably, expression was negative in the mesenchymal component. In cases where both components were intermingled, expression limited to the epithelial component was confirmed by a double stain for L1CAMand keratin. Expression of L1CAMdid not relate to overall or disease-free survival. Our findings suggest L1CAMis either not a marker for the mesenchymal phenotype in EMT, or UCS is not a good model for EMT