Methods for single- and double-target in situ hybridization (ISH) to, cells isolated from solid transitional cell carcinomas (TCC's) of the urinary bladder are described. Single cell suspensions were prepared from solid tumors of the urinary bladder by mechanical disaggregation and fixed in 70% ethanol. Using two DNA probes specific for the centromeres of chromosomes #1 and #18, ISH procedures were optimized for these samples. Human lymphocytes and cells from the T24 bladder tumor cell line were used as controls. In lymphocyte nuclei and metaphase chromosome spreads, ISH showed two major spots for each of the probes. About 80% of the nuclei from T24 cells showed three spots for both the chromosome #1 and #18 specific probes. When nuclei from TCC's were analyzed, often the number of spots for chromosome #1, and to a lesser extent for chromosome #18, differed from the number expected on basis of flow cytometric ploidy measurements. The double target-ISH method in all cases allowed the correlation of numerical aberrations for chromosomes #1 and #18 in one and the same cell. By such analyses a profound heterogeneity in chromosome number was detected in most tumors. In order to optimize the reproducibility of the method and the interpretation of the ISH-signals, criteria for their analysis have been determined. This procedure can now be applied on a routine basis to solid tumor specimens.

Cervical intraepithelial neoplasia (CIN I, II, and III) and cases of CIN III associated with micro-invasive cervical carcinoma (CIN III & mCA) were analysed for evidence of episomal or integrated human papillomavirus (HPV) 16/18 DNA by fluorescence in situ hybridization (FISH). In parallel, numerical aberrations of chromosomes 1, 17, and X were determined in these lesions as indicators of genomic instability. HPV 16/18 DNA was present in 2 of 12 CIN I, 19 of 23 CIN II/III, and 10 of 12 CIN III & mCA. None of the CIN I and only two of the 19 HPV 16/18-positive solitary CIN II/III showed an integrated HPV pattern. However, all ten cases of HPV-positive CIN III & mCA showed this pattern. Transition of CIN II/III to CIN III & mCA therefore correlates strongly with viral integration (p<0.001). Chromosomal aberrations were detected in 23 of 31 HPV 16/18-positive lesions (14 solitary CIN I-III and nine CIN III & mCA) and 5 of 16 HPV-negative lesions. Nine of 21 HPV 16/18-positive solitary CIN I-III showed tetrasomy for all chromosomes tested, while trisomies for a single chromosome were seen in a further five of these HPV-positive lesions. In eight of ten HPV-positive CIN III & mCA, predominantly aneusomies and/or polysomies were detected. A significant correlation (p<0.02) was found between the chromosome copy number and the physical status of HPV, indicating that in its episomal form HPV induces genomic changes such as tetrasomies and single trisomies, while HPV integration correlates with aneusomies and polysomies, predominantly detected in CIN III & mCA. These data indicate that integration of HPV 16/18 DNA is a pivotal step in the transition of CIN to micro-invasive carcinoma.

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Intermediate-sized filament proteins (IFP) are tissue specific in that antibodies to keratin, vimentin, desmin, glial fibrillary acidic protein (GFAP) and the neurofilament proteins can distinguish between cells of epithelial and mesenchymal origin as well as of myogenic and neural origin respectively. Malignant cells retain their tissue-specific IFP, which makes it possible to use these antibodies in tumour diagnosis. Carcinomas are exclusively detected by antibodies to keratin. Monoclonal antibodies to keratin have allowed the differentiation between subgroups of epithelial tumours until now between adenocarcinomas and squamous cell carcinomas. Lymphomas, melanomas and several soft tissue tumours are distinctly recognized by antibodies to vimentin. On the other hand, rhabdomyosarcomas and leiomyosarcomas are positive for desmin, while astrocytomas give a strong reaction with GFAP antibodies. Thus, antibodies to IFP are useful tools for differential diagnosis in surgical pathology.

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