Comparative FISH analysis of numerical chromosome 7 abnormalities in 5-μm and 15-μm paraffin-embedded tissue sections from prostatic carcinoma

Abstract

Interphase fluorescence in situ hybridization (FISH) was performed on 15-micron-thick paraffin sections from prostatic carcinomas using a chromosome 7-specific alpha-satellite DNA probe. A confocal laser scanning microscope (CLSM) was used for optical sectioning of the thick sections and reconstruction of 3D images. The number of FISH signals was determined by a gallery of optical sections evaluating only complete nuclei. To investiate the influence of section thickness and truncation and nuclei on scoring results, we compared the FISH data from 15-micron sections with signal counts obtained from 5-micron sections. The latter were evaluated by conventional fluorescence microscopy in the same tumor regions previously defined and marked on the slides. After statistical analysis of spot frequencies in tumor and non-tumorous cells (chi 2 test), we transferred the signal frequencies into a cytogenetic classification (-7, +7, polysomy 7). Based on this classification, most cases showed more than one chromosome 7 aberration type. Trisomy 7 (+7) became apparent in 15-micron thick sections in all 19 tumors, polysomy 7 (> 3 spots) in 18/19 cases, and monosomy 7 (-7) in 13/19 cases. In 5-micron sections, however, trisomy 7 and polysomy 7 were found in only 7/19 and 13/19 cases, respectively, and monosomy 7 in 7/19 cases. When comparing the classification results of tumor cells of the same tumor regions originating either from 5-micron or 15-micron sections, the following discrepancies were noted: in 15-micron sections exclusively, in 12/19 tumors, trisomy 7 was found; in 6/19 cases, polysomy 7; in 8/19 cases, monosomy 7. The high proportion of cases with tumor nuclei expressing only one hybridization signal of chromosome 7 in 15-micron sections could be confirmed as monosomy 7 in five selected cases by double-hybridization using centromere-specific probes for chromosomes 7 and 12. These results demonstrate that numerical chromosome 7 aberrations are more frequently observed in thick (15-micron) paraffin-embedded tissue sections by evaluating only complete nuclei. The use of routine sections (5-micron) for interphase cytogenetic analyses is compromised by a remarkable underestimation of the real chromosome copy numbers.

p53 Alteration and Chromosomal Instability in Prostatic High-Grade Intraepithelial Neoplasia and Concurrent Carcinoma: Analysis by Immunohistochemistry, Interphase In Situ Hybridization, and Sequencing of Laser-Captured Microdissected Specimens

“…These cutoff values were adopted from the available literature (32)(33)(34)(35)(36). In our study, as in others, no BPH specimens or normal prostate epithelium contained values that exceeded these criteria.…”

Chromosomal changes during development and progression of prostate adenocarcinomas

“…When the proportion of cells with nuclei without any signal exceeded 20%, the procedure was regarded as insufficient and therefore repeated. For a detailed description of the FISH method used as well as for the evaluation by confocal laser scanning mircoscopy see Aubele et al (1997).…”

Increasing chromosome 1 copy number parallels histological progression in breast carcinogenesis

“…FISH reactions were prepared with slight modifications of established protocols (Zitzelsberger et al, 1994;Aubele et al, 1997). The predigestion stages included: 70% formic acid for 15 min, incubation in 1 M NaSCN at 80°C, for 10 min, and Pronase digestion.…”

“…Fields of view were scanned with a confocal laser scanning microscope LSM 410 (Carl Zeiss, Jena) and a 100 × objective (Zeiss, PNF, NA 1.3, oil immersion) was used. A sequence of confocal optical sections was taken at axial distance of 0.5 µm, covering the full thickness of the histological section (Aubele et al, 1997). The FISH signals labelled with spectrum orange were taken using excitation at 543 nm and emission greater than 590 nm.…”