Comparative FISH analysis of numerical chromosome 7 abnormalities in 5-micron and 15-micron paraffin-embedded tissue...

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.