High-level detection of gene amplification and chromosome aneuploidy in extracted nuclei from paraffin-embedded tissue of human cancer using FISH: a new approach for retrospective studies

Abstract

A novel application of fluorescence in situ hybridization (FISH) to isolated nuclei is described. The method detects gene amplification and chromosome aneuploidy in extracted nuclei from paraffin-embedded tissue of human cancer with greater sensitivity and specificity than existing FISH methods. In this study, the method is applied to signal detection of the HER-2/neu (c-erbB-2) gene, whose amplification is one of the most common genetic alterations associated with human breast cancer. Nuclei were extracted and isolated from formalin fixed, paraffin embedded tissue of 43 different carcinomas (breast, ovary, endometrium, gastrointestinal stromal tumor and malignant mesothelioma). FISH was performed both on sections and extracted nuclei of each tissue using chromosome enumeration probes (CEP) for the centromeric regions of chromosomes 8 and 17, and a locus specific identifier (LSI) for the HER-2/neu oncogene. Differences between ploidy calculated in sections and extracted nuclei were seen in 3 breast carcinomas and 1 gastrointestinal stromal tumor (GIST). Furthermore, 1 breast cancer, previously considered to be borderline for HER-2/neu gene amplification turned out to be clearly amplified. Nuclei extraction and isolation bypass all the problems related to signal interpretation in tissue sections, and the adoption of this new technique, which improves the signal quality in several neoplastic samples, is suggested

Clinical value of DNA content assessment in endometrial cancer

“…Briefly, this method allows identification of the number of copies of a specific region of a chromosome, i.e., a gene or the centromere region, using specific probes, i.e., chromosome enumeration probes (22). Briefly, this method allows identification of the number of copies of a specific region of a chromosome, i.e., a gene or the centromere region, using specific probes, i.e., chromosome enumeration probes (22).…”

“…Another method used for analysis of DNA ploidy is fluorescent in situ hybridization (FISH). Briefly, this method allows identification of the number of copies of a specific region of a chromosome, i.e., a gene or the centromere region, using specific probes, i.e., chromosome enumeration probes (22). Hence, FISH can be used to detect chromosomal alterations such as loss or gain of specific chromosomes, and thus reflect aneuploidy (22)(23)(24).…”

“…Briefly, this method allows identification of the number of copies of a specific region of a chromosome, i.e., a gene or the centromere region, using specific probes, i.e., chromosome enumeration probes (22). Hence, FISH can be used to detect chromosomal alterations such as loss or gain of specific chromosomes, and thus reflect aneuploidy (22)(23)(24). The FISH method has been shown to be useful for evaluation of aneuploidy in endometrial carcinomas (24), but has no widespread clinical use yet.…”

Single-Nucleus RNA-Seq: Open the Era of Great Navigation for FFPE Tissue

“…In detail, FFPE sections were incubated in a pepsin dissociation solution (0.5% pepsin, 0.9% NaCl, 2N HCl) at 37 °C for 30 min to prepare a nuclei suspension. Since then, this protocol [ 17 ] has undergone several modifications and improvements to be suitable for paraffin-embedded materials, and applied to in situ hybridization (ISH) of DNA probes [ 18 ], as well as fluorescence in situ hybridization (FISH) [ 19 , 20 , 21 , 22 ]. In 1985, Schutte et al [ 23 ] dissociated the nuclei of FFPE tumor samples using a trypsin solution and determined the DNA ploidy levels by FCM.…”