Simultaneous imaging of membrane antigen and the corresponding chromosomal locus in pathology archives

Abstract

A new procedure for the simultaneous staining of membranous antigens, such as tyrosine kinase-type cell surface receptor HER2 (c-erbB2), and the corresponding chromosome (chromosome 17 for c-erbB2) in the same cell for use in examining pathology archives is presented. A multistep procedure involving microwave-assisted fluorescence in situ hybridization and immunofluorescence yielded cell images having c-erbB2 on the membrane and genomic signals from the chromosome 17 centromere and the c-erbB2 locus. Furthermore, a combination of microwave-assisted chromogenic in situ hybridization and immunohistochemistry found colorized signals from both chromosome 17 centromere in the nuclei and c-erbB2 on the membranes of individual cells. Quantitative image analysis further confirmed the presence of a significantly stronger c-erbB2 immunoreactivity on cells containing three or more signals from chromosome 17 than from those with less than three signals. It was possible to extend the constellation of cell surface markers and corresponding chromosomes or locus-specific makers to several other genes including CDH1. In this case, the disappearances of CDH1 expression, a CDH1 locus signal, and a centromere enumeration probe (CEP) 16 signal were simultaneously demonstrated in the less-adhesive tumor cells. Thus, it is believed that this procedure might pave the way for exploiting pathology archives for the genotype-phenotype analysis of individual cells.

PIK3CA mutation and amplification in human lung cancer

“…After fixing the tissue specimens in 80% ethanol or 3.8% formaldehyde and embedding them in paraffin, they were arrayed with tissue microarrayer KIN (Azumaya, Tokyo, Japan). 13 Sections on slides were dewaxed, re-hydrated, and then boiled in citrated buffer (0.01 mol/L, pH 6.0) to release closed chromosomal structures. 14,15 The sections were then hybridized with a Cy3-labeled probe for the PIK3CA locus (Bacterial artificial chromosome (BAC) number RP11-339F13) and fluorescein isothiocyanate-labeled centromere enumerating probe on chromosome 3 (CEP3; Vysis, Downers Grove, IL, USA).…”

Chromosomal numerical abnormalities in early stage lung adenocarcinoma

“…35 MW-assisted FISH using various bacterial artificial chromosome probes would also help this refinement. 36 CNA, both gains and losses, seem to occur both differently and concurrently in the temporal spectrum of lung carcinogenesis. In particular, after the tumor exhibits overt carcinoma (BAC and later), both gains and losses can occur, and we could not detect any unidirectional tendency.…”

“…Further analysis as to more specific changes covering the genome‐wide loci will refine the whole picture of this sequence, including chromosomal evolution 35 . MW‐assisted FISH using various bacterial artificial chromosome probes would also help this refinement 36 …”

Fluorescent in situ hybridization heating pretreatment: The key is temperature control

“…Notably, we were able to perform FISH successfully on immunostained slides from cases where paraffin blocks were exhausted (unpublished observations). This combination is very useful for diagnosis 23 …”