Fluorescent In Situ Hybridization
FISH, also a visualization method, is used for locating and mapping chromosomes, genes, and gene segments. It can be performed on dividing, nondividing, and formalin-fixed cells, and uses single-stranded DNA probe labeled with a fluorescent dye. The probe binds to its complementary DNA site, and its fluorescent tag depicts its location. Different FISH probes can be used for different types of gene interrogation such as finding genes, determining chromosome number, and examining chromosomal abnormalities.
One FISH procedure measures the ratio of genes to chromosome 17 in a 60-cell sample. In a normal cell, the HER2 gene per chromosome 17 ratio is 1:1. HER2 gene per chromosome 17 ratios ranging from 1.8 to 2.2 are borderline. Tumor samples with an average HER2 gene per chromosome 17 ratio greater than or equal to 2:1 are reported as HER2 positive. Another FISH procedure measures the average number copy number of HER2 genes per cell. If the average HER2 gene copies per cell are greater than four but fewer than six, results are borderline. Like the IHC test, FISH scoring is subjective.
Shelly Gunn, M.D., Ph.D., medical director, Combimatrix Molecular Diagnostics, states that, “the CAP/ASCO directives are meant to deal mainly with inconsistencies of HER2 testing due to preanalytical variables such as formalin-fixation times.” However, she does not think that the directives solve the inherent subjectivity of IHC and FISH. “Both tests rely totally on visualization of results, either by protein immunostaining or by fluorescent DNA probes, and have unreliable, inter-observer reproducibility.”
To address the CAP/ASCO directives, array CGH is gaining use in determining HER2 copy number and in helping direct the course of breast cancer treatment.
CGH was developed in 1992 to analyze cytogenetic changes in solid tumors. Though not initially designed as an array, it was an alternative to tumor karyotyping, which can be hampered by insufficient numbers of high-quality metaphase cells to study. CGH can use fixed tissue or fresh samples to map DNA CNVs as a function of chromosome location in the genome.
CNVs are DNA segment gains and losses ranging from 1 kb to several Mb observed by comparing two or more reference genomes. These quantitative structural variants can include one or more genes. In some cases, they can affect gene function profoundly; in other cases, they display no overt phenotype.
To design an array CGH for high- throughput testing, oligonucleotide microarrays are designed to span the entire genome of a species based on the reference sequence for that species. For human genomic testing, arrays are made using bacterial artificial chromosomes (BACs) that contain referenced regions of the human genome.
Test genomic DNA and a control are labeled differentially with fluorescent tags and are hybridized competitively to that microarray. The array is scanned and fluorescent ratios indicate relative CNVs of the given oligo probe for the test subject versus the control. Each oligo probe is mapped to the reference genome. These fluorescent ratios are processed through an algorithm to get CNVs.