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.
Global Genomic View
In addition to performing in high throughput, other advantages according to Dr. Gunn are that, “it is an objective and reproducible test that not only measures HER2 gene copy number, but gives a global genomic view of that patient’s tumor. This allows the clinician to design personalized treatment programs to give the patient an individualized prognosis based on the total profile of genomic aberrations present in the tumor.
“For example, many of the HER2-positive breast cancer genomes analyzed show co-amplification of the MYC gene on 8q24, which confers a less favorable prognosis and predicts decreased response to Herceptin.”
Array CGH can also complement IHC and FISH for subtyping newly diagnosed breast cancer. “IHC and FISH can be used in the original diagnostic workup of the patient to get a rough idea of key prognostic factors such as assessment of ER/PR (estrogen receptor per progesterone receptor), and HER2 status, which are critical for predicting response to systemic therapies,” Dr. Gunn explains.
“Using array CGH during the post-operative workup is best because fresh tissue is available, and the array can be used to confirm HER2 status and further subtype the tumor.”
There are some disadvantages to array CGH. It works best on fresh tissue, which may be difficult to get, and the technology is not yet widely available. As Dr. Gunn states, “it costs a little more than FISH analysis for HER2 gene status, but delivers a global view of the genome, which would be impossible to obtain using individual FISH probes.”
Though array CGH is seen as a rapid and cost-effective way of examining a whole genome of a subject for CNVs, Paul Kassner, Ph.D., principal scientist, lead discovery at Amgen, states that, “it is useful for finding the range of CNVs in the normal population that do not overtly cause disease, but that can influence phenotypes and impact disease. For example, extra copies of gene CC3L1 can reduce a person’s susceptibility to HIV infection and progression to AIDS.”
Some disadvantages that Dr. Kassner finds in working with mouse models are that arrays designed based on the reference genome for a given species can often be incomplete, thus, making array CGH results incomplete. “Array CGH is also unable to elucidate structure differences in the genome that do not result in copy number changes and is not able to tell us about translocations or inversions,” explains Dr. Kassner, who gave a presentation at the San Diego meeting.