HER2 Part 7: DNAarray HER2 PRO

Bottom line: CombiMatrix's DNAarray HER2 PRO test offers a more complete picture of HER2 amplification than FISH and can avoid some false negatives. It also shows other important chromosomal problems beyond HER2.

Understanding a breast tumor's HER2 status is critical for treatment planning. But what to do if a pathology report describes HER2 status as "equivocal" (FISH result between 1.8 and 2.2; IHC result of 2+) or if IHC and FISH results contradict one another and so are "discordant?" Inconclusive HER2 results happen about 10-15% of the time. So this is not a rare situation.

As a side note: HER2 testing guidelines for gastic cancer now require dual IHC and FISH testing, an acknowledgement of both the importance of, and concerns over current HER2 testing methods.

In the past few blogs we've written about newer tests based on technologies different than the ones used in IHC and FISH that can be used to make the determination. Today we'll discuss CombiMatrix's DNAarray HER2 PRO.

First, what does this test "look at?" The answer: the entire chromosome 17 in breast tumor cells in your biopsy sample. The HER2 gene is on chromosome 17. But FISH also looks at chromosome 17. So what's the difference?

It's pretty simple. FISH looks at a specific gene (HER2) by attaching ("hybridizing"... the "H" in FISH) fluorescent molecules to it ("fluorescence" is the "F" in FISH). The more HER2 DNA there is, due to gene amplification, the brighter the fluorescence that's emitted.

FISH determines whether the HER2 gene has been amplified by comparing the amount of fluorescence at the HER2 gene with the amount at a different part of the chromosome that FISH also targets. So the pathologist looks at two points on the chromosome using a fluorescence microscope.

If the amount of fluorescence at the HER2 gene is much greater than the amount at the centromere (pronounced sen'-tro-mere and more technically called CEP17), then the assumption is that the gene has been amplified. So the test is all about the ratio of fluorescence emitted at two different know points on chromosome 17. Pretty tricky.

Now DNAarray HER2 PRO. As previously mentioned, it looks at many more points on the chromosome using a technology you might have heard of in the news called a "DNA microarray" or "DNA chip." The generic term for the DNAarray test is array-based Comparative Genomic Hybridization, or aCGH. You don't have to know this. But I didn't want anyone to be confused if they happened to come across the term.

aCGH uses somewhat similar technology to FISH, but by using the DNA chip, it can simultaneously look at hundreds or thousands of points on the chromosome using fluorescence hybridization, not two. The chip is analyzed using an automated instrument. No one looks through a microscope, as with FISH.

Next question: Why does it matter to look at the whole chromosome? Mainly because one potential problem of FISH is that it can give a false negative result. This is a situation where the test says the tumor is HER2 negative even though there is amplification. This can occur if BOTH the HER2 gene and the centromere are amplified. Remember, FISH looks at the ratio between the two. If both are amplified, the amount of fluorescence signal could be about the same but the reason would be that they are BOTH amplified, NOT that neither is amplified! And centromere amplification is relatively common.

Second, and this goes beyond the issue of HER2, tumors that are more aggressive tend to have bigger problems than just HER2 gene amplification. They tend to have amplification elsewhere, changes in the numbers of chomosomes and other problems that can be summed up as "chromosomal chaos." Chromosomal chaos is easily visualized using aCGH. And if the DNAarray test shows big chromosomal problems on chromosome 17, then there's little doubt that the same thing has happened on other chromosomes. Knowing this, and thus that the tumor is aggressive, might alter treatment decisions.

DNAarray HER2 PRO costs $1,500. Reimbursement not certain, as it is with IHC and FISH. But for patients who either don't have a clear-cut HER2 status or who might be willing to pay for a high-tech method that might provide additional insights into the tumor's characteristics, then it's worth a look.

HER2 Part 4: How doctors determine if you're HER2 positive

Note: This is the blog of LATESTBreastCancer.com, where you can get personalized information about the latest in breast cancer treatment.

Remember what's happening in the cancer cells of patients with an overabundance of HER2: 1) there's amplification of the part of chromosome 17 containing the HER2 gene which creates more copies of the HER2 gene (DNA), 2) as a result there is an increase in the amount of HER2 mRNA produced, 3) the HER2 mRNAs make more HER2 proteins, 4) these HER2 proteins go to the cell membranes and there they have to pair up with each other with other HER family members to send a grow signal into the cell.

The two standard tests look at two different points in this process: the amount of HER2 protein on the cell surface or the amount of HER2 gene due to amplification.

These two tests are known by acronyms.

IHC (immunohistochemisty, and usually a commercial test called HercepTest) looks at the amount of HER2 protein.

FISH (fluorescence in situ hybridization, and usually either the PathVysion test or the HER2 FISH pharmDx test) looks at the amount of HER2 DNA (genes).

Normally IHC (HercepTest) is used as the primary test. A biopsy sample is sent to a pathology lab. Slices of the sample are stained with special antibody-based reagents that bind to HER2 molecules on cells. The stained slice is put under a microscope. A histopathologist looks at the level of staining and assigns a value based on the amount of staining seen from 0 to 3+. Zero and 1+ are considered HER2 negative. A value of 3+ is considered HER2 positive. A value of 2+ (weakly staining) is considered "equivocal," or in other words, unclear. (See image, the HER2 protein on the cells stains orange-brown).

In situations where the IHC result is equivocal, they then look at a different cell characteristic -- DNA amplification -- using FISH. Again they use a biopsy sample. In FISH, DNA is stained with fluorescently-labeled DNA (Note: there is a newer technique called CISH -- commercial test name SPoT-Light -- which uses something called chromogenic staining instead of fluorescent staining, but these are essentially equivalent).

Anyway, with FISH, HER2 genes stain red. A region of chromosome 17 that is never amplified is stained green. Using a microscope, the ratio of red to green fluorescence is determined. The higher the ratio, the higher the degree of HER2 gene amplification. Information about the amount of HER2 gene amplification is used to resolve an equivocal 2+ IHC test result.

The concerns about the accuracy of HER2 testing (and thus the appropriate use of Herceptin and other HER2 targeted drugs) have to do standardization, accuracy and reproducibility of these tests. Both are subjective and only semi-quantitative. It isn't always easy to grade IHC stained cells to begin with and, on top of that, artifacts due to lab technique can sometimes impact staining.

In 2007 the major medical associations in this field stated in their published guidelines that standardization of IHC and FISH was major problem. A study in a reputable journal in 2002 estimated that 20% of HER2 testing might be inaccurate. Other studies generally found that variations between different laboratories caused a 10-20% disagreement in results between IHC and FISH. And a study in 2006 found that agreement in test results between local and centralized laboratories for IHC was just 75-82%, and for FISH was 88%. (References supplied upon request.)

The issue of HER2 testing accuracy remains. This is a topic we'll be digging into deeper in future blogs.

Next: Newer tests for determining HER2 status.