FISHing for HER2 Stability on a Turbulent Chromosome 17

Have you ever wondered why so many high-risk breast cancer cases have HER2 equivocal results by FISH? That’s right, in an estimated 20% of the over 200,000 breast cancers diagnosed in the US every year, the FISH classification of the tumor’s HER2 gene copy number is reported as “EQUIVOCAL.” For clinicians trying to make decisions about whether (or not) to use anti-HER2 targeted therapies for their patients, the “Equivocal” result muddies the water instead of clarifying treatment options.

So what is wrong with FISH-based testing? Haven’t we been performing FISH using FDA approved kits for over a decade, and isn’t fluorescent in situ hybridization (FISH) a DNA based method that allows visualization of HER2 gene amplification on chromosome 17? The answer to all of these questions is yes! We’ve been performing FISH for many years and performing it correctly. We are getting the right answers about HER2 gene copy number.

The problem comes with the way HER2 FISH is reported in relationship to chromosome 17. When FISH was first developed as a test for HER2 gene amplification, the assumption was that the center portion of the chromosome between the p and q arms called (cep17) remained stable even when the HER2 gene was amplified. Thus to distinguish breast cancers with “true” HER2 gene amplification from those with an extra copy of chromosome 17, HER2 gene results are commonly reported as a ratio that looks like this:

If the HER2 gene and cep17 copy numbers are both in the normal range (< 4), the ratio should give a true negative result of < 2. If the HER2 gene is amplified to > 6 copies and cep17 is in the normal range, the result will be a true positive HER2 result of > 2. But if the HER2 gene copy number is > 6 and the ratio is less than 2, then something is wrong because this should be a positive ratio of > 2 (if cep17 is the expected 2.) We now know that cep17 region does not remain stable in breast cancer, and can amplify as much as the HER2 gene.

chromosome-17In fact, chromosome 17 is among the most unstable chromosomes in breast cancer. All regions can be gained, amplified, or deleted as you can see in this example of Chromosome 17 from a breast cancer patient with both cep17 (top arrow) and HER2 gene (bottom arrow) amplifications. Despite this biological reality, many labs are still reporting FISH results for HER2 based on assumed stability of the surrounding chromosome 17. Some laboratories are even using alternative probes from distant regions of chromosome 17 to try to establish a stable denominator. Yet as you can see from the chromosome example, there is no stability in this ocean.

It is time to update the analysis of HER2 gene copy number in breast cancer to reflect the reality of the chromosome 17 in which we FISH.

Shelly Gunn MD, PhD| Pathologist |October 29, 2016

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