HER2 Part 6: Agendia's TargetPrint Test

Bottom line: TargetPrint is a quantitative mRNA test that can be used as a second opinion to IHC or FISH testing or as a first line test to measure HER2, ER and PR.

There are three situations where it would be reasonable to consider some of the next generation, "non-standard-of-care" tests, like TargetPrint, that assess a breast tumor's HER2 status:

1) The first situation is when a basic IHC (immunohistochemistry) test like Dako's HercepTest gives an "equivocal" result (a score of 2+). Today FISH is most commonly used in this case. But other tests could be considered. Or multiple different tests could be used.

2) The second situation is where neither IHC or FISH deliver a clear positive or negative result, or if they give conflicting ("discordant") results.

3) Finally, if a tumor is designated HER2-negative but the disease is progressing faster than expected, then consider rechecking with another kind of test.

Agendia's TargetPrint test looks quantitatively at the amount of HER2 mRNA in tumor cells. This is different than standard IHC tests (like Dako's HercepTest) that look at the amount of protein on the tumor cell surface semi-quantitatively, or FISH tests (like Abbott's' PathVysion or Dako's HER2 FISH PharmDx) that look at the amount of DNA amplification.

Why might looking at mRNA be useful? Consider the biology. Within the cancer cells, a portion of chromosome 17 (home of the HER2 gene) is being amplified. Instead of the normal double helix of chromosome 17 DNA, the cell has aberrantly made many double helixes. There are too many HER2 genes so the cell naturally produces too much HER2 mRNA. These HER2 mRNAs float around the cell and are used to creates too many HER2 proteins.

In theory, the amount of DNA amplification, mRNA and protein should coincide. But biology is complex and scientists always have an imperfect picture of what's going on inside of the cell. In addition, there are strengths and weaknesses associated with different tests and lab techniques. Some tests are more subject to laboratory error, for example. So the idea of using different techniques to measure different aspects of biology (DNA, mRNA and proteins) and to cross check one against the other is a reasonable strategy.

In addition, analyzing mRNA as a means of understanding what's happening inside cancer cells has become important in recent years. mRNA analysis is the basis of Genomic Health's popular Oncotype Dx test and Agendia's better known test, MammaPrint test, both for assessing recurrence risk. So it is a growing diagnostic approach.

Back to TargetPrint. Yes, it analyzes HER2. But it also analyzes estrogen receptor (ER) and progesterone receptor (PR), also by looking at mRNA. Normally both are analyzed by IHC. Several recent studies show that ER testing is far from perfect. For example, a Yale study that just came out this week found that 10-20% of patients who are told they have ER-negative tumors really have ER-positive tumors. Another recent study found that patients test negative for ER by IHC more often on Fridays and Saturdays! So, as mentioned before, IHC is subject to lab and lab technician variability.

TargetPrint costs $1200 and it is covered by Medicare.

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.