In an IHC assay, a slice of tumor tissue is stained, along with a 3+ control specimen in a corner of the slide, and then the tumor sample in question is examined with a bright field microscope. The amount of observed staining correlates with the quantity of HER2 protein.
Subjective decisions in scoring a tissue specimen are opportunities for variability in an IHC HER2 assay. Even flawless technique and interpretation can still generate a FISH result in what pathologists call the “indeterminate” range of 1.8 to 2.2, which means another slice of the tumor tissue has to be tested using a FISH assay.
“With immunohistochemistry, the results can vary based on how long the tissue is fixed and what antibody you use for the staining. Many, many variables can influence the result,” says Paik, who nevertheless agrees with the majority of the ASCO/CAP expert panel that FISH is not superior to IHC. “You also have to judge the scoring based on how strong the staining is, and that can be quite subjective. You could have two pathologists looking at the same slide and one might call it 2+ positive staining and one might call it 3+ positive staining.”
Interpreting a FISH HER2 test, on the other hand, is a much more objective process, FISH proponents argue. With the FISH assay, the pathologist counts actual copies of HER2 genes, which appear as a red “signal” in a blue-stained cancer cell nucleus seen through the microscope.
“On a good day, I can count to 20 and I can tell whether there are two copies of a red signal in a blue nucleus or whether there are 20 red signals in a blue nucleus,” says Michael F. Press, MD, PhD, professor of pathology at the Keck School of Medicine, and coordinator of the Women’s Cancers Program at the Norris Comprehensive Cancer Center, University of Southern California.
Press and his group at USC, in collaboration with a group led by Dennis J. Slamon, MD, PhD, who led the research for trastuzumab and is now director of the Revlon/UCLA Women’s Cancer Research Program at Jonsson Comprehensive Cancer Center, in Los Angeles, and chief of the Division of Hematology/Oncology at UCLA’s Department of Medicine, have amassed more than 20 years of published research on the topic of HER2 testing and the accuracy of IHC and FISH assays. Overall, these studies unambiguously point to FISH as consistently more accurate.
In daily practice, however, between 80 and 90 percent of primary HER2 testing in the United States is done with IHC, while only 10 to 20 percent is done with FISH. Approximately 10 percent of IHC test results fall into the so-called “indeterminate” range, and those specimens are re-tested using FISH.
If you’re wondering why not just do the FISH test in the first place, you’re not alone.
“That would be my position exactly,” says Press, who then goes on to suggest several reasons why IHC outnumbers FISH in primary HER2 testing: Because pathologists are familiar with the assay (it’s been in use since the 1970s); because many pathologists believe that an IHC assay is just as accurate as a FISH assay; and because it’s fast and relatively inexpensive. Prices vary, but an IHC assay may cost $100 to $150, and a FISH assay may be double or triple that price.
“I do not think that immunohistochemistry done even in the best laboratories and with the best pathologists is good enough,” says ASCO/CAP guideline coauthor Michael F. Press, MD, PhD, “because the method is flawed.”
Price certainly is a consideration, but incorrect HER2 test results entail far greater economic and human costs. A 52-week course of chemotherapy plus trastuzumab based on a false positive assay exceeds $50,000 and comes with a grab bag of nasty side effects, including potential cardiotoxicity. Conversely, a false negative assay deprives a woman with HER2-positive breast cancer of therapy that can offer a total pathologically complete response to treatment (complete disappearance of tumors from both the breast and lymph nodes) in nearly half of patients.
Ross estimates that approximately 3 to 4 percent of IHC assays in the United States generate a false negative and are not followed by a FISH test. Of these 3,000 to 4,000 women, 1,500 to 2,000 whom otherwise might have benefited from trastuzumab therapy will relapse with breast cancer.
“Labs like mine believe the FISH test is more reliable and accurate, so we don’t bother with the immunohistochemistry and just do the definitive tests for all the patients,” say Ross.
The accuracy of an IHC assay also is more dependent than FISH on preanalytic variables, such as how long it takes before the tissue specimen is fixed, how long it remains in the fixative solution, and how it’s subsequently processed. In the United States, the majority of pathology specimens are fixed in formalin and embedded in paraffin.
“In 1989, we showed with molecularly characterized samples that immunohistochemistry has the potential for erroneously classifying tumors based on formalin-fixed, paraffin-embedded samples, whereas if one used a frozen tissue sample from the same patient, you got a relatively accurate result,” says Press. “Formalin fixing and paraffin embedding introduce a lot of artifacts that confound the assay results. It’s very hard to know whether you’re getting a good result or a flawed result.”
To be sure, FISH is not without its disadvantages. Cost again becomes a consideration because FISH requires a fluorescence microscope, a dark room in which to use it, and a board-certified pathologist who can tell the difference between the blue nuclei of cancer cells in the specimen and the blue nuclei of benign reactive cells.