College of American Pathologists
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Using FISH for primary testing

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September 2000
William Check, PhD

In 1993, at the outset of the Herceptin clinical program, practical considerations dictated the use of immunohistochemistry to guide patient selection. Says Robert Mass, MD, clinical team leader for Herceptin, "At the time, gene-based detection methods required fresh or frozen tissue, which was not practical for large-scale clinical work." Preclinical work suggested that HER2 receptor density greater than 100,000 to 200,000 receptors per cell would be required for activity. These were levels of HER2 expression typically seen with gene amplification and corresponded to an IHC score of 2+ (~500,000 receptors) or 3+ (~2,000,000) using the Clinical Trials Assay, or CTA.

"We needed to validate HercepTest by establishing concordance [with CTA results]. We couldn’t directly evaluate it on slides from trials patients because antigen had severely degraded over time," Dr. Mass says. They screened about 1,200 breast cancer blocks from the National Cancer Institute (the Cooperative Breast Cancer Tissue Resource) with the CTA to generate about 300 2+/3+ specimens and an equal number of 0/1+ specimens, which were then assayed with HercepTest. This analysis formed the basis of HercepTest’s approval as an aid in the selection of patients for Herceptin therapy.

At this year’s meeting of the American Society of Clinical Oncology, Dr. Mass presented results of FISH testing of a large subset of trials slides (unlike protein, DNA is stable) compared to original results with CTA. Gene amplification was seen in 89 percent of 3+ tissues, but only 24 percent of 2+ samples. More important, he presented data indicating Herceptin benefit is restricted to those patients demonstrating gene amplification. In the pivotal single-agent Herceptin study (reported by Cobleigh, et al), no patient responded in the FISH-negative group, including 17 patients demonstrating 3+ IHC scores by the CTA. All of the responses were in the FISH-positive group. In the pivotal chemotherapy trial (reported by Slamon, et al), there was no improvement in response rate by adding Herceptin to chemotherapy in the FISH-negative group; however, the response rate doubled, from 27 percent to 54 percent, when Herceptin was added to chemotherapy in the FISH-positive group.

"These data indicate that FISH testing is a more precise method compared to IHC to select patients for Herceptin therapy," says Dr. Mass. He points out that about 20 percent of gene-amplified patients will score as 0 or 1+ using IHC. "In our CTA/FISH concordance study, we confirmed the observations of others that IHC exhibits a false-negative risk. These data support using FISH for primary testing."

A tiered-testing strategy is not optimal, Dr. Mass says. "If pathologists are doing what many of them are doing, and that is screening with IHC and confirming 2+ results with FISH, they will miss substantial numbers of amplified patients that scored 0 or 1. Amplified patients are the group demonstrating benefit from the drug."

Data presented at ASCO show a 50 percent improvement in survival when Herceptin was added to chemotherapy in the FISH-positive group (18 months versus 27 months), he adds. "We believe that moving to primary FISH-based testing will reduce the number of patients treated, but will select patients most likely to benefit from Herceptin," he says.

Dr. Ann Thor, staff pathologist at Evanston (Ill.) Northwestern Healthcare, cautions, "The antibody used in the CTA is not even available anymore. People who have seen those slides say they have a very high background. So a comparison of that assay with FISH does not necessarily relate to the assay we are using today." She concludes, "We need to compare our current IHC methods to current FISH methods-with greater attention to assay and interpretive issues for each."

William Check, PhD