Using FISH for primary testing
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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