NEWS

Experts Debate Value Of HER2 Testing Methods

Nancy J. Nelson

In spite of the 1998 Food and Drug Administration approval of Herceptin for treating advanced breast cancer patients with HER2-positive tumors, there is still a controversy about the method of choice to determine whether a tumor is HER2 positive. To air the conflicting data and hopefully come up with new strategies, the National Cancer Institute brought together more than 100 experts for a day and a half last October.

"The general conclusion was essentially that we really don’t have an ideal way to detect HER2," said Thomas Davis, Ph.D., senior investigator at NCI’s Cancer Therapeutics Evaluation Program who organized the meeting. "We need to include multiple detection methods in future trials to establish which is a superior method."

Drug and Test Development

Overexpression of the HER2 protein occurs in 25% to 30% of breast cancers and to varying degrees in other tumors. The HER2 gene is amplified in about 90% of breast tumors that overexpress the HER2 gene protein product.

When Dennis Slamon, M.D., Ph.D., at the Jonsson Comprehensive Cancer Center at the University of California, Los Angeles and his colleagues found that human breast tumors that overexpress HER2 have high proliferation rates and are associated with poor prognosis and shortened disease-free survival, they decided to target the receptor for therapy by making antibodies to the HER2 protein product. The initial antibodies used in the preclinical studies were mouse monoclonal antibodies 4D5 and CB11. 4D5 was humanized (i.e., a part mouse, part human antibody molecule), and the resulting therapeutic agent was trastuzumab, or Herceptin, developed by Genentech Inc., South San Francisco, Calif. The trials that led to FDA approval in 1998 showed that Herceptin was effective in combination with chemotherapy as first-line therapy for treating women with HER2 overexpressing metastatic breast cancer.



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Dr. Dennis Slamon

 
Several techniques are available to assess HER2 status — those that measure gene amplification (Southern blot analysis, polymerase chain reaction, and fluorescence in situ hybridization or FISH), protein expression (immunohistochemistry or IHC and western blot analysis), or messenger RNA levels (northern blots). Three kits for testing HER2 status — one IHC and two FISH (see box, p. 294) — have been approved by the FDA.

One of the three kits, HercepTest (DAKO Corp., Glostrup, Denmark, and Carpinteria, Calif.), was approved in 1998 along with Herceptin as an aid to determine which patients are eligible for Herceptin treatment. Some of the confusion in the HER2 field arises from the fact that the two mouse monoclonal antibodies, 4D5 and CB11, used in the preclinical treatment studies were also used in the Herceptin clinical trials to test HER2 levels, but are not part of HercepTest. It contains a polyclonal goat antibody that was found to be nearly as effective as 4D5 and CB11 in detecting HER2 protein, but it was not used in the trials leading to the FDA approval of Herceptin.

HercepTest uses IHC, one of the two most common techniques to measure overexpression of the HER2 protein. The other, FISH, detects gene amplification. IHC is more widely used. (See sidebar on opposite page.)

No one knows yet which of the methods is best. Lynn Dressler of the University of North Carolina at Chapel Hill has found a strong association between PCR, FISH, and IHC as well as a very similar ability to predict clinical outcomes, but she thinks that not enough studies have assessed whether one method is superior to another.

"The clinical utility of any marker needs to be proven in randomized clinical trials and that hasn’t been done with any of the FDA-approved kits or any other methods out there," she said. "There is general agreement that this is exciting and that we’re headed in the right direction, but there are still gaps that will be filled by studies in progress or that need to be planned."

More Discrepancies

Shelia E. Taube, Ph.D., associate director of NCI’s Cancer Diagnosis Program, pointed to some of the confusion generated by the lack of a standardized test. She found that most prognostic studies showed that HER2 was a predictor of poorer outcome in patients with node positive tumors. However, Taube noted that in studies using gene amplification seven out of eight showed prognostic value, while in those using IHC only six out of 12 predicted poorer outcome.



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Dr. Shelia E. Taube

 
Not only is there a lack of concordance among different techniques, but researchers using the same technique often disagree on the outcome. Michael Press, M.D., Ph.D., at the University of Southern California School of Medicine, Los Angeles, in a 1994 paper in Cancer Research showed that part of the problem is the varying sensitivity of reagents. He reported that 18 out of the 28 antibodies commonly used by various laboratories detected the increase in HER2 levels when tissues were amplified by 5-fold or greater. However, at lower levels of amplification, 2 to 5-fold, only 12 of the 18 "good" antibodies stained the tumors and four stained virtually none of the tissue samples.

Different scoring systems used in IHC can also cause discrepancies. Researchers can choose the percentage of cells stained that deem the tumor "positive." This can vary from 10% to 50%, Taube said. "Obviously, this is going to make a difference in what your overall numbers are and whether or not you’re going to get statistical significance in your evaluations," she concluded.

A case in point is three studies that strongly suggest that tumors overexpressing HER2 respond better to doxorubicin therapy than tumors not overexpressing the receptor. In one (the Cancer and Leukemia Group B reported in the Journal in September 1998), researchers used the CB11 antibody and defined overexpression as 50% or more of cells showing positive expression.

In contrast, NSABP researchers (reported in the same issue of the Journal) used a cocktail of two antibodies, mAB-1 and pAb-1, a mouse monoclonal antibody and rabbit polyclonal serum, respectively. Any antibody staining was considered positive. In a third Southwest Oncology Group study, CB11 and mAB-1 antibodies were used separately to determine HER2 status, and mAb-1 was found to be a better predictor of response to therapy.

Some people at the conference, like Slamon, spoke emphatically that the lack of proven reagents make it impossible to interpret any of the data.

"Even in the hands of the best pathologists, experienced people, a bad reagent will not perform," said Slamon. "The best antibodies only detect 80% of the cases and miss 20% of the cases."

He is worried about the high false positive rate of the HercepTest antibodies and believes that they may have led to the appearance of "elevated" expression rates in lung, prostate, and colorectal tumors that, in fact, are really normal HER2 expression levels. "We have preliminary data that’s not published but has been submitted that shows FISH to be a more powerful technique by a lot. The DAKO HercepTest, I believe, is flawed."

Press, a pathologist, also argues in favor of FISH, not only because of the different sensitivity of the antibodies, but also because of disagreement among pathologists on how to score a positive result.

"Fifty percent of the time, pathologists cannot agree on how to interpret a test," he said. "I’m not talking about the quality of the test. What I mean is, once it’s finished, how it is interpreted."

Press showed in a 1999 paper in Seminars in Oncology that there was nearly complete concordance of the FISH with Southern/dot blot analysis. Using 140 breast cancer specimens, specificity was 100% and sensitivity nearly 100%; one of 50 specimens were falsely negative with FISH, but positive with Southern/dot blot analysis. Using FISH, he showed that HER2 status was an important predictor of early recurrence and overall survival of invasive breast cancers without lymph node involvement.

FISH also won out in the yet unpublished results Press presented at the conference comparing the levels of HER2 in 125 breast cancer cases that were first analyzed by southern dot blot hybridization and then by six other tests — four different antibodies and the two FDA-approved FISH assays.

"The FISH assays were the best, but the two in-house antibodies were close. The two worst tests were the two commercial antibodies — the HercepTest test, and Ventana’s CB11 antibody that is under consideration for approval by FDA."

Patient Perspective

Christine Brunswick from the National Breast Cancer Coalition put a more personal face on the meaning of the current state of HER2 testing. She pointed out the current 7% false positive rate of commonly used antibodies means that about 3,000 to 4,000 women will be treated with HercepTest with no known benefit. The 20% false negative rate means that 9,000 to 10,000 U.S. women who might benefit from the treatment will not be treated. "As patients and advocates, we believe that this is not good enough," she said.

In spite of the overwhelming dissatisfaction with the state of HER2 testing, there was a clear consensus that HER2 itself is a clinically important molecule. And several groups, including cooperative groups, Genentech, and DAKO, are working hard to get a handle on the best detection technique.

Peter M. Ravdin, M.D., of University of Texas Health Science Center at San Antonio, one of the conference organizers, stepped back a bit and put the lack of a standardized HER2 test in historical perspective by saying that he feels HER2 is one of the two central molecules driving breast cancer therapy; the estrogen receptor (ER) being the other. And he sees important analogies between them.



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Dr. Peter M. Ravdin

 
"In many ways the situation today is similar to that of 25 years ago, when early studies with ER suggested its utility but no well-accepted standard assay existed," he said. "Since that time, there have been several standardized methodologies developed for measuring ER status, as well as quality-assurance programs for validating the accuracy of the clinical laboratories. HER2 testing will probably follow a similar path."

In the March 1 News: A review of recently presented studies that look at the association between HER2 levels and response to hormone therapy and chemotherapy.


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