New ASCO/CAP Guideline Enhances HER2 Testing in Breast Cancer

Antonio C. Wolff, MD

The American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP) have released updated recommendations for HER2 testing in breast cancer. The new guideline endorses HER2 testing for every primary, recurrent, and metastatic tumor. Additionally, the report advocates retesting in patients who previously tested HER2-negative but demonstrate clinical behavior suggestive of HER2-positive or triple-negative disease at the time of recurrence.

The accurate identification of patients with HER2-positive breast cancer remains an area of high interest, since HER2-targeted therapies can substantially improve their survival. The updated guideline follows counsel from a joint committee that performed an extensive literature review of more than 70 publications that had been published since the National Comprehensive Cancer Network, in collaboration with ASCO/CAP, published the first guideline in 2007.

"This guideline update strengthens and clarifies recommendations for HER2 testing based on new evidence,” said Antonio C. Wolff, MD, co-chair of the ASCO/CAP HER2 Testing in Breast Cancer Panel and professor of Oncology at the Johns Hopkins Kimmel Comprehensive Cancer Center, in a statement. “Our ability to identify cancer subtypes that will lead to more individualized therapeutic decisions and that are shown to improve clinical outcomes is rapidly improving. Consequently, and more than ever before, society must demand access to high-quality cancer biomarker tests that can help cancer specialists match the right treatments with the right patients.”

The interpretation of HER2 test results follows an algorithm that was modernized for both immunohistochemistry (IHC) and in-situ hybridization (ISH) based assays. The refurbished criteria focus on HER2-positive status, defined via IHC as protein overexpression or via ISH as gene amplification by either HER2 gene copy number or HER2/CEP17 ratio.

At the time of the 2007 guidelines, a leading concern was the number of false-positive rates. However, in recent years, this has largely been reversed toward a concern regarding false-negative results. As such, the amended guideline places more emphasis on ensuring that HER2-positive disease is accurately detected.

For patients who test HER2-negative but demonstrate apparent histopathologic discordance, three levels of testing are suggested. The HER2 test can be performed on a core biopsy or on a surgical specimen from a patient with newly diagnosed breast cancer. If there is discordance, a section of the tumor from the excisional specimen should be tested. If this test is negative and concerns remain, the updated guideline recommends retesting in a different block from the patient’s tumor. If all three tests are negative, no additional testing is recommended (See table for full histopathological features).

Table. Histopathologic Features Suggestive

of Possible HER2 Test Discordance

Retest if the following histopathologic findings occur and the initial HER2 test was positive:

Histologic grade 1 carcinoma of the following types:

• Infiltrating ductal or lobular carcinoma, ER and PgR positive
• Tubular (at least 90% pure)
• Mucinous (at least 90% pure)
• Cribriform (at least 90% pure)
• Adenoid cystic carcinoma (90% pure) and often triple negative

If the core needle biopsy specimen is negative, a new HER2 test must be ordered on the excision specimen

If the following is true:

• Tumor is grade 3
• Amount of invasive tumor in the core biopsy is small
• Resection specimen contains high-grade carcinoma that is morphologically distinct from that in the core

• Core biopsy result is equivocal for HER2 after testing by both ISH and IHC
• There is doubt about the specimen handling of the core biopsy (long ischemic time, short time in fixative, different fixative) or the test is suspected by the pathologist to be negative on the basis of testing error

Do not retest if the following histopathologic findings occur and the initial HER2 test was negative:

Histologic grade 1 carcinoma of the following types:

• Infiltrating ductal or lobular carcinoma, ER and PgR positive
• Tubular (at least 90% pure)
• Mucinous (at least 90% pure)
• Cribriform (at least 90% pure)
• Adenoid cystic carcinoma (90% pure) and often triple negative

Sources: Adapted from Table 2 from Wolff AC et all. Recommendations for Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer: American Society of Clinical Oncology/College of American Pathologists Clinical Practice Guideline Update. Published in J Clin Oncol.

According to the modified algorithm, a tumor should be diagnosed as HER2-negative when IHC shows protein expression of 1+ in >10% (previously >30%) or 0 in ≤10% of cells, or when dual-probe ISH reveals a HER2 gene/CEP17 ratio of <2.0 (previously ≥2.2) with an average HER2 gene copy number of <4.0. Single-probe ISH tests are negative if the average HER2 copy number is <4.0.

IHC 3+ denotes a positive test if membrane staining is complete and intense in >10% cells that demonstrate circumferential membrane staining (a chicken wire-like pattern). For dual-probe ISH tests, a HER2/CEP17 ratio of ≥2.0 and an average HER2 gene copy number of ≥4.0 are positive. Additionally, if the HER2/CEP17 ratio is <2.0 and the average HER2 copy number is ≥6.0, the test is positive. In rare situations, a HER2/CEP17 ratio ≥2.0 and an average HER2 copy number of <4.0 are noted as being positive in the updated guideline. A single-probe ISH test is considered HER2-positive if the HER2 gene copy number is ≥6.0 signals per cell.

If results are equivocal, reflex testing should be performed using an alternative assay (IHC or ISH). In IHC testing, equivocal results are defined as HER2 protein expression of 2+ with incomplete or weak/moderate staining. Additionally, if the tumor protein expression is 2+ with complete or intense staining but only within ≤10% of invasive tumor cells, the test is equivocal. Dual-probe ISH results are equivocal if they show a HER2 gene/CEP17 ratio of <2.0 with an average gene copy number ≥4.0 and <6.0. By single probe, the results are equivocal if the average HER2 copy number is ≥4.0 and <6.0.

M. Elizabeth Hammond, MD

“The number of patients with equivocal HER2 test results used to be rather large. But evidence suggests that the quality of HER2 testing is improving and the frequency of equivocal and inaccurate results is decreasing. We believe that this is at least in part due to our earlier recommendations in 2007," according to M. Elizabeth H. Hammond, MD, co-chair of the ASCO/CAP HER2 Testing in Breast Cancer Panel and professor of Pathology at the University of Utah School of Medicine.

The original guideline focused on IHC and fluorescence ISH (FISH), whereas the updated guideline adds recommendations for a newer diagnostic technique known as bright-field ISH. This technique also evaluates for amplification of the HER2 gene, and uses a regular light microscope rather than a fluorescent microscope. Some sources of variability may be reduced with this technique as the invasive component can be more easily identified using bright-field microscopy.

To ensure the most accurate testing results, the updated guideline stresses the importance of fixing specimens used for HER2 testing within the first hour. The time frame for a sample to undergo processing was lengthened from 6 to 48 hours to 6 to 72 hours in the update.

"We hope the current guideline will resolve remaining challenges in the field, and ultimately result in better outcomes for all patients with breast cancer,” Hammond said in a statement.

The adjusted guideline continues to stress the importance of using CAP-accredited laboratories and emphasizes the need for ongoing competency assessments, as required under the Clinical Laboratory Improvement Amendments. Additionally, the joint Update Committee expressed concerns over the accuracy and utility of FDA-approved companion diagnostic tests and laboratory-developed tests and called for more clarity on the regulatory framework for these tests.

In addition to clinical guidelines, the updated recommendations provide key points for clinicians to discuss with patients regarding HER2 status. These include the importance of the marker and the types of therapies that are available, types of test and the interpretations of results, and the possible need to retest in new metastatic tumors.

2013 Recommended HER2 Assay Interpretation

HER2-Positive

IHC assay

3+ based on complete/intense circumferential membrane staining within >10% of invasive tumor cells*

ISH assay

Single probe: Average HER2 copy number ≥6.0†

Dual Probe: HER2/CEP17 ratio ≥2.0; average HER2 copy number ≥4.0†

Dual Probe: HER2/CEP17 ratio ≥2.0; average HER2 copy number <4.0†

Dual Probe: HER2/CEP17 ratio <2.0; average HER2 copy number ≥6.0†

HER2-Equivocal

Order reflex test on same specimen using alternative test or a new test (IHC or ISH) on a new specimen, if available

IHC assay

2+ based on incomplete or weak/moderate circumferential membrane staining and within >10% of invasive tumor cells*

2+ based on complete/intense circumferential membrane staining within ≤10% of invasive tumor cells*

ISH assay

Single probe: Average HER2 copy number ≥4.0 and <6.0†

Dual Probe: HER2/CEP17 ratio <2.0; average HER2 copy number ≥4.0 and <6.0†

HER2-Negative

IHC assay

1+ based on incomplete, faint/barely perceptible membrane staining and within >10% of invasive tumor cells*

0 based on no observed staining or incomplete, faint/barely perceptible membrane staining and within ≤10% of invasive tumor cells

ISH assay

Single probe: Average HER2 copy number <4.0 signals/cell

Dual Probe: HER2/CEP17 ratio <2.0; average HER2 copy number <4.0 signals/cell

Indeterminate

Technical issues prevent one or both tests from being reported; another specimen should be requested for testing

*Readily appreciated using a low-power objective and oberserved in a homogeneous and contiguous population

†Signals per cell oberserved in a homogeneous and contiguous population

Sources: Adapted from Table 1 from Wolff AC et all. Recommendations for Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer: American Society of Clinical Oncology/College of American Pathologists Clinical Practice Guideline Update. Published in J Clin Oncol.

Wolff AC, Hammond EH, Hicks DG, et al. Recommendations for Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer: American Society of Clinical Oncology/College of American Pathologists Clinical Practice Guideline Update. J Clin Oncol. Published online ahead of print October 7, 2013.