Updated (2018) ASCO/CAP Guidelines for HER2 Testing in Breast Cancer
Expires: July 2, 2021
Beiyun Chen, M.D., Ph.D., is a pathologist for the Division of Anatomic Pathology in the Department of Laboratory Medicine and Pathology (DLMP) in Rochester, Minnesota. She holds the academic rank of Associate Professor of Laboratory Medicine and Pathology.
Katherine Geiersbach, M.D., is a pathologist for the Division of Laboratory Genetics and Genomics in the Department of Laboratory Medicine and Pathology (DLMP) in Rochester, Minnesota. She holds the academic rank of Assistant Professor of Laboratory Medicine and Pathology.
Contact us: MMLHotTopics@mayo.edu.
Transcript and References
Hi, I’m Bobbi Pritt, Director of the Clinical Parasitology Lab and Vice Chair of Education in the Department of Laboratory Medicine and Pathology at Mayo Clinic. Did you know that 1 in 8 women in the United States will be diagnosed with breast cancer in her lifetime? In this month’s “Hot Topic,” my colleagues, Dr. Katherine Geiersbach and Dr. Beiyun Chen, will provide information about the 2018 Focused Update of the ASCO/CAP guidelines for HER2 testing in breast cancer. These updates include a refinement of the definition of equivocal, or “2+” HER2 immunohistochemistry results, a revision of the 2013 recommendations regarding the requirement for repeat HER2 testing, and new recommendations for follow-up immunohistochemistry testing on certain unusual categories of FISH results. I hope you enjoy this month’s Hot Topic, and I want to personally thank you for allowing Mayo Clinic the opportunity to be a partner in your patients’ health care.
Thank you for that introduction. Today we are going to talk about recent changes to the ASCO/CAP guidelines for HER2 testing in breast cancer.
We have no disclosures.
At the end of this presentation, the audience should be familiar with the following:
First, you should know the five Clinical Questions that form the core of the 2018 Focused Update of the HER2 Testing Guidelines for breast cancer, and you should be familiar with the ASCO/CAP response to these five Clinical Questions.
Second, you will be able to describe five categories of in situ hybridization, or “ISH,” results as defined by ASCO/CAP in the 2018 Focused Update.
Third, you will understand the appropriate work-up for three categories of results that represent less common patterns encountered in ISH testing for HER2 status in breast cancer.
HER2 Gene Amplification, HER2 Protein Overexpression, and Trastuzumab Binding to HER2
HER2, or ERBB2, is a gene that is amplified and its protein is over-expressed in approximately 15% to 20% of breast cancers. Cancers that over-express HER2 can be treated with monoclonal antibodies that target the HER2 protein on the surface of breast cancer cells. Trastuzumab, also known by the trade name, Herceptin, was the first drug approved by the U.S. Food and Drug Administration (FDA) in 1998 for treating HER2-positive metastatic breast cancer. Since that time, the treatment of HER2-positive breast cancer has evolved, with more treatment occurring in the neoadjuvant setting before the primary tumor is resected, and multiple other HER2-targeted therapies have also been introduced.
Laboratory testing for HER2 status in breast cancer in clinical laboratories in the United States is performed according to guidelines developed by an expert panel formed of members of the American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP). The ASCO/CAP guidelines were first published in 2007 and were updated in 2013. There are currently two approved methods for determining HER2 status in breast cancer: immunohistochemistry (IHC) and in situ hybridization (ISH). Several FDA-approved IHC and ISH assays are commercially available.
In November 2016, the ASCO/CAP Expert Panel convened to refine some controversial criteria in the 2007 and 2013 guidelines and to discuss standardizing the testing algorithm for several unusual categories of HER2 ISH results. The panel discussion focused on five clinical questions. My colleague, Beiyun Chen, M.D., Ph.D., will discuss the panel response to the first two questions, and I (Katherine Geiersbach, M.D.) will discuss the panel response to clinical questions 3 through 5.
IHC 0, 1+, 2+, and 3+ Results in Breast Cancer
Clinical Question 1: What is the most appropriate definition for IHC 2+ (IHC Equivocal)?
Answer: In the 2013 guidelines, the Expert Panel defined 2+ staining as “circumferential membrane staining that is incomplete and/or weak/moderate and within greater than 10% of tumor cells.” Following up on correspondence published in the Journal of Clinical Oncology in April 2015,1,2 the panel has refined the definition of 2+ staining to read as follows: “Weak to moderate complete membrane staining observed in greater than 10% of tumor cells.” This change to the definition of 2+ equivocal staining was primarily made because the descriptors “incomplete” and “circumferential” used in the 2013 definition were thought to be contradictory. The panel also added some unusual scenarios that should be considered 2+ equivocal, such as basolateral staining for HER2 in a rare subtype of breast cancer with micropapillary histology and circumferential staining that is intense but in less than 10% of the tumor cells. A breast cancer sample with 2+ equivocal results by IHC should have follow-up testing by ISH, if this method has not already been performed.
Histophatologic Features Suggestive of Possible HER2 Test Discordance
Clinical question 2: Must HER2 testing be repeated on a surgical specimen if initially negative test on core biopsy?
Answer: This question was also addressed in the correspondence published in the Journal of Clinical Oncology in April 2015.1,2 In the response to Rakha, et al., the Expert Panel cited the high concordance in HER2 testing between core and excisional biopsies.2 The 2018 Focused Update revised one of the recommendations provided in Table 2 of the 2013 guidelines (indicated with an arrow on the slide). According to the 2018 updated guidelines, repeat HER2 testing may be performed on the excisional biopsy if the tumor is grade 3, the amount of invasive tumor in the core biopsy is small, the resection contains morphologically distinct high-grade carcinoma, or there are quality concerns related to the HER2 testing performed on the core biopsy. The pathologist and oncologist should exercise their clinical judgment in deciding whether to repeat testing in these situations.
H&E Images of Typical Grade 1 Tubular Carcinoma, Mucinous Carcinoma, Cribriform Carcinoma, and Adenoid Cystic Carcinoma
As addressed in Table 2 of the 2013 guidelines, several histologic subtypes of breast carcinoma are usually HER2 negative: tubular carcinoma, mucinous carcinoma, cribriform carcinoma, and adenoid cystic carcinoma. The 2013 guidelines pertaining to these histologic subtypes are unchanged. When 90% or more of the tumor shows one of these histologic patterns and the tumor is grade 1, HER2 testing does not need to be repeated if the initial HER2 test is negative, but if the initial test is positive, HER2 testing should be repeated due to low frequency of HER2 amplification or overexpression observed in these histologic subtypes.
Graphical Representation of Group 1-5 FISH Patterns
The next three clinical questions addressed by the Expert Panel pertained to unusual patterns encountered in ISH testing. In the 2018 Focused Update, the Expert Panel recommended that dual-probe assays are preferred over single-probe assays. Shown here are five different categories of results that can be seen using a dual-probe assay. The most frequently encountered patterns in most laboratories are Group 1, HER2 positive with a copy number of 4 or greater and a positive ratio, or Group 5, HER2 negative with a copy number less than 4 and a negative ratio. The remaining patterns are less common and are discussed in the following slides. The panel deliberated over the appropriate handling of these uncommon patterns and ultimately determined that additional workup is required for Groups 2, 3, and 4. The overall prevalence of these unusual subgroups among all breast cancers undergoing HER2 testing is estimated to be about 5%, but within an individual laboratory, the frequency of Group 2, 3, or 4 ISH results is largely a function of the case mix. In the Cytogenetics Laboratory at Mayo Clinic, frequency of Group 2, 3, or 4 FISH results is estimated to be between 10% and 20%.
Group 2 FISH Result
Clinical question 3: Should invasive cancers with a HER2/CEP17 ratio greater than or equal to 2.0 but an average HER2 copy number less than 4.0 signals/cell be considered ISH positive?
Answer: Shown in this example is a dual-probe fluorescence in situ hybridization (FISH) slide where the tumor cells have an average of 3.2 red signals per cell indicating the average HER2 copy number and an average of 1.3 green signals per cell for the centromere, indicating copy number loss of chromosome 17. The ratio of HER2 to centromere signals is 2.5, making the ratio “positive.” This result was considered HER2 positive according to the 2013 ASCO/CAP guidelines. Described in the literature as “monosomy” using various thresholds, copy number loss affecting the centromere control probe labeled here in green can inflate the HER2/centromere ratio, leading to a “HER2 amplified,” or positive, result despite a low absolute HER2 copy number.. In the 2018 Focused Update, the Expert Panel has formalized the definition of this category as “Group 2” and has recommended additional workup by immunohistochemistry in the same institution that performed the ISH testing. If the immunohistochemistry result is 2+ equivocal, then the ISH slide must be reanalyzed in parallel with the IHC slide, with an additional observer scoring at least 20 cells in the areas with equivocal staining.
Group 3 FISH Result
Clinical question 4: Should invasive cancers with an average HER2 copy number greater than or equal to 6.0 signals/cell but a HER2/CEP17 ratio less than 2.0 be considered ISH positive?
Answer: This example shows a dual-probe FISH slide where the tumor cells have an average of 6.5 red signals per cell for HER2 and an average of 5.0 green signals per cell for the centromere. The ratio of HER2 to centromere signals is 1.3, making the ratio “negative.” Although the copy number for HER2 is fairly high, such cases may not show HER2 overexpression by IHC, as demonstrated in a retrospective study of clinical trial data published in 2016 in the Journal of Clinical Oncology.3 Therefore, this pattern, termed “Group 3,” may not be consistent with HER2-positive breast cancer despite the relatively high HER2 copy number. The Expert Panel has recommended additional workup using IHC, with IHC-guided rescoring of the FISH slide if the IHC remains 2+ equivocal.
Group 4 FISH Result
Clinical question 5: What is the appropriate diagnostic workup for invasive cancers with an average HER2 copy number greater than or equal to 4.0 but less than 6.0 signals/cell and a HER2/CEP17 ratio less than 2.0 and initially deemed to have an equivocal HER2 ISH test result?
Answer: This example shows a dual-probe FISH slide where the tumor cells have an average of 5.2 red signals per cell for HER2 and an average of 4.3 green signals per cell for the centromere. The ratio of HER2 to centromere signals is 1.2, making the ratio “negative.” A HER2 copy number between 4 and 6 was defined as equivocal for single-probe ISH assays in the 2007 guidelines, and in 2013, the panel opted to incorporate dual-probe ISH assays into this definition as well, when the HER2 copy number was between 4 and 6 but the ratio was negative. A dual-probe result in this equivocal range has been categorized as “Group 4.” In the 2013 guidelines, the panel recommended that follow-up testing occur for these cases due to the possibility of co-amplification of HER2 and the centromere, with the suggestion that labs performing ISH could re-test the same sample using an alternative control probe targeting another gene region on chromosome 17. However, this approach was never clinically validated or standardized. The ASCO/CAP Expert Panel deliberated over the appropriate workup for Group 4 results. The panel recommended against use of alternative control probes to resolve HER2 status by ISH following an equivocal result. As for “Group 2” and “Group 3” ISH results, “Group 4” results should be followed by IHC testing in the same institution performing the ISH testing, in an attempt to resolve HER2 status. If the IHC is not clearly negative or positive for HER2 over-expression but remains 2+ equivocal, then the ISH slide must be reanalyzed in parallel with the IHC slide, with an additional observer scoring at least 20 cells in the areas with equivocal staining.
Algorithm Following IHC-guided ISH Re-scoring
The final ISH result for Groups 2, 3, or 4 is therefore determined by integrated review of the IHC and ISH results. If rescoring of the ISH slide is required, an independent observer must rescore the ISH slide within areas of invasive tumor that demonstrate equivocal (2+) staining. The final interpretation is determined by the final ISH results as shown in this diagram.
To summarize, we have described the ASCO/CAP response to five clinical questions in the 2018 Focused Update of the HER2 Testing Guidelines for breast cancer. We described five categories of in situ hybridization (ISH) results defined in the 2018 Focused Update and the additional workup required for three of these categories, known as “Group 2,” “Group 3,” and “Group 4.”
- Rakha EA, Pigera M, Shaaban A, et al. National guidelines and level of evidence: comments on some of the new recommendations in the American Society of Clinical Oncology and the College of American Pathologists human epidermal growth factor receptor 2 guidelines for breast cancer. J Clin Oncol. 2015;33:1301-2.
- Wolff AC, Hammond ME, Hicks DG, et al. Reply to E.A. Rakha et al. J Clin Oncol. 2015;33:1302-4.
- Press MF, Sauter G, Buyse M, et al. HER2 gene amplification testing by fluorescent in situ hybridization (FISH): comparison of the ASCO-College of American Pathologists guidelines with FISH scores used for enrollment in breast cancer international research group clinical trials. J Clin Oncol. 2016;34(29):3518-3528.
- Wolff A, Hammond EH, Allison K, et al: Human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline focused update. Arch Pathol Lab Med epub 2018. doi: 10.5858/arpa.2018-0902-SA.