Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer: American Society of Clinical Oncology/College of American Pathologists Clinical Practice Guideline Focused Update

This 2018 Focused Update addresses five clinical questions raised after the publication of the 2013 Guideline Update:

What is the most appropriate definition for IHC 2+ (IHC equivocal)?

Must HER2 testing be repeated on a surgical specimen if initially negative test on core biopsy?

Should invasive cancers with an HER2/chromosome enumeration probe 17 (CEP17) ratio of ≥2.0 but an average HER2 copy number of <4.0 signals per cell be considered ISH positive?

Should invasive cancers with an average HER2 copy number of ≥6.0 signals per cell but a HER2/CEP17 ratio of <2.0 be considered ISH positive?

What is the appropriate diagnostic workup for invasive cancers with an average HER2 copy number of ≥4.0 but <6.0 signals per cell and an HER2/CEP17 ratio of <2.0, and initially deemed to have an equivocal HER2 ISH test result?

Clinical Questions 1 and 2 were formally addressed in correspondence from the ASCO/CAP HER2 testing Guideline Expert Panel published in JCO¹⁴ in 2015 in response to correspondence by Rakha et al,¹⁷  and this Focused Update contains a revised Figure 1 (Clinical Question 1) and a revised Table 2 (Clinical Question 2).

Clinical Questions 3, 4, and 5 regarding dual-probe (dual-signal) ISH testing (Figures 3 to 6) were addressed by the Expert Panel in a meeting at ASCO headquarters on November 28 and 29, 2016, and in subsequent conference calls and electronic communications. Figure 2 (single-probe ISH) from the 2013 Guideline Update includes a new footnote with a recommendation that concomitant IHC review become part of the interpretation of single-probe ISH results. Table 1 contains a summary of the recommendations of the 2013 Guideline Update and the 2018 Focused Update. Figure 7, describing the number of laboratories participating in predictive marker proficiency testing for HER2, has been updated.

This systematic review-based guideline product was developed by a multidisciplinary Expert Panel, which included a patient representative and ASCO guidelines staff with health research methodology expertise. PubMed and the Cochrane Library were searched for randomized controlled trials, systematic reviews, meta-analyses, and clinical practice guidelines for the period from January 1, 2013, through May 11, 2017. The disease and intervention search terms were those that were used for the 2013 Guideline Update. The updated search was guided by the signals¹⁸  approach that is designed to identify only new, potentially practice-changing data (signals) that might translate into revised practice recommendations. The approach relies on targeted routine literature searching and the expertise of ASCO Expert Panel members to help identify potential signals. Additional information about the literature search strategy string and results, as well as a discussion of the ASCO signals approach to guideline updating, are available in the 2018 Data Supplement and 2018 Methodology Supplement, respectively (see supplemental digital content at www.archivesofpathology.org in the November 2018 table of contents). A QUOROM diagram of the updated search and the clinical questions are provided (Data Supplement). In addition to the literature search, a research survey was distributed to gather additional real-world data from laboratories from before and after implementation of the ASCO/CAP HER2 Testing in Breast Cancer 2013 Update. Additional information regarding this survey process is available in the Data Supplement.

The Expert Panel met during a 2-day in-person meeting in November 2016 to consider the evidence for each of the recommendations contained in this 2018 Focused Update. Laboratories that shared with the Expert Panel their clinical experience with HER2 testing since the publication of the 2013 Guideline Update participated in the open session of the meeting. The guideline was circulated in draft form to the Expert Panel. Draft recommendations were released to the public for an open comment period between May 22 and June 19, 2017. ASCO's Clinical Practice Guidelines Committee reviewed and approved the final document. For CAP, an independent review panel was assembled to review and approve the guideline. The independent review panel was masked to the Expert Panel and was vetted through the conflict of interest process.

Only recommendations relating to the updated clinical questions have changed. The Data Supplement provides clinical questions corresponding to all recommendations from the 2013 Guideline Update.

This ASCO/CAP Clinical Practice Guideline Focused Update provides select recommendations with a comprehensive discussion of the relevant literature from January 1, 2013, to May 11, 2017, for these specific recommendations. The full guideline, which this revision applies to, and additional information are available at www.asco.org/breast-cancer-guidelines. The complete list of recommendations, including the updated recommendations, is in Table 1. All funding for the administration of the project was provided by ASCO and CAP.

The clinical practice guidelines and other guidance published herein are provided by the American Society of Clinical Oncology Inc (“ASCO”) to assist providers in clinical decision-making. The information therein should not be relied upon as being complete or accurate, nor should it be considered as inclusive of all proper treatments or methods of care or as a statement of the standard of care. With the rapid development of scientific knowledge, new evidence may emerge between the time information is developed and when it is published or read. The information is not continually updated and may not reflect the most recent evidence. The information addresses only the topics specifically identified therein and is not applicable to other interventions, diseases, or stages of diseases. This information does not mandate any particular course of medical care. Further, the information is not intended to substitute for the independent professional judgment of the treating provider, as the information does not account for individual variation among patients. Recommendations reflect high, moderate, or low confidence that the recommendation reflects the net effect of a given course of action. The use of words like “must,” “must not,” “should,” and “should not” indicates that a course of action is recommended or not recommended for either most or many patients, but there is latitude for the treating physician to select other courses of action in individual cases. In all cases, the selected course of action should be considered by the treating provider in the context of treating the individual patient. Use of the information is voluntary. ASCO provides this information on an “as is” basis and makes no warranty, express or implied, regarding the information. ASCO specifically disclaims any warranties of merchantability or fitness for a particular use or purpose. ASCO assumes no responsibility for any injury or damage to persons or property arising out of or related to any use of this information or for any errors or omissions.

The Expert Panel was assembled in accordance with ASCO's Conflict of Interest Policy Implementation for Clinical Practice Guidelines (“Policy,” found at www.asco.org/rwc) as agreed upon with CAP. All members of the Expert Panel completed ASCO's disclosure form, which requires disclosure of financial and other interests, including relationships with commercial entities that are reasonably likely to experience direct regulatory or commercial impact as a result of promulgation of the guideline. Categories for disclosure include employment; leadership; stock or other ownership; honoraria, consulting or advisory role; speaker's bureau; research funding; patents, royalties, other intellectual property; expert testimony; travel, accommodations, expenses; and other relationships. In accordance with the Policy, the majority of the members of the Expert Panel did not disclose any relationships constituting a conflict under the Policy.

All recommendations regarding each of the five clinical questions are predicated on the assumption that the cases have been properly fixed, processed, and tested in a laboratory that follows ASCO/CAP HER2 testing guideline recommendations, especially those related to IHC and ISH interpretation and reporting.

In the 2013 Guideline Update, the workup of cases in the less common dual-probe ISH categories (groups 2 to 4) addressed in Clinical Questions 3, 4, and 5 (Figure 3) included only ISH. In this 2018 Focused Update, we recommend that these cases be worked up by considering both the IHC and the dual-probe ISH results together. Many publications since the 2013 Guideline Update have referenced the value of adjudicating ISH results in these uncommon categories using IHC.^{12,16,19–21}  These tests should be performed on the same tissue sample using sections from the same block. Ideally, adjacent tissue levels from the same block should be tested and then reviewed together. If IHC has already been performed, it should be used to guide the selection of the areas to be counted during ISH such that areas with the strongest protein expression can be included in ISH scoring. This is common practice among laboratories performing both testing procedures. If the ISH laboratory only performs ISH, it is recommended that an adjacent section in the same block be assessed at a companion IHC laboratory, and then the slides from both ISH and IHC be reviewed together to guide the selection of areas to score by ISH. Local practice considerations will dictate the best procedure to accomplish this concomitant review.

What is the most appropriate definition for IHC 2+ (IHC equivocal)?

IHC 2+ (equivocal) was defined in Figure 1 of the 2013 HER2 Testing Update as invasive breast cancer showing “circumferential membrane staining that is incomplete and/or weak/moderate and within > 10% of tumor cells or complete and circumferential membrane staining that is intense and within ≤ 10% of tumor cells.”

In the revised Figure 1, the revised definition of IHC 2+ (equivocal) is invasive breast cancer with “weak to moderate complete membrane staining observed in > 10% of tumor cells” (type: evidence based; evidence quality: high; strength of recommendation: strong).

IHC 2+ (equivocal) had been defined in the 2013 Guideline Update (2013 figure 1: Algorithm for evaluation of HER2 protein expression by IHC assay of the invasive component of a breast cancer specimen) as invasive breast cancer showing “circumferential membrane staining that is incomplete and/or weak/moderate and within > 10% of tumor cells or complete and circumferential membrane staining that is intense and within ≤ 10% of tumor cells.” However, many pathologists expressed concern that the terms “circumferential” and “incomplete” were confusing, could not be reconciled when used together in the IHC interpretation of HER2 expression, and could lead to many IHC 1+ (HER2-negative) tumors being called IHC 2+ (HER2 equivocal) and submitted for reflex testing.

In the same figure 1 of the 2013 Guideline Update, the statement “complete and circumferential membrane staining that is intense and within ≤10% of tumor cells” referred to an unusual pattern that did not need to be specified in the main portion of the figure. This information has now been moved to the footnote of Figure 1, which will now read as follows:

Consequently, the revised definition of IHC 2+ (HER2 equivocal) in this 2018 Focused Update (Figure 1) reflects a commonly accepted definition of invasive breast cancer that now reads “weak to moderate complete membrane staining observed in > 10% of tumor cells.”²³  During the open comment period, pathologists requested guidance about the uncommon scenario of cases in which “intense circumferential membrane staining is observed in ≤ 10% of tumor cells.” As described in the footnote in Figure 1, such cases may be considered IHC 2+ equivocal, although additional samples may reveal different percentages of HER2-positive staining. These revisions were previously communicated in a 2015 correspondence from the ASCO/CAP HER2 testing Expert Panel published in JCO.¹⁴ 

Must HER2 testing be repeated on a surgical specimen if initially negative test on core biopsy?

In Table 2 in the 2013 HER2 Testing Update, it was stated that, on the basis of some criteria (including a tumor grade 3), “If the initial HER2 test result in a core needle biopsy specimen of a primary breast cancer is negative, a new HER2 test must be ordered on the excision specimen ….”

In the revised Table 2, it is now stated that, on the basis of some criteria (including a tumor grade 3), “If the initial HER2 test result in a core needle biopsy specimen of a primary breast cancer is negative, a new HER2 test may be ordered on the excision specimen …” (type: evidence based; evidence quality: high; strength of recommendation: strong).

The auxiliary verb “must” was used in the 2013 Guideline Update (Table 2)¹  to indicate that, on the basis of some criteria (including a tumor grade 3), “If the initial HER2 test result in a core needle biopsy specimen of a primary breast cancer is negative, a new HER2 test must be ordered on the excision specimen ….”

The panel had previously indicated in the Data Supplement of the 2013 Guideline Update that:

Several data sets originally referenced in 2013 showed excellent concordance for HER2 testing in paired samples (core biopsy specimen and excision) using IHC as the initial test, such as a 98.8% concordance observed among 336 patients in the Royal Marsden experience.²⁴ 

Rakha et al,¹⁷  in their 2015 correspondence to JCO, described their own institutional experience and that of other groups, including many previously described in the 2013 Guideline Update. In its 2015 response¹⁴  to the correspondence by Rakha et al,¹⁷  the panel agreed that, in view of the greater clinical experience that confirmed the high concordance in HER2 testing between core and excisional biopsies, it was appropriate to allow the pathologist and oncologist to exercise clinical judgment and that grade 3 alone did not suffice as a criterion for mandatory retesting.

Therefore, in the revised Table 2, the auxiliary verb “must” has been replaced by “may” to indicate that, “If the initial HER2 test result in a core needle biopsy specimen of a primary breast cancer is negative, a new HER2 test may be ordered on the excision specimen ….” These changes are the same as those previously communicated in a 2015 correspondence by the authors of the 2013 Guideline Update and published in JCO.¹⁴ 

Should invasive cancers with an HER2/CEP17 ratio of ≥2.0 but an average HER2 copy number of <4.0 signals per cell be considered ISH positive?

Cases in which the HER2/CEP17 ratio is ≥2.0 with an average HER2 signals per cell of <4.0 were considered ISH positive.

If a case has an HER2/CEP17 ratio of ≥2.0 but the average HER2 signals per cell is <4.0, a definitive diagnosis will be rendered based on additional workup.

If not already assessed by the institution or laboratory performing the ISH test, IHC testing for HER2 should be performed using sections from the same tissue sample used for ISH, and the slides from both ISH and IHC should be reviewed together to guide the selection of areas to score by ISH (local practice considerations will dictate the best procedure to accomplish this concomitant review):

• •

  If the IHC result is 3+, diagnosis is HER2 positive.

• •

  If the IHC result is 2+, recount ISH by having an additional observer, blinded to previous ISH results, count at least 20 cells that include the area of invasive cancer with IHC 2+ staining:

• • o

    If reviewing the count by the additional observer changes the result into another ISH category, the result should be adjudicated per internal procedures to define the final category.

  • o

    If the count remains an average of <4.0 HER2 signals per cell and the HER2/CEP17 ratio is ≥2.0, diagnosis is HER2 negative with a comment.

• •

  If the IHC result is 0 or 1+, diagnosis is HER2 negative with a comment.

The Expert Panel recommends the following comment: evidence is limited on the efficacy of HER2-targeted therapy in the small subset of cases with an HER2/CEP17 ratio of ≥2.0 and an average HER2 copy number of <4.0 per cell. In the first generation of adjuvant trastuzumab trials, patients in this subgroup who were randomly assigned to the trastuzumab arm did not seem to derive an improvement in disease-free or overall survival, but there were too few such cases to draw definitive conclusions. IHC expression for HER2 should be used to complement ISH and define HER2 status. If the IHC result is not 3+ positive, it is recommended that the specimen be considered HER2 negative because of the low HER2 copy number by ISH and the lack of protein overexpression (Type: evidence based; evidence quality: intermediate; strength of recommendation: strong). An algorithm for Clinical Question 3 is presented in Figures 3 and 4.

Members of the HER2 testing Expert Panel in 2013 had expressed concern about describing an invasive breast cancer as HER2 positive on the basis of a single HER2 ISH test that showed an HER2/CEP17 ratio of ≥2.0 but an average HER2 copy number of <4.0 signals per cell (Figure 3, group 2) and recommended additional testing of such cases. Members of the 2013 Guideline Update Panel also expressed their view that using the HER2/CEP17 ratio alone could be misleading in cases with CEP17 gains or losses and could lead to an underestimation or overestimation of HER2 amplification. However, the eligibility criteria for the first adjuvant trials of trastuzumab generally followed US Food and Drug Administration criteria (IHC 3+ or ISH ratio ≥2.0 regardless of average HER2 copy number based on HER2 signals per cell), and the panel in 2013 ultimately opted to consider these rare group 2 patients as having HER2-positive disease.

Since then, investigators have further reported on the outcome of patients with a group 2 dual-probe ISH test result. Greater experience and a more refined collection of test results in the past few years confirmed that such cases are infrequent (Table 3) and represented a small number of patients enrolled in the initial adjuvant trastuzumab trials. Among 4340 patients (41.5% of 10 468) screened by dual-probe fluorescent ISH (FISH) for trials Breast Cancer International Research Group (BCIRG) 005 (HER2-negative trial) and BCIRG-006 (HER2-positive trial) and found to have a dual-probe ISH ratio of ≥2.0, only 71 (0.7% of 10 468) had an average number of HER2 signals per cell of <4.0.¹⁰  Furthermore, in 35 of these 71 patients who were also tested later by IHC, only 3 were IHC 2+ and none were IHC 3+. A retrospective assessment of potential benefit from trastuzumab in group 2 patients produced an observed hazard ratio estimate of slightly >1.0 (favoring no trastuzumab benefit), but the sample size was insufficient to statistically rule out a benefit from adjuvant trastuzumab in this group; nor could it be established statistically whether group 2 patients not treated with trastuzumab had outcomes different from patients with HER2-negative disease treated with just chemotherapy.¹⁰  In the HERA trial, group 2 patients were also uncommon (0.8%) among all patients centrally screened for eligibility (Table 3), whereas group 1 patients (ISH ratio ≥2.0 and average HER2 signals per cell of ≥4.0) represented 55% of all tested cases (48.7% ≥6.0 and 6.3% ≥4.0 and <6.0).¹⁵  In summary, the panel concluded that these group 2 cases should no longer be considered HER2 positive unless IHC 3+ overexpressed.

Repeat testing of other tissue samples from the patient may also be appropriate in this setting, and in particularly challenging cases or if the results are in question, expert consultation may be appropriate.

Should invasive cancers with an average HER2 copy number of ≥6.0 signals per cell but an HER2/CEP17 ratio of <2.0 be considered ISH positive?

Cases in which the HER2/CEP17 ratio is <2.0 with an average of ≥6.0 HER2 signals per cell were considered ISH positive.

If a case has an average of ≥6.0 HER2 signals per cell with an HER2/CEP17 ratio of <2.0, formerly diagnosed as ISH positive for HER2, a definitive diagnosis will be rendered based on additional workup.

If not already assessed by the institution or laboratory performing the ISH test, IHC testing for HER2 should be performed using sections from the same tissue sample used for ISH, and the slides from both ISH and IHC should be reviewed together to guide the selection of areas to score by ISH (local practice considerations will dictate the best procedure to accomplish this concomitant review):

• •

  If the IHC result is 3+, diagnosis is HER2 positive.

• •

  If the IHC result is 2+, recount ISH by having an additional observer, blinded to previous ISH results, count at least 20 cells that include the area of invasion with IHC 2+ staining:

• • o

    If reviewing the count by the additional observer changes the result into another ISH category, the result should be adjudicated per internal procedures to define the final category.

  • o

    If the HER2/CEP17 ratio remains <2.0 with ≥6.0 HER2 signals per cell, diagnosis is HER2 positive.

• •

  If the IHC result is 0 or 1+, diagnosis is HER2 negative with a comment.

The Expert Panel recommends the following comment: there are insufficient data on the efficacy of HER2-targeted therapy in cases with an HER2 ratio of <2.0 in the absence of protein overexpression because such patients were not eligible for the first generation of adjuvant trastuzumab clinical trials. When concurrent IHC results are negative (0 or 1+), it is recommended that the specimen be considered HER2 negative (type: evidence based; evidence quality: intermediate; strength of recommendation: strong). An algorithm for Clinical Question 4 is presented in Figures 3 and 5.

Based on available data, samples with ISH results in this category (ratio <2.0 and mean HER2 signals per cell ≥6.0) are uncommon, only representing between 0.4% and 3.0% of cases sent for dual-probe FISH testing (Table 3). These ISH cases have increases in both HER2 and control centromere signals, resulting in ratio results of <2.0. At the time of the pivotal HER2 trials, cases with these results were considered to have duplication of CEP17 (polysomy) and were most often excluded because they were considered negative for HER2 gene amplification, although the BCIRG-006 (Clinicaltrials.gov identifier: NCT00021255) allowed patients to be enrolled if the HER2 copy number was ≥10 and central IHC testing was 3+. Subsequent studies examining multiple regions of chromosome 17 supported that the majority of cases with these results have HER2 amplifications that include regions encompassing the centromere rather than true polysomy for the entire chromosome 17 (coamplification of control and HER2 signals).^25–31  Based on these data, the 2013 Guideline Update clarified that cases with an average HER2 copy number of ≥6.0 HER2 signals per cell ISH results (by either single- or dual-probe assays) should be reported as HER2 positive by gene amplification. However, it was acknowledged that data on the clinical response of this group to HER2-targeted therapies were limited.

Since the 2013 update, additional data have been published including concurrent IHC results for this ISH category, and they show that this group can be heterogeneous. Data from a reanalysis of the HERA trial identified a small number of cases (21 total) originally considered negative due to ratios of <2.0 but with an average of ≥6.0 HER2 signals per cell.¹⁵  All of these cases had >3 mean CEP17 signals per cell, and 75% of them (15 of 20) had HER2 overexpression by IHC.

In a combined study of three major academic medical centers performing HER2 FISH and IHC, similar results were seen with 63 cases in this ISH category; 31.7% were IHC 3+ for HER2 by IHC, 55% were IHC 2+, and 13.7% were IHC 0 or 1+.¹⁶  This study also reported a higher frequency of Nottingham grade 3 cancers with these ISH results than with other ISH result categories. Published data from a reference laboratory at the University of Southern California described 48 cases with the same ISH characteristics and found that only 8.3% were IHC 3+, while 14.6% were IHC 2+ and 77% were IHC 0 or 1+.¹²  Additional analysis of these cases identified a highly amplified subgroup (eight total cases) with an average of 12.3 HER2 signals per cell that correlated well with HER2 IHC 2+ or 3+ (75%). This subgroup differed significantly from the other subgroup (40 total cases) that had a lower average of 6.8 HER2 signals per cell and 87.5% IHC negative (0 or 1+) results. Similarly, in the BCIRG central testing clinical trial data, of the limited cases (nine total) with IHC data and ISH results in this category, one was IHC 3+ positive, one was IHC 2 +, and seven were IHC negative.¹⁰  Taken together, these results suggest that cases in this ISH category form a heterogeneous group that is best discriminated by the combination of IHC and ISH.

Due to the rarity of cases with these ISH results, there is still limited clinical evidence regarding benefit from HER2-targeted therapy. The BCIRG-005 data (no HER2-targeted treatment) indicated a worse disease-free and overall survival for this ISH category than for cases with both an HER2/CEP17 ratio of <2.0 and <4.0 signals per cell (ISH nonamplified).¹⁰  However, the few cases enrolled in the BCIRG-006 adjuvant trastuzumab trial with these ISH results were insufficient to assess whether there was benefit from HER2-targeted therapy, and statistical analysis was not attempted.

Overall, the absence of robust clinical data to guide decisions, and the variability in IHC data, support the concept that protein expression results should be used concurrently in this setting to aid in determining the significance of ISH results. In summary, group 3 cases are uncommon and heterogeneous. Based on available data, the ratio may not be a reliable indicator of the true gene amplification status.^25–31  Given the evidence that some group 3 cases have true HER2 amplification rather than polysomy for chromosome 17, particularly when the HER2 copy number is high, the Expert Panel ultimately favored continuing to classify these cases as HER2 positive unless the concurrent IHC result is clearly negative (0 or 1+).^25–31 

Repeat testing of other tissue samples from the patient may also be appropriate in this setting, and, in particularly challenging cases or if the results are in question, expert consultation may be appropriate and include alternative probes or other genetic methods.¹¹  However, alternative probes should not be used as standard practice in view of the absence of outcome data.

What is the appropriate diagnostic workup for invasive cancers with an average HER2 copy number of ≥4.0 but <6.0 signals per cell and an HER2/CEP17 ratio of <2.0, and initially deemed to have an equivocal HER2 ISH test result?

Cases in which the HER2/CEP17 ratio is <2.0 with an average HER2 copy number of ≥4.0 and <6.0 signals per cell were considered ISH equivocal, and additional workup was required (“Must order a reflex test [same specimen using IHC], test with alternative ISH chromosome 17 probe, or order a new test [new specimen if available, ISH or IHC]”).

If the case has an average HER2 signals per tumor cell of ≥4.0 and <6.0 and the HER2/CEP17 ratio is <2.0, formerly diagnosed as ISH equivocal for HER2, a definitive diagnosis will be rendered based on additional workup.

If not already assessed by the institution or laboratory performing the ISH test, IHC testing for HER2 should be performed using sections from the same tissue sample used for ISH, and the slides from both ISH and IHC should be reviewed together to guide the selection of areas to score by ISH (local practice considerations will dictate the best procedure to accomplish this concomitant review):

• •

  If the IHC result is 3+, diagnosis is HER2 positive.

• •

  If the IHC result is 2+, recount ISH by having an additional observer, blinded to previous ISH results, count at least 20 cells that include the area of invasion with IHC 2+ staining:

• • o

    If reviewing the count by the additional observer changes the result into another ISH category, the result should be adjudicated per internal procedures to define the final category.

  • o

    If the count remains an average of ≥4.0 and <6.0 HER2 signals per cell with an HER2/CEP17 ratio of <2.0, diagnosis is HER2 negative with a comment.

• •

  If the IHC result is 0 or 1+, diagnosis is HER2 negative with a comment.

The Expert Panel recommends the following comment: It is uncertain whether patients with an average of ≥4.0 and <6.0 HER2 signals per cell and an HER2/CEP17 ratio of <2.0 benefit from HER2-targeted therapy in the absence of protein overexpression (IHC 3+). If the specimen test result is close to the ISH ratio threshold for positive, there is a high likelihood that repeat testing will result in different results by chance alone. Therefore, when IHC results are not 3+ positive, it is recommended that the sample be considered HER2 negative without additional testing on the same specimen (Type: evidence based; evidence quality: intermediate; strength of recommendation: strong). An algorithm for Clinical Question 5 is presented in Figures 3 and 6.

Cases with an average of ≥4.0 and <6.0 HER2 signals per cell and an HER2/CEP17 ratio of <2.0 were considered equivocal in the 2013 Guideline Update. This category (group 4 cases) has been reconsidered by the Expert Panel based on published literature since then, and was discussed by the representatives of expert laboratories and Expert Panel members during the open portion of the November 2016 in-person meeting. In many published studies, the incidence of equivocal cases has changed since the 2013 update, when more stringent requirements for ISH interpretation were described.^1,4  The number of such cases within a laboratory varies based on the patient population referred for ISH testing, but it seems to be approximately 5% of cases (range, 1% to 16%).^{15,19–21,32–42}  The use of alternative probes to adjudicate these cases has also increased since 2013.

Data from a central reference laboratory at Mayo Clinic included FISH data in a population of patients that is enriched from those with HER2 IHC 2+ results based on the original IHC testing performed locally by the referring laboratories (1922 patients; 85% IHC 2+). Among these cases tested by FISH at Mayo, 14% of patients had ISH equivocal results and one half became HER2 positive by ratio when a locally developed and analytically validated 17p arm probe (D17S122; Mayo Clinic, Rochester, MN) was combined with the HER2 probe (Abbott Molecular, Abbott Park, IL) for additional FISH testing. However, clinical information about benefit from HER2-targeted therapy in such patients is not available and may not exist because these patients would not have been eligible for the original pivotal trials.¹¹  The reference laboratory experience reported by Press et al¹²  involving a different patient population found 4.6% of patients among 7,526 cases with equivocal results when using the 2013 criteria, while 89% of these cases were IHC HER2 0 or 1+, 10% were IHC HER2 2+, and only 0.9% were IHC HER2 3+ positive. Another academic laboratory experience that combined results from three laboratories had a similar frequency of numbers of specimens with equivocal results (5.2%) among 8068 patients. Similar clinical characteristics were observed in patients with an average of ≥4.0 and <6.0 HER2 signals per cell, regardless of whether the ratio was above or below 2.0, and most of these cases were HER2 negative by IHC and more likely to be estrogen receptor positive.¹⁶ 

Group 4 cases reported as HER2 equivocal since the 2013 Guideline Update have posed a challenge to oncologists and patients due to a perceived ambivalence about whether to recommend HER2-targeted therapy. In the absence of an unequivocally positive or negative test result, multiple testing of the same tissue sample has been performed frequently, and many laboratories have relied exclusively on alternative probe testing to resolve cases that are more difficult. This has often included ISH testing using multiple chromosome 17 probes at once, many not analytically or clinically validated. Such indiscriminate testing often results in four or more ISH ratios being described in a single test report and a final designation of HER2 gene amplified if just a single ratio is ≥2.0. After careful consideration of this practice and available data, the 2018 Expert Panel strongly recommends against this as a routine testing strategy. When the HER2 ratio score is near a decision threshold (positive or negative), based on random variation in scoring, a subsequent test may result in a positive or negative score barely crossing the threshold (on either side). In such cases, repeated ISH testing may therefore not result in higher confidence in the final result.

Clinical correlation with other factors in a particular case (such as grade and special histologic subtypes) or repeat testing of other tissue samples from the patient may also be appropriate in this setting. In particularly challenging cases or if the results are in question, expert consultation may be appropriate and may include alternative probes or other genetic methods.¹¹  However, alternative probes should not be used as standard practice due to limited data on outcomes for this subset of patients.³³ 

Additional information, including data supplements, evidence tables, and clinical tools and resources, can be found at www.asco.org/breast-cancer-guidelines. Patient information is available there and at www.cancer.net.

• Use of Biomarkers to Guide Decisions on Adjuvant Systemic Therapy for Women With Early-Stage Invasive Breast Cancer⁴³  (http://ascopubs.org/doi/10.1200/JCO.2017.74.0472)

• Selection of Optimal Adjuvant Chemotherapy Regimens for Human Epidermal Growth Factor Receptor 2 (HER2)–Negative and Adjuvant Targeted Therapy for HER2-Positive Breast Cancer⁴⁴  (http://ascopubs.org/doi/10.1200/JCO.2016.67.0182)

• Role of Patient and Disease Factors in Adjuvant Systemic Therapy Decision Making for Early-Stage Operable Breast Cancer⁴⁵  (http://ascopubs.org/doi/10.1200/JCO.2015.65.8609)

• Use of Biomarkers to Guide Decisions on Systemic Therapy for Women With Metastatic Breast Cancer⁴⁶  (http://ascopubs.org/doi/10.1200/JCO.2015.61.1459)

• Chemotherapy and Targeted Therapy for Women With Human Epidermal Growth Factor Receptor 2–Negative (or unknown) Advanced Breast Cancer⁴⁷  (http://ascopubs.org/doi/10.1200/JCO.2014.56.7479)

• Systemic Therapy for Patients With Advanced Human Epidermal Growth Factor Receptor 2-Positive Breast Cancer⁴⁸  (http://ascopubs.org/doi/10.1200/JCO.2013.54.0948)

• Recommendations on Disease Management for Patients With Advanced Human Epidermal Growth Factor Receptor 2–Positive Breast Cancer and Brain Metastases⁴⁹  (http://ascopubs.org/doi/10.1200/JCO.2013.54.0955)

• Immunohistochemical Testing of Estrogen and Progesterone Receptors in Breast Cancer⁵⁰  (http://ascopubs.org/doi/10.1200/JCO.2009.25.6529)

Manuscript writing: All authors

Final approval of manuscript: All authors

Accountable for all aspects of the work: All authors

The Expert Panel thanks Gary Lyman, Cynthia Anderson, the Clinical Practice Guidelines Committee, and the CAP Advisory and Independent Review Panels for their thoughtful reviews and insightful comments on this guideline. The Expert Panel also wishes to thank Neil Goldstein, Allen Gown, Erin Grimm, Christine Houston, Loralee McMahon, Dylan Miller, Bill Wyatt, and Hadi Yaziji for sharing their routine clinical experience with HER2 testing in breast cancer through the research survey and open session of the meeting, and those who participated in the online open comment period for providing feedback and suggestions for improving the guideline recommendations.

The following represents disclosure information provided by authors of this manuscript. All relationships are considered compensated.

Relationships are self-held unless noted. I = immediate family member; Inst = my institution. Relationships may not relate to the subject matter of this manuscript. For more information about ASCO's conflict of interest policy, please refer to www.asco.org/rwc or ascopubs.org/jco/site/ifc.

Research Funding: Pfizer (Inst)

Patents, Royalties, Other Intellectual Property: Antonio C. Wolff has been named as inventor on one or more issued patents or pending patent applications relating to methylation in breast cancer and has assigned his rights to Johns Hopkins University, and participates in a royalty-sharing agreement with Johns Hopkins University.

No relationship to disclose

Expert Testimony: Kaiser Permanente

No relationship to disclose

No relationship to disclose

Honoraria: Oncology Education

Consulting or Advisory Role: Insight Genetics, BioNTech AG, Due North, bioTheranostics

Research Funding: NanoString Technologies, BioNTech, Agendia NV, Thermo Fisher Scientific

Patents, Royalties, Other Intellectual Property: Five pending patents. (1) January 2017: Methods and Devices for Predicting Anthracycline Treatment Efficacy; US application, 15/325472; EP application, 3169815A1; Canada application, not yet assigned. (2) January 2017: Systems, Devices and Methods for Constructing and Using a Biomarker; US application, 15328108; EP application, 3172362A4; Canada application, not yet assigned. (3) October 2016: Histone gene module predicts anthracycline benefit, PCT/CA2016/0002474. (4) December 2016: 95-Gene Signature of Residual Risk Following Endocrine Treatment, PCT/CA2016/0003045. (5) December 2016: Immune Gene Signature Predicts Anthracycline Benefit, PCT/CA2016/000305 (Inst)

Honoraria: Hoffmann-La Roche

Consulting or Advisory Role: Hoffmann-La Roche

Travel, Accommodations, Expenses: Hoffmann-La Roche

Employment: Source BioScience PLC

Stock or Other Ownership: Source BioScience PLC

Stock or Other Ownership: Johnson & Johnson, Pfizer, Medtronic, Merck

No relationship to disclose

Patents, Royalties, Other Intellectual Property: Royalties from Abbott Molecular; royalties from Genome Diagnostics

Honoraria: Biocartis, Halozyme Therapeutics, Puma Biotechnology, Cepheid, Ventana Medical Systems, Eli Lilly, Karyopharm Therapeutics, ADC Therapeutics, Scripps Health

Consulting or Advisory Role: Biocartis, Halozyme Therapeutics, Puma Biotechnology, Cepheid, Ventana Medical Systems, ADC Therapeutics, Eli Lilly, Karyopharm Therapeutics, Brogent International

Consulting or Advisory Role: Pfizer

Travel, Accommodations, Expenses: Roche/Genentech

Expert Testimony: Washington Mutual Insurance

Honoraria: MSD

Consulting or Advisory Role: Dako, Roche/Genentech, AstraZeneca, Bristol-Myers Squibb, Astellas Pharma

Speakers' Bureau: Pfizer

Travel, Accommodations, Expenses: Roche, Celgene

No relationship to disclose

Honoraria: Novartis, Pfizer, Myriad Genetics

Consulting or Advisory Role: Radius Health

Research Funding: Pfizer (Inst), Radius Health (Inst)

Other Relationship: Institute of Cancer Research

The information shown above reflects disclosures collected in the course of preparing this article, using the forms and protocols of the American Society of Clinical Oncology (ASCO). This information appears here in print for the convenience of our readers, while conforming to our joint publication agreement with ASCO.