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

The HER2 testing Update Committee (Appendix Table A1, online only at www.asco.org/guidelines/her2) met 3 times via Webinars coordinated by its Steering Committee to review the data published from January 2006 to January 2013 and to revise the recommendations. Additional data were gathered from in-press publications and personal correspondence with researchers to address the issue of mandatory testing if a test result is 0 or 1+. Draft manuscripts were circulated by e-mail, and the Update Committee approved the final manuscript. This guideline was reviewed by external reviewers and approved by the ASCO Clinical Practice Guideline Committee and relevant CAP entities.

The MEDLINE and the Cochrane Collaboration Library electronic databases were searched with the date parameters of January 2006 through January 2013 for articles in English. The MEDLINE search terms are included in Data Supplement 3, and a summary of the literature search results is provided in Data Supplement 4.

Articles were selected for inclusion in the systematic review of the evidence if they met the following criteria: (1) the study compared, prospectively or retrospectively, fluorescent ISH (FISH) and immunohistochemistry (IHC) results or other tests; described technical comparisons across various assay platforms; examined potential testing algorithms for HER2 testing; or examined the correlation of HER2 status in primary versus metastatic tumors from the same patients; (2) the study population consisted of patients with a diagnosis of invasive breast cancer; or (3) the primary outcomes included the negative predictive value (NPV) or positive predictive value (PPV) of ISH and IHC assays used to determine HER2 status, alone and in combination; negative and positive concordance across platforms; and accuracy in determining HER2 status and benefit from anti-HER2 therapy and in determining sensitivity and specificity of individual tests. Consideration was given to studies that directly compared results across assay platforms.

Studies were not limited to randomized controlled trials but also included other study types, including cohort designs, case series, evaluation studies, and comparative studies. The Update Committee also reviewed other testing guidelines and proficiency strategies of various US and international organizations, including unpublished data. Letters, commentaries, and editorials were reviewed for any new information. Case reports were excluded. The clinical questions addressed in the update are available in Data Supplement 5.

This information was used to help the Update Committee develop new algorithms (for pathologists and oncologists) for testing, specify testing requirements and exclusions, and facilitate the necessary quality assurance monitoring that will make HER2 testing less variable and ensure more analytic consistency between laboratories. The term ratio, as used in the guideline recommendations and algorithms, always applies to the HER2/CEP17 ratio, which means the ratio of HER2 signals per cell (numerator) over CEP17 signals per cell (denominator).

The clinical practice guideline and other guidance published herein are provided by ASCO to assist practitioners in clinical decision making. The information herein should not be relied on 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 herein and is not applicable to other interventions, diseases, or stages of diseases. This information does not mandate any particular course of medical care. Furthermore, the information is not intended to substitute for the independent professional judgment of the treating physician, because 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 terms like must, must not, should, and should not indicate 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 physician 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.

Clinical practice guidelines reflect the best available evidence and expert consensus supported in practice. They are intended to assist physicians and patients in clinical decision making and to identify questions and settings for further research. With the rapid flow of scientific information, new evidence may emerge between the time a practice guideline or consensus statement is developed and when it is published or read. Guidelines and statements are not continually updated and may not reflect the most recent evidence. Guidelines and statements address only the topics specifically identified therein and are not applicable to other interventions, diseases, or stages of diseases. Furthermore, guidelines and statements cannot account for individual variation among patients and cannot be considered inclusive of all proper methods of care or exclusive of other treatments. It is the responsibility of the treating physician, relying on independent experience and knowledge, to determine the best course of treatment for the patient. Accordingly, adherence to any practice guideline or consensus statement is voluntary, with the ultimate determination regarding its application to be made by the physician in light of each patient's individual circumstances and preferences. CAP makes no warranty, express or implied, regarding guidelines and statements and specifically excludes any warranties of merchantability and fitness for a particular use or purpose. CAP assumes no responsibility for any injury or damage to persons or property arising out of or related to any use of this statement or for any errors or omissions.

The Update Committee was assembled in accordance with CAP and ASCO Conflicts of Interest Management Procedures for Clinical Practice Guidelines (ASCO procedures are summarized at http://www.asco.org/guidelinescoi). Members of the Update Committee completed the ASCO disclosure form, which requires disclosure of financial and other interests that are relevant to the subject matter of the guideline, including relationships with commercial entities that are reasonably likely to experience direct regulatory or commercial impact as the result of promulgation of the guideline. Categories for disclosure include employment relationships, consulting arrangements, stock ownership, honoraria, research funding, and expert testimony. In accordance with the procedures, the majority of the members of the Update Committee did not disclose any such relationships.

What is the optimal testing algorithm for the assessment of HER2 status?

The Update Committee found more than 70 new publications that informed a revision of the testing algorithms contained in the original 2007 guideline. At the time of the original guideline, significant concern existed about false-positive HER2 test results. Guideline recommendations emphasized those changes that would mitigate false positives, particularly relating to issues of specimen fixation and pathologist interpretation.^39,43–47  Preliminary data from an ongoing prospective study seem to suggest that the frequency of false-positive test results may have diminished, in that the concordance between local testing in laboratories throughout the United States and confirmatory central HER2 testing at the Mayo Clinic (Rochester, MN) for the ALTTO (Adjuvant Lapatinib and/or Trastuzumab Treatment Optimization HER2 Adjuvant Trial) trial showed that less than 6% of patients initially considered eligible were not subsequently centrally confirmed as being HER2 positive.⁴⁸ 

On the other end of the spectrum, clinical experience and recent literature have indicated that false-negative HER2 test results must also be considered. The Update Committee was sensitive to the concerns that surfaced after the publication of the 2007 guideline about the very small number of patients potentially affected by the recommendation to consider as HER2 positive only those tumors with more than 30% of cells (or >10% to ≤30% if HER2 amplified by FISH) with diffuse and intense circumferential staining.⁴⁹  Therefore, the Update Committee decided to revert to the previously used IHC criterion of more than 10% cells staining for HER2, which had been used as an entry criterion for eligibility for the first generation of prospective randomized trials of adjuvant trastuzumab.^{18,22,49–53}  The rationale for this recommendation by the Update Committee is detailed in Data Supplement 1. Aside from the very small number of patients affected (as few as 0.15% of all newly diagnosed patients, as previously discussed),⁵  the Update Committee was also of the opinion that improvements in analytic performance of HER2 testing in clinical practice since 2007 have further reduced the already small number of patients potentially at risk of receiving a false-negative test result.

Testing is now recommended for primary, recurrent, and metastatic tumors.^{19,35,45,54–63,64}  Tissue from the primary tumor can be obtained through a core needle biopsy, as well as from an incisional and excisional surgical procedure.⁶⁵  Metastases can be biopsied from chest wall, regional lymph nodes, or distant organs.^66–74  It is essential to ensure that time to fixation (cold ischemic time) and time in fixative (which has increased from 6 to 48 hours to 6 to 72 hours in this update on the basis of available data and to conform with the ASCO/CAP estrogen receptor [ER]/progesterone receptor [PgR] testing guideline^75,76 ) are recorded and considered in defining the test result. More detail about preanalytic issues is available in Data Supplement 6.

In summary, if available, perform the first test in the core biopsy specimen in a patient with newly diagnosed breast cancer. If the test result is clearly positive or clearly negative as defined in Table 1, no retesting is needed. If the test is negative and there is apparent histopathologic discordance (Table 2), or if specimen handling has not been in accordance with guideline recommendations, a section of the tumor from the excisional specimen should be tested. If this result is positive, no further testing is needed. However, if the test is negative and there remains significant clinical concern about the result after consultation between the pathologist and the medical oncologist, it may be appropriate to repeat the test in a different block from the patient's tumor. If all three tests are negative, no additional testing is recommended.

Data Supplement 7 is a table of IHC Interpretation Criteria, and Data Supplement 8 provides ISH Interpretation Criteria. Both of these Data Supplements expand on details provided in Table 1.

The Update Committee clarified several issues in the update on the basis of recently published literature. The recommendations in Table 1 reflect the Update Committee's interpretation of the new data on polysomy, heterogeneity in ISH, types of assays, and methods of analysis ^{10–14,19–21,45,67,69,79–135}  for inclusion in this update. See Data Supplement 2 for an extensive discussion of these issues.

A list of US Food and Drug Administration (FDA) approved assays is available at http://www.accessdata.fda.gov/scripts/cdrh/devicesatfda/index.cfm?start_search=1&search_term=HER2&approval_date_from=&approval_date_to=07/14/2013&sort=approvaldatedesc&pagenum=10 (last checked July 14, 2013). The product package inserts for trastuzumab and pertuzumab prepared by the FDA indicate that “HER2 testing should be performed using US Food and Drug Administration-approved tests by laboratories with demonstrated proficiency.”^77,78 

Each assay type has diagnostic pitfalls to be avoided. The Update Committee agreed that there were situations in which one assay type was preferred because of assay or sample considerations. Exclusion criteria to perform or interpret an IHC or any ISH assay for HER2 are unchanged but can be viewed in the original guideline.^1,2  The pathologist who reviews the histologic findings should determine the optimal assay (IHC or ISH) for determination of HER2 status.

Algorithms for evaluation of HER2 protein expression by IHC and HER2 amplification by single-probe or dual-probe ISH are presented in Figures 1, 2, and 3.

What strategies can help ensure optimal performance, interpretation, and reporting of established assays?

The Update Committee reviewed new papers and reports on strategies to ensure optimal performance, interpretation, and reporting of assays.^{16,22,100,136,137}  Most new HER2 assays have been submitted to the FDA for premarket approval review as class III devices in view of their use for therapy selection. Although a new HER2 assay ideally should have its clinical utility validated using specimens from prospective therapeutic trials that tested the effects of anti-HER2 therapy, the Update Committee recognizes that the rarity of these valuable specimens requires that new HER2 assays be approved on the basis of concordance studies comparing them with other established HER2 tests. Consequently, it is important that tissues selected for such concordance studies come from datasets that include a broad representation of patients with breast cancer in whom HER2-positive status will be observed in approximately 15% to 20%.

The Update Committee urges ongoing competency assessment as a part of every laboratory's internal quality assessment program. The competency of the laboratory professionals and pathologists interpreting assays must be continuously addressed as required under the Clinical Laboratory Improvements Amendments (CLIA 88). The acceptable performance standard for such competency tests remains the same as in the original guideline.

Data Supplements 9 and 10 are tables of reporting elements for IHC and reporting elements for ISH, respectively. Some changes have been made to the reporting elements for IHC and ISH to ensure that they are in accordance with the revised recommendations. In addition, a disclaimer statement is required if the specimen handling requirements are not met.

New interpretation requirements relate to the definition of tumor samples with genomic heterogeneity as well as the examination of specimens and interpretation of results in these samples. No specific requirements were added for designation of polysomy by ISH. Laboratories should maintain documentation of their quality assurance practices and ensure that such documentation is available for inspection.

The regulatory framework remains the same as discussed in the original guideline. At the current time, the FDA exercises enforcement discretion over laboratory-developed tests (LDTs) that are generated and performed within an individual laboratory under CLIA 88. CLIA 88 provides stringent quality standards for highly complex tests, which include all predictive cancer factor assays. This legislation also requires biannual surveys of laboratories that perform highly complex tests, with defined criteria and actions required when performance is deficient. However, CLIA certification does not require that the tests performed have been shown with a high level of evidence to have clinical utility.^138,139  Moreover, FDA approval of devices, which includes in vitro diagnostic tests such as those discussed in this guideline, does not necessarily require demonstration that use of the assay results in improved clinical outcomes compared with not using the assay. The Update Committee expresses concern about the need for greater clarity in the regulatory environment with regard to companion diagnostic tests and LDTs for higher-risk tumor biomarker tests, such as HER2. Some of this has been discussed by the Evaluation of Genomic Applications in Practice and Prevention (EGAPP) Initiative and endorsed by the Institute of Medicine Committee in regard to omics-based tests, as well as others,¹³⁹  and the Update Committee understands the FDA is developing a risk-based framework to address concerns about test accuracy and clinical utility.

External proficiency testing is a mandatory requirement for CAP-accredited laboratories, beginning with the 2007 guideline. External proficiency testing challenge failure requires investigation and corrective action before the laboratory can continue to offer HER2 testing.

CAP modified its laboratory accreditation program to include more careful scrutiny of HER2 testing, thus creating a mandatory and expanded proficiency testing program to evaluate laboratory performance. The systematic review revealed many new papers on quality assurance, quality improvement, proficiency testing, and establishment of concordance between local and central laboratories, both in the United States and internationally.^{40,51,136,140–146}  A revised table addressing proficiency testing is contained in Data Supplement 11, which describes statistical requirements for proficiency testing. Examples of international external quality assurance schemas are included in Data Supplement 12.

The number of laboratories participating in predictive marker proficiency testing for HER2 and ER as part of the CAP laboratory improvement program since 2004 is shown in Figure 4, and the program is described at http://www.cap.org/apps/cap.portal?_nfpb=true&_pageLabel=accreditation (last checked July 14, 2013).

CAP has undertaken comprehensive efforts to educate pathologists about ways to improve laboratory performance of HER2, ER, and PgR assays. Numerous live and online educational offerings are available from CAP and other organizations. Examples in North America include the American Society of Clinical Pathology (ASCP) and United States and Canadian Academy of Pathology (USCAP). CAP provides varied live and online education focused on HER2 and ER/PgR testing elements of relevance to pathologists in meeting the original ASCO/CAP HER2 and ER/PgR guidelines and updates. In follow-up surveys, participants routinely report they made changes to their practice as a result of the educational experience. Many of these learning opportunities have a scored assessment component, allowing participants to test their knowledge as part of completing the courses, and can be used to meet the American Board of Pathology (ABP), the US pathologist certifying organization, Maintenance of Certification requirements. More information can be found at the CAP learning portal (http://www.cap.org) and in the original guideline. CAP has also created a listing of competencies in breast pathology, compiled by experts and available for pathologist self-assessment. After taking this self-assessment, pathologists are prompted to learning offerings that target those areas of self-reported educational deficiency. A listing of the courses is available online at http://www.cap.org via the learning portal.

Whether in the context of trastuzumab clinical trials or of studies comparing HER2 testing platforms, interpretation of the literature in the field of HER2 testing is still complicated by a lack of standardization across trials in assay utilization and interpretation, presence or absence of confirmatory testing, and local versus central laboratory testing, among other considerations. Although FDA-approved assays have been carefully validated, not all LDTs may have, which complicates direct comparisons across trials and platforms, and we maintain that this situation leaves open the possibility that a substantial percentage of some patients with breast cancer could be either over- or undertreated with HER2-targeted therapies.

An important gap in the literature identified by the Update Committee concerns those patients with test results reported as equivocal. The decision to treat with specific therapies like trastuzumab is by necessity dichotomous (yes or no) and will not be informed by an equivocal diagnosis with respect to HER2 status without repeat testing, if possible. However, HER2 test results are derived from a continuous variable, which can be expected to lead to some results falling into a gray area. Adding to this confusion is the fact that there is variability in the reporting definitions of the equivocal ranges for both bright-field ISH and FISH assays.

The literature is lacking evidence on response to HER2-targeted therapy in the subgroup of patients with equivocal results, and there are limited efficacy data in the subgroup tested with both high quality IHC and FISH and found to have a discordant result between these two tests. Patients with such results constitute poorly studied subsets for which there is less confidence in the scores and actual benefit from trastuzumab therapy. Because the retrospective evaluation of the benefit from trastuzumab in patients with apparent discordance between IHC and FISH who were enrolled onto the first generation of trastuzumab trials included only a small number of patients in each of the discordant subsets, patients who would have qualified for enrollment in those trials should be considered for HER2-targeted therapy.

The Update Committee's goal was to address the most common clinical situations encountered by pathologists and oncologists in routine clinical practice. Specifically in regard to ISH assays, it expected that additional but rare categories of HER2 status by ISH could be created that are not covered by the definitions illustrated in Figures 2 and 3. Data Supplement 2E addresses a narrower set of scenarios that may on occasion be observed with dual-signal ISH assays.

For patients with low levels of HER2 expression that do not reach the threshold for HER2-positive disease, the Update Committee encourages enrollment of such patients, if eligible, onto prospective clinical trials that aim to address the value of adjuvant HER2-targeted therapies in patients whose breast cancers show low levels of HER2 expression, like the NSABP B-47 (National Surgical Adjuvant Breast and Bowel Project B-47) trial (NCT01275677). The Update Committee also supports participation in studies evaluating other cutoffs and other technologies to optimize eligibility for HER2-targeted therapies.

Patients (and family members or caregivers) should be educated about the results of pathology tests and how they are used to develop a treatment plan tailored to the biology of their cancers. Because many newly diagnosed patients are under emotional stress and/or may be unaccustomed to complex medical terminology, the use of easily understood language (at an educational level that the patient can understand) is key to clear communication. Asking patients to repeat back key pieces of information, providing written or recorded notes, and using visual aids can help ensure information is effectively communicated.

Patients should be given a copy of their pathology report and HER2 test results. The clinician should review the results with the patient, discuss any issues with the test interpretation or performance, and ask if he or she has any additional questions about the results.

Patients should understand that the most common biologic tests are those for ER, PgR, and HER2 and that testing for these markers is important to select an appropriate treatment. The overall percentage of patients with HER2-positive breast cancer is between 15% and 20%. Observed numbers may vary depending on the population being tested by individual laboratories.

Patients should understand that HER2 status determines whether certain drugs (eg, trastuzumab, lapatinib, pertuzumab, T-DM1) are recommended. They should also understand that the HER2 gene is important in tumor cell growth and that tumors that have increased levels of HER2 (as measured by HER2 gene amplification or HER2 protein overexpression) usually have a higher growth rate and a more aggressive clinical behavior.

Patients should understand the type of tissue used for HER2 testing (eg, core biopsy, excisional biopsy).

Patients should understand that there are different FDA-approved testing methods that detect HER2 protein overexpression or the presence of HER2 gene amplification.

Patients should understand that although most HER2 test results are definitively positive or negative, there are equivocal results that require additional testing using an alternative test or using the same or alternative test on a different portion of the same specimen (different block). Sometimes, the oncologist or pathologist may recommend additional testing using a different type of tumor specimen (eg, surgical excision v core biopsy), if available. Patients should be informed about which test or tests were performed and the expected turnaround time for these tests. Unfortunately, some results remain indeterminate or inconsistent with other histopathologic findings. In such cases, a final treatment decision to consider treatment with HER2-targeted therapy should be made after consultation between the pathologist and oncologist and a discussion with the patient.

Patients should understand that HER2 status may occasionally be different (discordant) when comparing a previous primary tumor and a site of recurrence or in the setting of multiple simultaneous metastatic sites. In some cases, it is not possible to fully differentiate between a true biologic change, tumor heterogeneity, or variability in the performance of the assay.

Patients should be assured that HER2 testing guidelines were followed. Refer patients to the ASCO/CAP guideline update at www.asco.org/guideline/her2 and/or http://www.cap.org and to www.cancer.net for additional patient-focused information.

Although ASCO clinical practice guidelines present recommendations on the best practices in diagnosis and disease management to provide the highest level of cancer diagnosis and care, it is important to note that some racial/ethnic minority patients have limited access to optimal medical care and/or accredited pathology laboratories. At the same time, some Medicaid or uninsured patients may have access to accredited pathology laboratories by virtue of receiving some or all of their care in an academic medical center.^147–150 

Disparities clearly exist in the likelihood of receiving HER2 testing. In the United States, Lund et al¹⁵¹  used data from the National Cancer Institute Metropolitan Atlanta SEER Registry in conjunction with the Georgia Comprehensive Cancer Registry to examine HER2 testing among all cases of primary invasive breast cancer diagnosed among female residents during 2003 to 2004. Overall, 90.1% of women had evidence of HER2 testing. Rates of HER2 testing did not vary significantly based on socioeconomic status (based on the percent living below the federal poverty level) and were similar between black (91.3%) and white (89.8%) women. This is in agreement with other reports showing similar or greater rates of HER2 testing among black versus white women with breast cancer.¹⁵²  However, in the Lund et al study, Hispanic women were significantly less likely to receive HER2 testing (79.3%), as were women diagnosed with stage IV (80.7%) or unknown stage (71.7%) disease. In addition, the mean age of women who received HER2 testing (58.8 years) was significantly younger than that of women who did not receive testing (61.3 years). Other studies have also reported that older women^153,154  and those with distant disease are significantly less likely to have documentation of HER2 testing. Stark et al¹⁵⁵  also reported that women with capitated insurance (versus fee-for-service insurance) were significantly more likely to be tested for HER2 status. Awareness of possible disparities in access to care should be considered in the context of this clinical practice guideline, and health care providers should strive to deliver the highest level of cancer care to these vulnerable populations.

Data Supplements, including evidence tables, and clinical tools and resources can be found at www.asco.org/guidelines/her2. Information for patients is available at http://www.cancer.net.

Although all authors completed the disclosure declaration, the following author(s) and/or an author's immediate family member(s) indicated a financial or other interest that is relevant to the subject matter under consideration in this article. Certain relationships marked with a “U” are those for which no compensation was received; those relationships marked with a “C” were compensated. For a detailed description of the disclosure categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors.

Employment or Leadership Position: Donald C. Allred, Clarient-GE Healthcare (C) Consultant or Advisory Role: Mitch Dowsett, Roche (C); Donald C. Allred, Clarient (U); John M.S. Bartlett, Roche Canada (C), Roche UK (C); Michael Bilous, Roche (C); Wedad Hanna, Roche (C); Michael F. Press, Roche (C); Giuseppe Viale, Roche (C), Genomic Health (C), Dako (C) Stock Ownership: None Honoraria: Mitch Dowsett, Roche, Dako; John M.S. Bartlett, Roche Canada, Roche UK; Michael Bilous, Roche; Wedad Hanna, Roche; Giuseppe Viale, Roche Research Funding: Mitch Dowsett, Roche; John M.S. Bartlett, Roche UK; Wedad Hanna, Roche; Michael F. Press, Roche Expert Testimony: None Patents: None Other Remuneration: John M.S. Bartlett, Roche UK; Wedad Hanna, Roche; Robert B. Jenkins, Abbott.

AUTHOR CONTRIBUTIONS

Administrative support: Antonio C. Wolff, M. Elizabeth H. Hammond, David G. Hicks, Mitch Dowsett, Lisa M. McShane, Pamela B. Mangu, Daniel F. Hayes

Manuscript writing: All authors

Final approval of manuscript: All authors