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 This is an original JCO publication from 2013. Please visit the JCO website to access the full article.


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


 

 Authors

Antonio C. Wolff,* M. Elizabeth H. Hammond,* David G. Hicks,* Mitch Dowsett,* Lisa M. McShane,* Kimberly H. Allison, Donald C. Allred, John M.S. Bartlett, Michael Bilous, Patrick Fitzgibbons, Wedad Hanna, Robert B. Jenkins, Pamela B. Mangu, Soonmyung Paik, Edith A. Perez, Michael F. Press, Patricia A. Spears, Gail H. Vance, Giuseppe Viale, and Daniel F. Hayes*

*Steering Committee member

This guideline is currently in the process of being updated.

Notice: Both ASCO and CAP are committed to maintaining and updating guidelines as new evidence becomes available. ASCO and CAP will reconvene the HER2 Testing Update Committee to develop and publish a focused update on recent issues brought forth. Further information may be found in the March 9th early release of the letter to the editor and reply in the Journal of Clinical Oncology. A separate editorial will be forthcoming in the Archives of Pathology and Laboratory Medicine. 

 

THE BOTTOM LINE

Intervention

  • Recommendations for HER2 testing in breast cancer

Target Audience

  • Medical oncologists, pathologists, and surgeons

Key Recommendations for Oncologists

  • Must request HER2 testing on every primary invasive breast cancer (and on metastatic site, if stage IV and if specimen available) from a patient with breast cancer to guide decision to pursue HER2-targeted therapy. This should be especially considered for a patient who previously tested HER2 negative in a primary tumor and presents with disease recurrence with clinical behavior suggestive of HER2-positive or triple-negative disease.
  • Should recommend HER2-targeted therapy if HER2 test result is positive, if there is no apparent histopathologic discordance with HER2 testing (Tables 1 and 2), and if clinically appropriate. If the pathologist or oncologist observes an apparent histopathologic discordance after HER2 testing, the need for additional HER2 testing should be discussed. 
  • Must delay decision to recommend HER2-targeted therapy if initialHER2test result is equivocal. Reflex testing should be performed on the same specimen using the alternative test if initial HER2 test result is equivocal or on an alternative specimen (Tables 1 and 2).
  • Must not recommend HER2-targeted therapy if HER2 test result is negative and if there is no apparent histopathologic discordance with HER2 testing (Tables 1 and 2). If the pathologist or oncologist observes an apparent histopathologic discordance after HER2 testing, the need for additional HER2 testing should be discussed. 
  • Should delay decision to recommend HER2-targeted therapy if HER2 status cannot be confirmed as positive or negative after separate HER2 tests (HER2 test result or results equivocal). The oncologist should confer with the pathologist regarding the need for additional HER2 testing on the same or another tumor specimen. 
  • If the HER2 test result is ultimately deemed to be equivocal, even after reflex testing with an alternative assay (ie, if neither test is unequivocally positive), the oncologist may consider HER2-targeted therapy. The oncologist should also consider the feasibility of testing another tumor specimen to attempt to definitely establish the tumor HER2 status and guide therapeutic decisions. A clinical decision to ultimately consider HER2-targeted therapy in such cases should be individualized on the basis of patient status (comorbidities, prognosis, and so on) and patient preferences after discussing available clinical evidence.

Key Recommendations for Pathologists

  • Must ensure that at least one tumor sample from all patients with breast cancer (early-stage or metastatic disease) is tested for either HER2 protein expression (IHC assay) or HER2 gene expression (ISH assay) using a validated HER2 test.  
  • In the United States, the ASCO/CAP Guideline Update Committee preferentially recommends the use of an assay that has received FDA approval, although a CLIA-certified laboratory may choose instead to use a laboratory-developed test (LDT). In this case, the analytic performance of the LDT must be prospectively validated in the same clinical laboratory that will perform it, and the test must have documented analytic validity (CAP guidance document). Bright-field ISH assays must be initially validated by comparing them with an FDA-approved FISH assay.
  • Must report HER2 test result as positive if: (a) IHC 3positive or (b) ISH positive using either a single-probe ISH or dual-probe ISH (Table 1; Figs 1 to 3). This assumes that there is no apparent histopathologic discordance observed by the pathologist (Table 2). 
  • Must report HER2 test result as equivocal and order reflex test on the same specimen (unless the pathologist has concerns about the specimen) using the alternative test if: (a) IHC 2 equivocal or (b) ISH equivocal using single-probe ISH or dual-probe ISH (Table 1; Figs 1 to 3). This assumes that there is no apparent histopathologic discordance observed by the pathologist (Table 2).
    Note that there are some rare breast cancers (eg, gland-forming tumors, micropapillary carcinomas) that show IHC 1 staining that is intense but incomplete (basolateral or U shaped) and that are found to be HER2 amplified. The pathologist should consider also reporting these specimens equivocal and request reflex testing using the alternative test.
  • Must report HER2 test result as negative if a single test (or all tests) performed in a tumor specimen show: (a) IHC 1 negative or IHC 0 negative or (b) ISH negative using single-probe ISH or dual-probe ISH (Table 1; Figs 1 to 3). This assumes that there is no apparent histopathologic discordance observed by the pathologist (Table 2).
  • Must report HER2 test result as indeterminate if technical issues prevent one or both tests (IHC and ISH) performed in a tumor specimen from being reported as positive, negative, or equivocal. This may occur if specimen handling was inadequate, if artifacts (crush or edge artifacts) make interpretation difficult, or if the analytic testing failed. Another specimen should be requested for testing, if possible, and a comment should be included in the pathology report documenting intended action.
  • Must ensure that interpretation and reporting guidelines for HER2 testing are followed (Table 1; Data Supplements 7, 8, 9, and 10). 
  • Should interpret bright-field ISH on the basis of a comparison between patterns in normal breast and tumor cells, because artifactual patterns may be seen that are difficult to interpret. If tumor cell pattern is neither normal nor clearly amplified, test should be submitted for expert opinion. 
  • Should ensure that any specimen used for HER2 testing (cytologic specimens, needle biopsies, or resection specimens) begins the fixation process quickly (time to fixative within 1 hour) and is fixed in 10% neutral buffered formalin for 6 to72 hours and that routine processing, as well as staining or probing, is performed according to standardized analytically validated protocols.
  • Should ensure that the laboratory conforms to standards set for CAP accreditation or an equivalent accreditation authority, including initial test validation, ongoing internal quality assurance, ongoing external proficiency testing, and routine periodic performance monitoring. 
  • If an apparent histopathologic discordance is observed in any HER2 testing situation (Table 2), the pathologist should consider ordering additional HER2 testing and conferring with the oncologist, and should document the decision-making process and results in the pathology report. As part of the HER2 testing process, the pathologist may pursue additional HER2 testing without conferring with the oncologist.
  • Although categories of HER2 status by IHC or ISH can be created that are not covered by these definitions, in practice they are uncommon and if encountered should be considered IHC equivocal or ISH equivocal.

Methods

  • Systematic review and analysis of the medical literature were conducted by the 2013 Update Committee.

Additional Information

  • The revised recommendations and a brief summary of the literature and analysis are provided in this article. Data Supplements including clinical tools and resources can be found at http://www.asco.org/guidelines/her2 and at http://www.cap.org. Patient information is available at http://www.cancer.net. ASCO and CAP believe that cancer clinical trials are vital to inform medical decisions and improve cancer care, and that all patients should have the opportunity to participate.

 

 

SUMMARY OF RECOMMENDATIONS

Topic

2007 Recommendation

2013 Recommendation

Specimens to be tested

All primary breast cancer specimens and metastases should have at least one HER2 test performed

All newly diagnosed patients with breast cancer must have a HER2 test performed.  Patients who then develop metastatic disease must have a HER2 test performed in a metastatic site, if tissue sample is available.

Optimal algorithm for HER2 testing

Positive for HER2 is either IHC HER2 3+ (defined as uniform intense membrane staining of > 30% of invasive tumor cells) or FISH amplified (ratio of HER2 to CEP17 of > 2.2 or average HER2 gene copy number > six signals/nucleus for those test systems without an internal control probes

Must report HER2 test result as Positive for HER2 if: a,b

  • IHC 3+ based on circumferential membrane staining that is complete, intense c,d
  • ISH Positive based on:
    • Single Probe average HER2 copy number ≥6.0 signals/cell.c,d
    • Dual Probe HER2/CEP17 ratio ≥ 2.0;c,e with an average HER2 copy number ≥4.0 signals/cell

Dual Probe HER2/CEP17 ratio ≥ 2.0;c,e; with an average HER2 copy number < 4 signals/cellb

Dual Probe HER2/CEP17 ratio < 2.0;c,e with an average HER2 copy number ≥ 6.0 signals/cell

 

Equivocal for HER2 is defined as: IHC 2+ or

 

FISH HER2/CEP17 ratio of 1.8-2.2 or average HER2 gene copy number 4-6 HER2 signals/nucleus for test systems without an internal control probe

Must report HER2 test result as equivocal and order reflex test (same specimen using the alternative test) or new test (new specimen, if available, using same or alternative test) if: a,b

  • IHC 2+ based on circumferential membrane staining that is incomplete and/or weak/moderatef and within >10% of the invasive tumor cells;d or complete and circumferential membrane staining that is intense and within ≤10% of the invasive tumor cells d
  • ISH equivocal based on:
    • Single-probe ISH average HER2 copy number ≥4.0 and <6.0 signals/celle,f
    • Dual-probe HER2/CEP17 ratio <2.0 with an average HER2 copy number ≥4.0 and <6.0 signals/celle,f

 

Negative for HER2 is defined as:

  • IHC HER2 0: no staining
  • IHC HER2 1+: Weak incomplete membrane staining in any proportion of tumor cells or weak, complete membrane staining in <10% of cells
  • FISH HER2/CEP17 ratio of < 1.8 or average HER2 gene copy number of < 4 signals/nucleus for test systems without an internal control probe

Must report a HER2 test result as negative if a single test (or both tests) performed show:a,b

  • IHC 1+ as defined by incomplete membrane staining that is faint/barely perceptible and within >10% of the invasive tumor cellsd 
  • IHC 0 as defined by no staining observedd or membrane staining that is incomplete and is faint/barely perceptible and within ≤10% of the invasive tumor cellsd
  • ISH negative based on:
    • Single-probe average HER2 copy number <4.0 signals/cell
    • Dual-probe HER2/CEP17 ratio <2.0

 

Indeterminate for HER2

Must report HER2 test result as indeterminate if technical issues prevent one or both tests (IHC and ISH) from being reported as positive, negative, or equivocal. Conditions may include:

  • Inadequate specimen handling
  • Artifacts (crush or edge artifacts) that make interpretation difficult
  • Analytic testing failure

Another specimen should be requested for testing to determine HER2 status. Reason for indeterminate testing should be noted in a comment in the report.

ISH rejection criteria

Test is rejected and repeated if 

  • Controls are not as expected
  • Observer cannot find and count at least two areas of invasive tumor
  • > 25% of signals are unscorable due to weak signals
  • > 10% of signals occur over cytoplasm
  • Nuclear resolution is poor
  • Autofluorescence is strong

Same and report HER2 test result as Indeterminate as per parameters described above.

 

ISH interpretation

Interpretation done by counting at least 20 cells; a pathologist must confirm that counting involved invasive tumor criteria followed

The pathologist should scan the entire ISH slide prior to counting at least 20 cells or use IHC to define the areas of potential HER2 amplification.

If there is a second population of cells with increased HER2 signals/cell and this cell population consists of more than 10% of tumor cells on the slide (defined by image analysis or visual estimation of the ISH or IHC slide), a separate counting of at least 20 nonoverlapping cells must also be performed within this cell population and reported.

For brightfield ISH, counting requires comparison between patterns in normal breast and tumor cells because artifactual patterns may be seen that are difficult to interpret. If tumor cell pattern is neither normal nor clearly amplified, test should be submitted for expert opinion.

Acceptable [IHC and] ISH testsg

 

Should preferentially use an FDA-approved IHC, brightfield ISH, or FISH assay.g,h

IHC rejection criteria

Test is rejected and repeated or tested by FISH if:

  • Controls are not as expected
  • Artifacts involve most of sample
  • Sample has strong membrane staining of normal breast ducts (internal controls)

Same

IHC interpretation criteria

Positive HER2 result requires homogeneous, dark circumferential (chicken wire) pattern in > 30% of invasive tumor

Interpreters have method to maintain consistency and competency

Should interpret IHC test using a threshold of more than 10% of tumor cells that must show homogeneous, dark circumferential (chicken wire) pattern to call result 3+, HER2 positive.

Reporting requirements for all assay types

Report must include guideline-detailed elements

Same except for changes to reporting requirement and algorithms defined in this table. (Data Supplements 9 and 10)

 

 

Optimal tissue handling requirements

Time from tissue acquisition to fixation should be as short as possible; samples for HER2 testing are fixed in 10% neutral buffered formalin for 6–48 hours; cytology specimens must be fixed in formalin.

Samples should be sliced at 5-mm to 10-mm intervals after appropriate gross inspection and margins designation and placed in sufficient volume of neutral buffered formalin

Duration of fixation has been changed from 6-48 hours to 6-72 hours.  Any exceptions to this process must be included in report.

 

 

Optimal tissue sectioning requirements

Sections should ideally not be used for HER2 testing if cut > 6 weeks earlier; this may vary with primary fixation or storage conditions

Same.

Optimal internal validation procedure

Validation of test must be done before test is offered

 

Same

Data Supplement12 lists examples of various external quality assurance schemes.

Optimal initial test validation

Initial test validation requires 25-100 samples tested by alternative validated method in the same laboratory or by validated method in another laboratory

Laboratories performing these tests should be following all accreditation requirements, one of which is initial testing validation. The laboratory should ensure that initial validation conforms to the published 2010 ASCO/CAP Recommendations for IHC Testing of ER and PgR guideline validation requirements with 20 negative and 20 positive for FDA-approved assays and 40 negative and 40 positive for LDTs. This requirement does not apply to assays that were previously validated in conformance with the 2007 ASCO/CAP HER2 testing guideline, and who are routinely participating in external proficiency testing for HER2 tests, such as the program offered by the CAP (Data Supplement 12).

 

Proof of initial testing validation in which positive and negative HER2 categories are 90% concordant with alternative validated method or same validated method for HER2

Laboratories are responsible for ensuring the reliability and accuracy of their testing results, by compliance with accreditation and proficiency testing requirements for HER2 testing assays. Specific concordance requirements are not required. (Data Supplement 11)

 

Optimal monitoring of test concordance between methods

Concordance testing must be done prior to initiation of testing, optimally as the form of testing validation. If concordance is below 95% for any testing category, that category of test result of either FISH or IHC must be automatically flexed to alternative method before final interpretation.

See text below under “Optimal Laboratory Accreditation”

Optimal internal QA procedures

 

Should review and document external and internal controls with each test and each batch of tests.

 

 

Ongoing quality control and equipment maintenance

Same.

 

Initial and ongoing laboratory personnel training and competency assessment

Same.

 

Use of standardized operating procedures including routine use of control materials

Same.

 

Revalidation of procedure if changed

Same.

 

Ongoing competency assessment and education of pathologists

Should perform ongoing competency assessment and document the actions taken as a part of the laboratory record.

 

Optimal external proficiency assessment

Participation in and successful completion of external proficiency testing program with at least two testing events (mailings) a year

Same.

 

Satisfactory performance requires at least 90% correct responses on graded challenges for either test

  • Unsatisfactory performance will require laboratory to respond according to accreditation agency program requirements

Same

Optimal laboratory accreditation

Onsite inspection every other year with annual requirement for self-inspection

  • Reviews laboratory validation, procedures, QA results and processes, results and reports
  • Unsatisfactory performance results in suspension of laboratory testing for HER2 for that method

Same (Data Supplement 11).

 


 

 

 

 

ASCO Guideline Disclaimer: The clinical practice guidelines and other guidance published herein are provided by the American Society of Clinical Oncology, Inc. ("ASCO") to assist practitioners 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 physician, 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" 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.


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