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©2013 UpToDate ® Print Email Summary of the joint ASCO/CAP guideline recommendations for assessment of HER2 status Optimal algorithm for HER2 testing Positive for HER2 is either IHC HER2 3+ (defined as uniform intense membrane staining of >30 percent of invasive tumor cells) or FISH amplified (ratio of HER2 to CEP17 of >2.2 or average HER2 gene copy number >6 signals/nucleus for those test systems without an internal control probe) Equivocal for HER2 is defined as either IHC 2+ or FISH ratio of 1.8-2.2 or average HER2 gene copy number four to six signals/nucleus for test systems without an internal control probe Negative for HER2 is defined as either IHC 0-1+ or FISH ratio of <1.8 or average HER2 gene copy number of <4 signals/nucleus for test systems without an internal control probe These definitions depend on laboratory documentation of the following: 1. Proof of initial testing validation in which positive and negative HER2 categories are 95 percent concordant with alternative validated method or same validated method for HER2 2. Ongoing internal QA procedures 3. Participation in external proficiency testing 4. Current accreditation by valid accrediting agency Optimal FISH testing requirements Fixation for fewer than six hours or longer than 48 hours is not recommended Test is rejected and repeated if: Controls are not as expected Observer cannot find and count as least two areas of invasive tumor >25 percent of signals are unscorable due to weak signals >10 percent of signals occur over cytoplasm Nuclear resolution is poor Autofluorescence is strong Interpretation done by counting at least 20 cells; a pathologist must confirm that counting involved invasive tumor Sample is subjected to increased counting and/or repeated if equivocal; report must include guideline-detailed elements Optimal IHC testing requirements Fixation for fewer than six hours or longer than 48 hours is not recommended 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) Interpretation follows guideline recommendation Positive HER2 result requires homogeneous, dark circumferential (chicken wire) pattern in >30 percent of invasive tumor Interpreters have method to maintain consistency and competency Sample is subjected to confirmatory FISH testing if equivocal based on initial results Report must include guideline-detailed elements

Interpretation follows guideline recommendation Positive HER2 result requires homogeneous, dark circumferential (chicken wire) pattern in >30 percent of invasive tumor Interpreters have method to maintain consistency and competency Sample is subjected to confirmatory FISH testing if equivocal based on initial results Report must include guideline-detailed elements Optimal tissue handling requirements Time from tissue acquisition to fixation should be as short as possible; samples from HER2 testing are fixed in neutral buffered formalin for 6 to 48 hours; samples should be sliced at 5 to 10 mm intervals after appropriate gross inspection and margins designation and placed in sufficient volume of neutral buffered formalin Sections should ideally not be used for HER2 testing if cut >6 weeks earlier; this may vary with primary fixation or storage conditions Time to fixation and duration of fixation if available should be recorded for each sample Optimal internal validation procedure Validation of test must be done before test is offered Initial test validation requires 25 to 100 samples tested by alternative validated method in the same laboratory or by validated method in another laboratory Proof of initial testing validation in which positive and negative HER2 categories are 95 percent concordant with alternative validated method or same validated method for HER2 Ongoing validation should be done biannually Optimal internal QA procedures Initial test validation Ongoing quality control and equipment maintenance Initial and ongoing laboratory personnel training and competency assessment Use of standard operating procedures including routine use of control materials Revalidation of procedure if changed Ongoing competency assessment and education of pathologists Optimal external proficiency assessment Participation in external proficiency testing program with at least two testing events (mailings)/year Satisfactory performance requires at least 90 percent correct responses on graded challenges for either test Unsatisfactory performance will require laboratory to respond according to accreditation agency program requirements 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

Unsatisfactory performance will require laboratory to respond according to accreditation agency program requirements 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 HER2: human epidermal growth factor receptor 2; IHC: immunohistochemistry; FISH: fluorescent in situ hybridization; QA: quality assurance. Reproduced with permission from Wolff AC, Hammond EH, Schwartz JN. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. J Clin Onc 2007; 25:1. Copyright © 2007 the American Society of Clinical Oncology.