| Optimal FISH testing requirements |
| 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 unfavorable due to weak signals |
| >10 percent of signals occur over cytoplasm |
| Nuclear resolution is poor |
| Autofluorescence is strong |
| The entire ISH slide should be scanned 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 consists of more than 10 percent of tumor cells on the slide, a separate counting of at least 20 non-overlapping cells must also be performed with this cell population and reported |
| Counting should compare patterns in normal breast and tumor cells if bright-field ISH is used; if the tumor cell pattern is neither normal nor clearly amplified, expert opinion should be sought |
| Optimal IHC testing requirements |
| 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 >10 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 within one hour; samples from HER2 testing are fixed in 10 percent neutral buffered formalin for 6 to 72 hours; any exceptions to this process must be included in reporting |
| 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 |
| Laboratories performing these tests should be following all accreditation requirements |
| Initial testing validation should conform to the published 2010 ASCO/CAP recommendations for IHC testing of ER and RT guidance validation requirements with 20 negative and 20 positive FDA-approved assays and 40 negative and 40 positive for laboratory-developed tests; laboratories are responsible for ensuring the reliability and accuracy of their testing results |
| 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 |
