[Lani]

IHC is performed by staining for a certain protein and looking at the tissue under the microscope for both the quantity of stain taken up and for certain patterns of staining (eg staining of the nucleus vs staining of the cytoplasm)

FISH detects the number of copies of the her2 gene, rather than the her2 protein.

IHC is done as a preliminary to avoid performing the more expensive and more technically demanding FISH test ie, if the staining is 0 or 1+ there is no need to proceed to the FISH test.

FISH rather than IHC is the test most indicative of response to (effectiveness of) herceptin. From the official guidelines:

Positive HER2 test. Based on a literature review of clinical trials, international studies and protocols, expert consensus, and US Food and Drug Administration Panel findings, a positive HER2 test is defined as either IHC result of 3+ cell surface protein expression (defined as uniform intense membrane staining of > 30% of invasive tumor cells) or FISH result of amplified HER2 gene copy number (average of > six gene copies/nucleus for test systems without internal control probe) or HER2/CEP 17 ratio of more than 2.2, where CEP 17 is a centromeric probe for chromosome 17 on which the HER2 gene resides. The 30% criteria for a positive IHC is further discussed in Appendix G. The original FISH test results were defined as either positive or negative, but an intermediate range (from hereon referred to as equivocal range) has since been described and the clinical significance of this observation remains unclear.34-36 This strategy classifies patients as having HER2-positive disease based on positive results with either test. It is recognized that current data are insufficient to define whether these patients represent true- or false-positives. Although the large prospective randomized clinical trials of trastuzumab were not prospectively designed to answer these questions, we anticipate and recommend that such analyses will be forthcoming as correlative studies.

Equivocal HER2 test. Much of the confusion about HER2 testing has resulted from the need to define trastuzumab treatment (yes or no) based on test results that represent a continuous rather than a categoric variable. Furthermore, there is significant variation in the intermediate (equivocal) ranges for both the IHC and FISH assays. The equivocal range for IHC consists of samples scored 2+, and this may include up to 15% of samples.29 An equivocal result (2+) is complete membrane staining that is either nonuniform or weak in intensity but with obvious circumferential distribution in at least 10% of cells. Very rarely, in the experience of panel members, invasive tumors can show intense, complete membrane staining of 30% or fewer tumor cells. These are also considered to be equivocal in this guideline. Some but not all of these samples may have HER2 gene amplification and require additional testing to define the true HER2 status (Figs 1 and 2).54,55

The equivocal range for FISH assays is defined as HER2/CEP 17 ratios from 1.8 to 2.2 or average gene copy numbers between 4.0 and 6.0 for those systems without an internal control probe.34-36 Note, however, that patients with a HER2/CEP17 FISH ratio between 2.0 and 2.2 were formerly considered HER2-positive and were eligible for treatment in the adjuvant trastuzumab trials. Therefore, available efficacy data do not support excluding them from therapy with trastuzumab. This group is much smaller, probably fewer than 3% of samples.56 Polysomy 17 is observed in approximately 8% of all specimens, mostly among cases with four to six HER2 gene copies (equivocal range).57,58 There is no accepted definition of what constitutes polysomy and authors have used different criteria to define it. If polysomy 17 is defined as three or more copies of CEP17, most are not associated with protein or mRNA overexpression57 and the same has been observed in tumors with a HER2 gene copy number between 4 and 6.35

Discordant results (IHC 3+/FISH-negative or IHC < 3+/FISH-positive) have also been described, and were observed in approximately 4% among 1,503 patients screened centrally (LabCorp, Burlington, NC) with both methods for eligibility for a clinical trial with trastuzumab.59 However, clinical outcome data for these two groups are not yet available. We anticipate and recommend that such analyses will be forthcoming as correlative studies of the large prospective randomized clinical trials of trastuzumab.

It is also clear from the panel discussion and literature review that patients with equivocal HER2 test results constitute a poorly studied subgroup with uncertain association of test scores to benefit from HER2-directed therapy.60 The panel suggested that further studies of this patient group would be promoted by defining these test results as equivocal or borderline. The panel elected to use the term equivocal to avoid confusion with borderline positive and borderline negative terminology which is sometimes used in the interpretation of FISH assays. Equivocal results of a single test require additional action which should be specified in the initial report. Equivocal IHC samples must be confirmed by FISH analysis of the sample. Equivocal FISH samples are confirmed by counting additional cells or repeating the FISH test. If FISH remains equivocal after additional cells counted or assay repeated, confirmatory IHC is recommended so that HER2 protein expression is known for the sample with true equivocal gene amplification status.