Comparison of Quantitative Immunofluorescence With Conventional Methods for HER2/neu Testing With Respect to Response to Trastuzumab Therapy in Metastatic Breast Cancer

Archives of Pathology & Laboratory Medicine, Oct 2008 by Giltnane, Jennifer M, Molinaro, Annette, Cheng, Huan, Robinson, Andrew, Turbin, Dmitry, Gelmon, Karen, Huntsman, David, Rimm, David L

Context.-Selection for trastuzumab therapy depends on a companion diagnostic assessment of HER2 by either immunohistochemistry (IHC) for protein overexpression or fluorescence in situ hybridization (FISH) to detect gene amplification. Although many studies have compared IHC to FISH, few have compared the tests to the true gold standard, tumor response.

Objective.-To compare HER2 testing by FISH and IHC along with a third immunofluorescence-based assay (automated quantitative analysis-tissue microarray [AQUATMA]) and to assess the value of each test for prediction of response to trastuzumab.

Design.-Immunohistochemistry and FISH assays were done on both whole slides (IHC-WS and FISH-WS) and on TMAs (IHC-TMA and FISH-TMA). AQUA was only done on TMAs (AQUA-TMA). Response was assessed according to modified Response Evaluation Criteria in Solid Tumors.

Results.-AQUA-TMA scores showed a significant linear relationship to both the FISH signal ratio and IHC scores on whole sections and TMAs. Assay assessment by outcome showed no association between response and FISH-WS ratio (P = .96), FISH-TMA (P = .55), IHC-WS (P = .75), or IHC-TMA (P = .06), but a significant relationship between AQUA score and categoric response was observed (P = .01). Assessed as a function of outcome using models of logistic regression, both AQUA-TMA and IHC-TMA were equally significant (P = .01). FISH-WS was the most sensitive assay, with a significantly higher true-positive fraction than all other tests except AQUA-TMA, although it was the least specific. IHC-TMA was the most specific assay. The lowest misclassification rate was achieved using AQUA-TMA (0.30).

Conclusions.-Both AQUA-TMA and IHC-TMA were substantially more predictive than the FISH or IHC-WS tests. Although these results are derived from a small retrospective series, they suggest that accurate measurement of protein expression and unbiased selection of tissue for measurement may be key factors in prediction of response.

(Arch Pathol Lab Med. 2008;132:1635-1647)

Selection for trastuzumab therapy depends on a companion diagnostic assessment, the classification of a patient's breast cancer as HER2/neu-positive by standard assays of immunohistochemistry (IHC) for protein overexpression or fluorescence in situ hybridization (FISH) to detect gene amplification.1 Although the reproducibility and accuracy of these methods have improved over time, controversy continues over which test is best. Both IHC and FISH suffer from errors of reproducibility across testing laboratories, although data from several studies of small sample sizes suggest that FISH yields more consistent results for the same samples across multiple test centers. 2-4 However, FISH costs more than four times the cost of IHC,5 and it requires more expertise to perform and measure results.6 The value of both tests is further complicated by the site at which the tests are performed. A report of local and central laboratory testing of more than 2500 patients from the N9831 clinical trial of adjuvant trastuzumab treatment showed 88.1% concordance for FISH and 81.6% for IHC,7 suggesting each test may be subject to between 12% and 20% error. This observation has recently been further clouded by a report from Paik and colleagues8 that showed response to therapy in patients whose central tests were negative as equivalent to response to therapy for patients whose central tests were positive.

A number of studies have been published comparing IHC to FISH for reproducibility against biochemical assays (radioimmunohistochemisty, Southern blotting, or Western blotting) of HER2 expression, and most have concluded that FISH-based testing is more accurate than standard IHC.9,10 However, only 2 studies have compared these methods using response to trastuzumab as a metric of "true-positive" HER2/neu breast cancer, and in both of these, the impact of the FISH assay was tested in a patient population selected by only IHC.11,12 Many oncologists now rely on the results of both assays before making the clinical decision to start trastuzumab.

A reliable and accurate assay to predict response is critical now that indications for trastuzumab have moved to the adjuvant setting. Three major recent clinical trials of trastuzumab for postsurgical treatment of HER2+ breast cancer have shown its effectiveness.13,14 They showed an average of around 50% reduction in risk of relapse during trastuzumab therapy.1 However, this result is expensive, as costs to administer trastuzumab to 1 breast cancer patient often exceed $100 000 a year, and the risk of treatment includes potentially fatal cardiotoxicity.1 When the low response rate is considered in that context, it is clear that improved pharmacodiagnostic assays are needed to improve the cost-benefit ratio of trastuzumab therapy.

Although there have been a range of efforts to find a better test with a variety of methods, immunofluorescence- based assays have not yet been evaluated. Here, we describe the assessment of an immunofluorescence-based method for in situ assessment of protein concentration called AQUA (automated quantitative analysis; HistoRx, New Haven, Conn). AQUA is an automated immunofluorescence technique for quantifying compartmentalized protein expression patterns9 that produces a continuous scale of fluorescent intensities, as opposed to the traditional 4-point ordinal scale associated with subjective, manual scoring approaches to IHC. This assay was performed on a series of 152 cases treated with trastuzumab in the metastatic setting collected by a group at the British Columbia Cancer Agency (BCCA). In each case, HER2/ neu copy number was measured by FISH, and protein expression was measured by IHC. These patients were previously selected for therapy based on IHC and/or FISH using standard assay protocols, and so all assays were repeated for the current study in tissue microarray (TMA) format. The purpose of this retrospective study was to estimate the diagnostic accuracy of AQUA and compare its diagnostic accuracy to FISH and IHC using Response Evaluation Criteria in Solid Tumors (RECIST)-based patient response15 to trastuzumab as the criterion standard.