Human epidermal growth factor 2 (HER2) amplification is observed in approximately 20% of all breast cancers and is associated with poor clinical outcome.1 Determination of HER2 status defines the eligibility for trastuzumab therapy and significantly improves the clinical outcome in a subset of patients who test positive for HER2.1-3 While assessment of HER2 status by fluorescent in situ hybridization (FISH) methodology remains a “gold standard” practice, the use of immunohistochemistry (IHC) has also become an accepted practice.4-6 A positive HER2 result is determined by a ratio of >2.2 of HER2 to CEP17 signals measured across 20 or more randomly selected cancer cells by FISH and a strong circumferential staining (IHC score 3+) based on IHC. The majority of laboratories employ IHC as a first-line test and reflex the weakly positive (IHC score 2+) cases to a FISH test.4-6
Figure 1. Dual in situ hybridization (DISH) demonstrating (1a) discrete signals containing a single copy of HER2 or CEP17; (1b, 1d) multiple discrete signals of HER2 or CEP17; (1c) overlapping signals (clusters) of HER2.
Introducing a third test
Both FISH and IHC tests have been approved by the U.S. Food and Drug Administration (FDA) for clinical testing of HER2 status.7,8 Recently the FDA also approved a dual in situ hybridization (DISH) test for the determination of HER2 status.9 The DISH methodology was also recently approved by ASCO/CAP as one of the three accepted tests for its determination. Though both in situ methods (FISH and DISH) are performed on paraffin-embedded tissue, DISH utilizes light microscopy in assessment of HER2/CEP17 signals.7
Specifically, the DISH assay is designed to determine HER2 gene status by enumeration of the ratio of the HER2 gene to chromosome 17. The HER2 and chromosome 17 probes are detected by using two-color chromogenic in situ hybridization (ISH) in formalin-fixed, paraffin-embedded breast cancer tissue. One of the principal drawbacks of standard FISH tests is the absence of visualization of tumor tissue; this is overcome by the DISH test, which performs visualization with light microscopy. Additionally, the specimen result can be archived and retrieved indefinitely.
There are a few considerations to keep in mind with DISH methodology. Reflex testing to IHC or FISH is recommended in the presence of heterogeneity of signals, staining issues, or clusters. Also, caution should be used when working with decalcified specimens, as DISH fails in the majority of these cases.
Comparing the data: FISH, IHC, and DISH
Currently, the literature on the specificity of DISH testing is still emerging. Comparative data with other test methods, including FISH and IHC, have been published.8, 10-13 However, because the FDA only recently approved the first DISH test, data on the performance of DISH in pathology laboratories as a routine test have not been widely published. We recently reported the results and performance of the referenced DISH assay in our validation cases as well as our experience of incorporating the test in routine practice.
From January 2013 to August 2014, the DISH test was performed on 490 breast cancer tissues (including 30 cases for initial validation). The specimens included core biopsies, excision specimens, as well as metastatic specimens. Five cases were eliminated; 11% (49/452) were amplified; 2% (7/452) showed borderline amplification; 87% (396/452) were non-amplified (NA). Among the NA cases, 2% (6/396) showed aneusomy. In a subset of cases, the comparison of DISH to FISH ratio showed a high concordance rate (p value = 0.2451), though DISH showed slightly lower HER2 copy numbers. Technical difficulties were encountered in 3% (13/452) of cases, and these were repeated and reflexed to an alternate test (FISH/IHC).
Although the established methodologies of FISH and IHC are preferred by many labs, difficulties can arise with interpretation, as tissue architecture appears to be obscured by FISH methodology, potentially leading to some misinterpretation. On the other hand, the DISH methodology has the advantage of assessment of morphology and gene copy numbers on the same glass slide. The benefits of the DISH methodology include the ability to perform the test on archival paraffin embedded tissue and the use of a conventional lightmicroscope to interpret the results.
The current ASCO/CAP guidelines for FISH assessment of HER2 status can be extended to interpretation of DISH as well. Using the CAP/ASCO guidelines, the concordance rate of DISH versus FISH has been shown to be ~ 98%. We have also observed that DISH underscores the HER2 copy numbers in some cases as reported in other studies.10 There were no significant discrepancies in our cases, although seven (2%) of the cases demonstrated failure. The most common reasons for the failure of DISH in our cases were related to decalcification and technical issues. None of our cases showed co-amplification with HER2/CEP-17.
In the metastatic setting, at this time we do not recommend performing DISH on bony samples after decalcification. We also recommend that the core sample be thoroughly examined for heterogeneity and be reflexed to an alternate test.
Providing support for test interpretation
Evidence to date indicates that DISH is a valid test and comparable to FISH or IHC. Based on our experience, we have developed an algorithmic guide and have enumerated several helpful hints for practicing pathologists, to support them in the routine interpretation of a DISH test. There are undeniable advantages to utilizing the DISH method, though minimal disparities exist. We believe that identifying these disparities and applying both our algorithm and strict adherence to clinical practice guidelines help ensure successful implementation in routine practice.
- Slamon DJ, Clark GM, Wong SG, et al. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science. 1987;235:177-182.
- Slamon DJ, Eiermann W, Robert n et al. Adjuvant Trastuzumab in HER2 positive breast cancer. NEJM. 2011;365:1273-1283.
- Baselga J, Swain SM; CLEOPATRA Study Group. Pertuzumab plus trastuzumab plus docetaxel for metastatic breast cancer. NEJM. 2012 Jan 12; 366(2): 109-119.
- Wolff AC, Hammond ME, Hicks DG, Dowsett M et al. American Society of Clinical Oncology; College of American Pathologists. 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. Clin Oncol. 2013;Nov 1; 31(31):3997-4013.
- Wolff AC, Hammond ME et al. American Society of Clinical Oncology; College of American Pathologists. 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. Clin Oncol. 2007;131:18-43.
- Owens MA, Horten BC, DeSilva MM. HER 2 amplification ratios by flouroscence in situ hybridization and correlation with immunohistochemistry in a cohort of 6556 breast cancer tissues. Clin Breast Cancer. 2004;5-63-69.
- Grogan T, McElhinny A, Loftin I et al. Interpretation guide Ventana INFORM HER 2 Dual ISH. DNA Probe Cocktail Assay.
- Lim SJ, Cantillep A, Carpenter PM. Validation and workflow optimization of human epidermal growth factor receptor 2 testing using INFORM HER2 dual-color in situ hybridization. Hum Pathol. 2013;44(11): 2590-2596.
- INFORM HER2 Dual ISH DNA Probe Cocktail, Roche/Ventana.
- Mansfield AS, Sukov WR, Eckel-Passow JE, Sakai Y, Walsh FJ, Lonzo M, Wiktor AE, Dogan A, Jenkins RB. Comparison of fluorescence in situ hybridization (FISH) and dual-ISH (DISH) in the determination of HER2 status in breast cancer. Am J Clin Pathol. 2013;139(2):144-150.
- Gao FF, Dabbs DJ, Cooper KL, Bhargava R. Bright-field HER2 dual in situ hybridization (DISH) assay vs fluorescence in situ hybridization (FISH): focused study of immunohistochemical 2+ cases. Am J Clin Pathol. 2014;141(1):102-110.
- Brügmann A, Lelkaitis G, Nielsen S, Jensen KG, Jensen V.Testing HER2 in breast cancer: a comparative study on BRISH, FISH, and IHC.Appl Immunohistochem Mol Morphol. 2011;19(3):203-211.
- Hartman AK, Gorman BK, Chakraborty S, Mody DR, Schwartz MR. Determination of HER2/neu Status: A Pilot Study Comparing HER2/neu Dual In Situ Hybridization DNA Probe Cocktail Assay Performed on Cell Blocks to Immunohistochemisty and Fluorescence In Situ Hybridization Performed on Histologic Specimens. Arch Pathol Lab Med. 2014;138(4):553-558.
- Chivukula M, Metkus A, Brown J,Borofsky H,Cuff J, Bulusu G,Duncan K. Are we ready for implementation of Bright field HER2 In situ Hybridization (DISH) Assay in routine HER2 testing. USCAP Annual meeting, 2015 Modern Pathology. Vol 28, Suppl 2, March 2015, Abstract 143.