How Results are Interpreted

Interpretation of IHC relies on a qualitative scoring system on a scale of 0 to 3+. A tumor biopsy is scored as 0 (negative), 1+ (negative), 2+ (borderline), or 3+ (positive) on an IHC test based on the reviewer's interpretation of staining intensity and completeness of membrane staining. 5 With FISH testing, the results are quantitative instead of qualitative; tumors are interpreted as HER2 "negative" or "positive" by enumerating the HER2/neu gene copy number. 3

Qualitative interpretation of IHC restults - slide images

What does it mean if a patient is weakly positive by IHC?

Patients whose tumors are weakly positive by IHC have weak or moderate intensity staining (see IHC 2+ slide below).5

IHC 2+ slide - weakly positive

This borderline group is the most difficult to score consistently by IHC and has a high rate of interobserver variability among pathologists.9 Analysis by FISH may be useful for accurate determination of HER2 status in this group. NCCN guidelines recommend confirming an IHC result of 2+ with FISH. 4

FISH* results are presented as a quantitative score of the level of gene amplification. FISH testing measures the HER2/neugene copy number against a standard internal chromosomal control (CEP 17). Results are expressed as a ratio of the number of HER2 gene copies (orange) per number of chromosome 17 copies (green). 3

Interpretation of FISH test - slide images

A normal ratio is less than 2 (FISH-). 3

A ratio greater than or equal to 2 HER2/neugene copies per chromosome 17 is gene-amplified (FISH+). 3

HER2 gene amplification and protein overexpression

HER2 gene amplification is a permanent genetic change. The excess copies of the HER2 gene result in the continuous overexpression of the HER2 protein on the cell surface. 6

*Vysis PathVysion®

If A Patient is IHC 2+ or 3+ and FISH-, are they HER2+?

Overexpression of the HER2 protein rarely occurs in the absence of gene amplification.10 FISH analysis reveals that some patients with apparent protein overexpression (IHC 2+ or 3+) do not have gene amplification (FISH-), suggesting that these patients may be "false positives." Approximately 2%-4% of patients who demonstrated HER2 protein overexpression by molecular techniques did not have gene amplification.10,11 In current laboratory testing, variability in pre-analytical tissue processing, reagent variability, antigen retrieval, and scoring may result in IHC false-positives.7,8

HER2 testing algorithms1,2

ASCO/CAP consensus guidelines for HER2 testing recommend that a final positive or negative HER2 result be achieved using the testing algorithms for IHC and FISH

HER2 testing algorithms

*Patients with strong complete membrane staining in more than 10% of tumor cells were eligible for the adjuvant trastuzumab trials. Users should refer to the package inserts of specific assay kits for information on the validation and performance of each assay.

  • References:
  • 1. Wolff AC, Hammond EH, Schwartz JN, et al. American Society of Clinical Oncology/College of American Pathologists Guideline Recommendations for Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer. JClin Oncol. 2007; 25: 118-145.
  • 2. Wolff AC, Hammond ME, Schwartz JN, et al. American Society of Clinical Oncology/College of American Pathologists Guideline Recommendations for Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer. Arch Pathol Lab Med.2007; 131: 18-43.
  • 3. PathVysion® HER-2 DNA Probe Kit Package Insert, Vysis, Inc.; November, 2006.
  • 4. NCCN® Practice Guidelines in Oncology - v.2.2005: Breast Cancer. National Comprehensive Cancer Network. Available at: http://www.nccn.org/professionals/physician_gls/PDF/breast.pdf. Accessed September 2, 2008.
  • 5. DAKO HercepTest® Information Web site. Welcome to HercepTest™ e-Learning. Available at: http://www.dako.com/prod_productrelatedinformation?url=support_herceptest_elearning.htm. Accessed November 3, 2008.
  • 6. Sliwkowski MX, Lofgren JA, Lewis GD, et al. Nonclinical studies addressing the mechanism of action of trastuzumab (Herceptin).Semin Oncol. 1999;26 (suppl 12):60-70.
  • 7. Paik S, Bryant J, Tan-Chiu E, et al. Real-world performance of HER2 testing -National Surgical Adjuvant Breast and Bowel Project experience. J Natl Cancer Inst. 2002;94:852-854.
  • 8. O'Leary TJ. Standardization of immunohistochemistry. Appl Immunohistochem Mol Morphol. 2001;9:3-8.
  • 9. Bose S, Mohammed M, Shintaku P, Rao PN. Her-2/neu gene amplification in low to moderately expressing breast cancers: possible role of chromosome 17/Her-2/neu polysomy. Breast J. 2001;7:337-344.
  • 10. Pauletti G, Godolphin W, Press MF, Slamon DJ. Detection and quantitation of HER-2/neu gene amplification in human breast cancer archival material using fluorescence in situ hybridization. Oncogene. 1996;13:63-72.
  • 11. Kallioniemi O-P, Kallioniemi A, Kurisu W, et al. ERBB2 amplification in breast cancer analyzed by fluorescence in situ hybridization. Proc Natl Acad Sci U S A. 1992;89:5321-5325


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