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.

  • Adjuvant indications
  • Herceptin is indicated for adjuvant treatment of HER2-overexpressing node-positive or node-negative (ER/PR-negative or with one high-risk feature)† breast cancer:
  • As part of a treatment regimen containing doxorubicin, cyclophosphamide, and either paclitaxel or docetaxel
  • With docetaxel and carboplatin
  • As a single agent following multi-modality anthracycline-based therapy

† High-risk is defined as ER/PR positive with one of the following features: tumor size >2 cm, age <35 years, or tumor grade 2 or 3.

  • Metastatic indications
  • Herceptin is indicated:
  • In combination with paclitaxel for first-line treatment of HER2-overexpressing metastatic breast cancer
  • As a single agent for treatment of HER2-overexpressing breast cancer in patients who have received one or more chemotherapy regimens for metastatic disease

Boxed WARNINGS and Additional Important Safety Information

  • Cardiotoxicity
  • Herceptin administration can result in sub-clinical and clinical cardiac failure manifesting as congestive heart failure (CHF) and decreased left ventricular ejection fraction (LVEF).
    • The incidence and severity of left ventricular cardiac dysfunction was highest in patients who received Herceptin concurrently with anthracycline-containing chemotherapy regimens.
    • Discontinue Herceptin treatment in patients receiving adjuvant therapy for breast cancer and strongly consider discontinuation of Herceptin in patients with metastatic breast cancer who develop a clinically significant decrease in left ventricular function.
  • Herceptin can cause left ventricular cardiac dysfunction, arrhythmias, hypertension, disabling cardiac failure, cardiomyopathy, and cardiac death.
    • In one adjuvant clinical trial, cardiac ischemia or infarction occurred in the Herceptin-containing regimens.
    • Herceptin can also cause asymptomatic decline in LVEF.
  • Cardiac Monitoring
  • Candidates for treatment with Herceptin should undergo a thorough baseline cardiac assessment, including history, physical examination, and an assessment of LVEF by echocardiogram or MUGA scan.
    • Patients should undergo frequent monitoring for decreased left ventricular function during and after Herceptin treatment.
    • More frequent monitoring should be employed if Herceptin is withheld in patients who develop significant left ventricular cardiac dysfunction.
  • Infusion reactions and Pulmonary Toxicity
  • Serious infusion reactions and pulmonary toxicity have occurred; fatal infusion reactions have been reported.
    • In most cases, symptoms occurred during or within 24 hours of administration of Herceptin. Herceptin infusion should be interrupted for patients experiencing dyspnea or clinically significant hypotension.
    • Patients should be monitored until signs and symptoms completely resolve.
    • Discontinue Herceptin for infusion reactions manifesting as anaphylaxis, angioedema, interstitial pneumonitis, or acute respiratory distress
  • Infusion reactions consist of a symptom complex characterized by fever and chills, and on occasion include nausea, vomiting, pain (in some cases at tumor sites), headache, dizziness, dyspnea, hypotension, rash, and asthenia.
    • In postmarketing reports, serious and fatal infusion reactions have been reported. Discontinue Herceptin in all patients with severe or life-threatening infusion reactions.
  • Herceptin use can result in serious and fatal pulmonary toxicity, which includes dyspnea, interstitial pneumonitis, pulmonary infiltrates, pleural effusions, non-cardiogenic pulmonary edema, pulmonary insufficiency and hypoxia, acute respiratory distress syndrome, and pulmonary fibrosis.
    • Such events can occur as sequelae of infusion reactions.
    • Patients with symptomatic intrinsic lung disease or with extensive tumor involvement of the lungs, resulting in dyspnea at rest, appear to have more severe toxicity.
  • Neutropenia
  • Exacerbation of chemotherapy-induced neutropenia has also occurred
    • In controlled clinical trials, severe neutropenia and febrile neutropenia occurred more frequently in metastatic breast cancer patients receiving Herceptin with myelosuppressive chemotherapy compared to chemotherapy alone.
    • The incidence of septic death was not significantly increased.
  • Pregnancy Category D
  • Herceptin can cause fetal harm when administered to a pregnant woman.
  • Post-marketing reports suggest that Herceptin use during pregnancy increases the risk of oligohydramnios during the second and third trimester.
  • Most Common Adverse Events
  • The most common adverse reactions associated with Herceptin use were fever, nausea, vomiting, infusion reactions, diarrhea, infections, increased cough, headache, fatigue, dyspnea, rash, neutropenia, anemia, and myalgia.

Please see the Herceptin full Prescribing Information including Boxed WARNINGS and additional important safety information.

  • 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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