This document discusses HER-2 testing in breast cancer and updates guidelines. It notes that HER-2 positive breast cancer is a heterogeneous disease that can be targeted by specific drugs like Herceptin. Accurate HER-2 testing is important to identify patients who will benefit from Herceptin therapy, as false results could deny patients effective treatment or cause patients to receive unnecessary treatment. The document reviews HER-2 testing methods and provides guidelines on confirming HER-2 status in ambiguous cases.

1. UPDATE ON HER-2 TESTING Pathmanathan Adjunct Professor MonashMedical School ManipalMedical School Senior Consultant Pathologist, SDMC

5. HER receptors HER2 HER1 HER3 HER4 Cell membrane 5

6. ErbB-2 HER2/neu Does not need ligands activation occurs through heterodimerization with another ErbB family member or homidimerization when HER2 is overexpressed. ErbB-2 is the preferred dimerization partner of the other 3 ErbB family members. Heterodimers including ErbB-2 exhibit increased stability and prolonged activation HER2 is a poor prognosis factor in breast cancer

7. Trastuzumab: targeting HER2 Attacks HER2-positive tumours via 5 distinct mechanisms of action Activation of antibody-dependent cellular cytotoxicity (ADCC) Prevention of the formation of p95HER2, a truncated and very active form of HER2 Degradation of HER2 dimers Inhibition of cell proliferation by preventing HER2-activated intracellular signalling Inhibition of HER2-regulated angiogenesis 3

8. Herceptinis an effective drug For HER2 + positive patients (importance of the quality of testing In the metastatic setting in combination with taxanes, other CT agents + / -antiaromatase (ER+) Herceptin action Potentialization of cytotoxic drugs and hormonal treatment HER2 targeting

9. HER-2 Positive state shortens survival Median survival from first diagnosis: HER2 positive  3 years HER2 normal  6 - 7 years Slamon DJ et al. Science 1987;235: 177-182

10. HER2+ is a heterogeneous disease Up to 50% of human epidermal growth factor receptor 2 (HER2)-positive breast cancers are also oestrogen receptor (ER) positive Evidence of crosstalk between HER2 and ER signalling pathways Simultaneous targeting of both pathways may improve outcomes over monotherapy Vogel et al 2001;Penault-Llorca et al 2002; Piccart-Gebhart et al 2005

11. Herceptin® is indicated for HER2-positive breast cancer HER2 positivity is the criterion to select patients for Herceptin® therapy strong overexpression of the HER2 protein on the cell surface HER2 gene amplification

12. HER2 TESTING HER2 PROTEIN OVEREXPRESSION IHC FISH OR CISH HER2 GENE AMPLIFICATION

13. 0 ou 1+ FISH or CISH IHC + 2+ 3+ – FISH or CISH + – Aneuploidy or ambiguous case Tester by IHC Anti her2 treatment Anti her2 treatment 2+ or - 3+ Breast tumor Anti her2 treatment Anti her2 treatment ASCO, CAP Guidelines 2006

14. Importance of accurate testing Accurate testing is essential to identify those patients who will benefit from Herceptin® false-negative assessment:denies patients life-extending treatment false-positive assessment: patients will not benefit from Herceptin® Important requirements for the pathology laboratory standardisation and regular validation of testing quality control measures and quality assurance minimum number of cases (>150 per year) detailed documentation

15. Normal Normal Abnormal low Abnormal high amplification amplification Abnormal 2+ Abnormal 3+ Normal 0 Normal 1+ ErbB-2/HER2 in Breast Cancer

18. Published in 2007 Problem of tumour heterogeneity apparent at that time Group consensus meeting in 2008 to discuss this problem Vetted through CAP / American College of Medical Cytogenetics Resource Committee ASCO / CAP guidelines

19. Well documented Represents subclonal diversity Incidence varies from 5 – 30 % Increases subjectivity of HER-2 interpretation by pathologist Intratumoral heterogeneity

24. Definition > 5 % but < 50 % of infiltrating tumour cells have ratio higher than 2.2 HER-2 genetic heterogeneity (GH)

25. If 20 cells are counted and at least one cell is identified with a HER2/ CEP17 ratio of > 2.2, the specimen has GH If 60 cells are counted, > 3 cells show a ratio of 2.2 , GH exists These definitions based on published works, agreed by consensus

26. Polyploidy 17 In about 19.5 % of cases tested with FISH which show an equivocal result by absolute copy number About 1.3 % of patients showing equivocal result by HER2/ CEP17 ratio Polysomy 17 in Breast Cancer

27. Polysomy, PathVysion™ kit The >2 green signals (CEP17) and 2 orange signals (HER2 genes) per nucleus indicate polysomy

28. Polysomy 17 on its own Not associated with HER2 overexpression Not associated with increased levels of HER2 mRNA on RT-PCR Not associated with high grade tumours Not associated with ER negativity Not associated with reduced disease free survival May not benefit from Herceptin therapy MORE STUDIES NEEDED Bempt et.al (2008) J ClinOncol 26: 30, pp 4869- 4874

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