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 TESTINGPathmanathanAdjunct Professor MonashMedical SchoolManipalMedical SchoolSenior Consultant Pathologist, SDMC

2. Introduction For many years, breast cancer has been considered as a unique disease and treated as a unique disease based on clinical and pathological parameters

3. During the last 20 years, extended knowledge of breast cancer biology, has put some light in our understanding of breast cancerHigh throughput technologies such as expression profiling recently introduce new classifications of IBC

4. Some cellular targets, such as HER2, have been identified and drugs have been designed to specifically fight themHER2 + breast cancer represents a heterogeneous disease targeted by specific drugs and is a hallmark of strategic treatment

5. HER receptorsHER2HER1HER3HER4Cell membrane5

6. ErbB-2 HER2/neuDoes not need ligandsactivation 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 HER2Attacks HER2-positive tumours via 5 distinct mechanisms of actionActivation of antibody-dependent cellular cytotoxicity (ADCC)Prevention of the formation of p95HER2, a truncated and very active form of HER2Degradation of HER2 dimersInhibition of cell proliferation by preventing HER2-activated intracellular signallingInhibition of HER2-regulated angiogenesis3

8. Herceptinis an effective drugFor HER2 + positive patients (importance of the quality of testingIn the metastatic setting in combination with taxanes, other CT agents + / -antiaromatase (ER+)Herceptin actionPotentialization of cytotoxic drugs and hormonal treatmentHER2 targeting

9. HER-2 Positive state shortens survivalMedian survival from first diagnosis:HER2 positive  3 yearsHER2 normal  6 - 7 yearsSlamon DJ et al. Science 1987;235: 177-182

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

11. Herceptin® is indicated for HER2-positive breast cancerHER2 positivity is the criterion to select patients for Herceptin® therapystrong overexpression of the HER2 protein on the cell surfaceHER2 gene amplification

12. HER2TESTINGHER2 PROTEIN OVEREXPRESSIONIHCFISH OR CISHHER2 GENE AMPLIFICATION

13. 0 ou 1+FISH or CISHIHC+2+3+–FISH or CISH+–Aneuploidy or ambiguous caseTester by IHCAnti her2 treatmentAnti her2 treatment2+ or -3+Breast tumorAnti her2 treatmentAnti her2 treatmentASCO, CAP Guidelines 2006

14. Importance of accurate testingAccurate 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 laboratorystandardisation and regular validation of testingquality control measures and quality assuranceminimum number of cases (>150 per year)detailed documentation

15. Normal Normal Abnormal lowAbnormal highamplificationamplificationAbnormal 2+Abnormal 3+Normal 0Normal 1+ErbB-2/HER2 in Breast Cancer

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

19. Well documentedRepresents subclonal diversityIncidence varies from 5 – 30 %Increases subjectivity of HER-2 interpretation by pathologistIntratumoral heterogeneity

24. Definition> 5 % but < 50 % of infiltrating tumour cells have ratio higher than 2.2HER-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 GHIf 60 cells are counted, > 3 cells show a ratio of 2.2 , GH existsThese definitions based on published works, agreed by consensus

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

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

28. Polysomy 17 on its ownNot associated with HER2 overexpressionNot associated with increased levels of HER2 mRNA on RT-PCRNot associated with high grade tumoursNot associated with ER negativityNot associated with reduced disease free survivalMay not benefit from Herceptin therapy MORE STUDIES NEEDEDBempt et.al (2008) J ClinOncol 26: 30, pp 4869- 4874

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