UPDATE ON HER-2 TESTING

PathmanathanAdjunct Professor

Monash Medical SchoolManipal Medical School

Senior Consultant Pathologist, SDMC

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

• During the last 20 years, extended knowledge of breast cancer biology, has put some light in our understanding of breast cancer

• High throughput technologies such as expression profiling recently introduce new classifications of IBC

• Some cellular targets, such as HER2, have been identified and drugs have been designed to specifically fight them

HER2 + breast cancer represents a heterogeneous disease targeted by specific drugs and is a hallmark of

strategic treatment

HER receptors

HER2

HER1 HER3 HER4

Cell membrane

5

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

Trastuzumab: targeting HER2

Attacks HER2-positive tumours via 5 distinct mechanisms of action1. Activation of antibody-dependent cellular

cytotoxicity (ADCC)2. Prevention of the formation of p95HER2,

a truncated and very active form of HER23. Degradation of HER2 dimers4. Inhibition of cell proliferation by preventing

HER2-activated intracellular signalling5. Inhibition of HER2-regulated angiogenesis

3

Herceptin is 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

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

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

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

HER2TESTING

HER2 PROTEIN OVEREXPRESSION IHC

HER2 GENE AMPLIFICATION

FISH OR CISH

Anti her2 treatment

Breast tumor

FISH or CISH

+ –

IHC

2+ 3+

0 ou 1+

ASCO, CAP Guidelines 2006

Anti her2 treatmentFISH or CISH

– +

Anti her2 treatment

Aneuploidy or ambiguous case

Tester by IHC

2+ or -3+

Anti her2 treatment

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

Abnormal 2+ Abnormal 3+Normal 0 Normal 1+

ErbB-2/HER2 in Breast Cancer

Normal Normal Abnormal lowamplification

Abnormal highamplification

METHOD ADVANTAGES DISADVANTAGES

IHC Ab to detect HER2 protein expression on cell membrane

Easy, quick, cheap, most labs can do itMorphology preservedLong storage

Affected by many variablesScoring subjective

FISH Fluorescent DNA probe to detect HER2 gene amplification

Robust , less affected by pre-analytical factorsObjective scoring systemGold standard

CostlySignals fade with timePathologists need special trainingDifficult to assess areas if invasion

CISH Digoxigenin labeled DNA probe to detect HER2 gene amplication

Less affected by pre-analytical factorsQuickUses standard light microscopyStable stainingSimultaneously assess cell morphology

New technologyDifficult to interpretCEP17 not assessed simultaneously

SISH Dinitrophenol-labeled DNA probe to detect HER2 gene amplication

AutomatedSimilar to CISH

As for CISH

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

Well documented Represents subclonal diversity Incidence varies from 5 – 30 % Increases subjectivity of HER-2

interpretation by pathologist

Intratumoral heterogeneity

Definition◦ > 5 % but < 50 % of infiltrating tumour cells have

ratio higher than 2.2

HER-2 genetic heterogeneity (GH)

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

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

Polysomy, PathVysion™ kit

The >2 green signals (CEP17) and 2 orange signals (HER2 genes) per nucleus indicate polysomy

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 Clin Oncol 26: 30, pp 4869- 4874

Tubbs RR, Hicks DG, Cook J, et al. Diagn Mol Pathol. 2007;16:207– 210.

Lewis JT, Ketterling RP, Halling KC, et al. Am J Clin Pathol. 2005;124:273–281.

Fujii H, Marsh C, Cairns P, Sidransky D, Gabrielson E. Cancer Res. 1996;56:1493–1497.

Miller DV, Jenkins RB, Lingle WL, et al. 2004 ASCO Annual Meeting Proceedings. J Clin Oncol. 2004;22(14S):568.

Glockner S, Buurman H, Kleeberger W, Lehmann U, Kreipe H. Lab Invest. 2002;82:1419–1426

References