HER2 testing
in breast cancer
The HER2 testing algorithm
Patient tumour sample
ISH
IHC
0
1+
2+
Retest
with ISH
–
3+
Eligible for
Herceptin®
(trastuzumab)
–
+
Eligible for
Herceptin®
(trastuzumab)
+
Eligible for
Herceptin®
(trastuzumab)
• If primary ISH testing is used, patients whose tumours overexpress
HER2 (i.e. IHC 3+) may not always be identified
• ISH detection mechanism can be fluorescent, chromogenic or silver
Hanna & Kwok 2006
Summary of HER2 testing
methods
IHC
Uses antibodies to detect HER2 protein
expression
FISH
Uses fluorescent DNA probe to detect
HER2 gene amplification
CISH
Uses digoxigenin-labelled DNA probe with
chromogenic detection (DAB signal) to detect
HER2 gene amplification
SISH
Uses DNP-labelled DNA probe with chromogenic
detection (silver signal) to detect HER2 gene
amplification
Dual
SISH
Uses DNP-labelled DNA and centromeric
probes with chromogenic detection (silver for
HER2 and Alk-Phos Red for chromosome 17)
CISH, chromogenic ISH; DNP, dinitrophenol; IHC, immunohistochemistry; ISH, in
situ hybridisation; FISH, fluorescence ISH; SISH, silver-enhanced ISH
Principles of IHC
Primary antibody binds to
HER2 receptor
Secondary antibody binds to
primary antibody and activates
the visualisation substrate
Interpretation of IHC results
IHC 0
No staining or membrane
staining in <10% of tumour cells
IHC 2+
Weak / moderate complete membrane
staining in ≥10% of tumour cells
IHC 1+
Barely perceptible membrane staining
in ≥10% of tumour cells;
cells only stained in part of membrane
IHC 3+
Strong complete membrane staining
in ≥10% of tumour cells
Images courtesy of Dako; Wolff et al, 2007
Principles of ISH
HER2 gene
Probe
Prepare
target DNA
Denature
target DNA
Probe binds to denatured
DNA and emits a signal
ISH: HER2 gene-amplification
detection mechanisms
FISH-positivea
Fluorescent DNA probe
CISH-positiveb
Digoxigenin-labelled DNA
probe with chromogenic
detection (DAB signal)
SISH-positivec
Dinitrophenol-labelled DNA
probe with chromogenic
detection (silver signal)
Dual SISH / Red
Amplified HER2c
Dinitrophenol-labelled DNA
and centromeric probes with
chromogenic detection
(silver and Alk-Phos Red)
bimage
aImage
courtesy of W Hanna;
from Invitrogen; cimage from Ventana
FISH interpretation
FISH-negative (no amplification)
Ratio of HER2 gene (orange) to CEP17 (green) signals
is <2.0*
FISH-positive (amplified)
Ratio of orange to green signals is ≥2.0*
Images courtesy of W Hanna using PathVysion
* Interpretation guide available at: http://www.dako.com/uk/00198_21apr10_her2_fish_
pharmdx_interpretation_guide_breast_cancer-10477.pdf
CISH interpretation
CISH-negative
(no amplification: 1–5 single
dots in >50% of tumour cells)*
CISH-positive
(≥6 dots, clusters or a mixture of
these in >50% of tumour cells)*
Images from Invitrogen SPoT-Light® HER2 CISH™ kit
* Interpretation guide available at: http://tools.invitrogen.com/content/sfs/manuals/PI840150A%
20HER2%20CISH%20Kit%20Appendix%20A%20Rev%200608.pdf
SISH interpretation
SISH-negative
SISH-positive
Images from Ventana INFORM™ HER2 DNA
Interpretation guide available at:
http://www.her2sish.com/ECO_SISH_Interp_Guide_N616B_F_w_cover.pdf
Dual SISH interpretation
Dual SISH non-amplified for
HER2 gene or centromere
Dual SISH amplified for HER2 gene
and non-amplified for centromere
Images from Ventana;
Interpretation guide available at: http://www.nordiqc.org/ERFA/VENTANA_Guide1010.pdf
Sources of variation in
HER2 testing
Scoring
system
Time to slicing
and fixation
Method of tissue
processing
Reporting
elements
Interpretation
criteria
Time of
fixation
Post-analytic
Use of
image
analysis
Pre-analytic
HER2-testing
variation
Assay
validation
Equipment
calibration
Assay
conditions
Control
materials
Type of
fixation
Analytic
Test
reagents
Type of antigen
retrieval
Laboratory
procedures
Staff
competence
Adapted from Wolff et al 2007
QA / QC procedures to minimise
variation
NordiQC
programme
Internal QC
External QA
Canadian QA
programme
UK NEQAS ICC and
ISH scheme
• Accredited laboratories follow guidelines developed
by these schemes to ensure standardisation of
HER2-testing procedures
NordiQC, Nordic Immunohistochemical Quality Control; ICC, immunocytochemistry
Summary
• Accurate HER2 testing identifies patients with
breast cancer who are most likely to benefit from
Herceptin treatment1–3
• Validated HER2 testing methods currently
routinely used are
• IHC, FISH, normal and dual-colour CISH and SISH4
• Accuracy of all methods can be affected by
tissue preparation and operator expertise3
• Internal QC and external QA are essential to
maintain HER2-testing accuracy and consistency3
1. Dowsett et al 2009;
2. Hofmann et al 2008;
3. Wolff et al 2007;
4. Hanna & Kwok 2006
References
• Hanna WM and Kwok K. Chromogenic in-situ hybridization: a viable
alternative to fluorescence in-situ hybridization in the HER2 testing
algorithm. Mod Pathol 2006; 19:481–487.
• Dowsett M, et al. Disease-Free Survival According to Degree of
HER2 Amplification for Patients Treated With Adjuvant
Chemotherapy With or Without 1 Year of Trastuzumab: The HERA
Trial. J Clin Oncol 2009; 27:2962–2969.
• Hofmann M, et al. Central HER2 IHC and FISH analysis in a
trastuzumab (Herceptin) phase II monotherapy study: assessment
of test sensitivity and impact of chromosome 17 polysomy.
J Clin Pathol 2008; 61:89–94.
• Wolff AC, et al. American Society of Clinical Oncology/College of
American Pathologists Guideline Recommendations for Human
Epidermal Growth Factor Receptor 2 Testing in Breast Cancer.
J Clin Oncol 2007; 25:118–145.