HER2 testing in breast cancer

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HER2 testingin breast cancer
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The HER2 testing algorithm
Patient tumour sample

• 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

Retest with ISH


Hanna Kwok 2006
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Summary of HER2 testing methods
CISH, chromogenic ISH DNP, dinitrophenol IHC,
immunohistochemistry ISH, in situ hybridisation
FISH, fluorescence ISH SISH, silver-enhanced ISH
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Principles of IHC
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Interpretation of IHC results
IHC 1 Barely perceptible membrane staining in
10 of tumour cells cells only stained in part
of membrane
IHC 0 No staining or membranestaining in lt10
of tumour cells
IHC 2 Weak / moderate complete membrane
staining in 10 of tumour cells
IHC 3 Strong complete membrane staining in
10 of tumour cells
Images courtesy of Dako Wolff et al, 2007
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Principles of ISH
HER2 gene
Probe
Prepare target DNA
Denature target DNA
Probe binds to denatured DNA and emits a signal
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ISH HER2 gene-amplification detection mechanisms
FISH-positivea
Fluorescent DNA probe
Digoxigenin-labelled DNA probe with chromogenic
detection (DAB signal)
CISH-positiveb
Dinitrophenol-labelled DNA probe with chromogenic
detection (silver signal)
SISH-positivec
Dinitrophenol-labelled DNA and centromeric probes
with chromogenic detection (silver and Alk-Phos
Red)
Dual SISH / Red Amplified HER2c
aImage courtesy of W Hanna bimage from
Invitrogen cimage from Ventana
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FISH interpretation
FISH-negative (no amplification) Ratio of HER2
gene (orange) to CEP17 (green) signals is lt2.0
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_ ph
armdx_interpretation_guide_breast_cancer-10477.pdf
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CISH interpretation
CISH-negative(no amplification 15 single dots
in gt50 of tumour cells)
CISH-positive(6 dots, clusters or a mixture of
these in gt50 of tumour cells)
Images from Invitrogen SPoT-Light HER2 CISH kit
Interpretation guide available at
http//tools.invitrogen.com/content/sfs/manuals/PI
840150A 20HER220CISH20Kit20Appendix20A20Rev
200608.pdf
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SISH interpretation
SISH-negative
SISH-positive
Images from Ventana INFORM HER2 DNA
Interpretation guide available at
http//www.her2sish.com/ECO_SISH_Interp_Guide_N616
B_F_w_cover.pdf
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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
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Sources of variation inHER2 testing
Time to slicing and fixation
Scoring system
Method of tissue processing
Reportingelements
Time of fixation
Interpretation criteria
Type of fixation
Post-analytic
Pre-analytic
Use ofimageanalysis
HER2-testing variation
Assayvalidation
Equipment calibration
Assayconditions
Analytic
Laboratoryprocedures
Control materials
Staff competence
Test reagents
Type of antigen retrieval
Adapted from Wolff et al 2007
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QA / QC procedures to minimise variation
Internal QCExternal QA
NordiQC programme
Canadian QAprogramme
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
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Summary

• Accurate HER2 testing identifies patients with
  breast cancer who are most likely to benefit from
  Herceptin treatment13
• 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
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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 19481487.
• 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 2729622969.
• 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 618994.
• 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 25118145.