Molecular Targeted Therapy for Cancer
Moderators: Dr. Robert Yoshiyuki Osamura and Dr. Allen M. Gown
Section 4 -
Her2/neu Testing in Breast Cancer
Chief, Anatomic Pathology
Sunnybrook Health Sciences Centre
Professor, Department of Laboratory Medicine and Pathobiology
University of Toronto
This presentation will cover the current status regarding Her2/neu testing in breast
cancer and highlight outstanding issues and future research opportunities.
The Her2/neu gene is one of four members in the Her family which encodes a 185 kilodalton
glycoprotein with tyrosine kinase activity. To date the ligand for activation of the receptor has not
been identified. Activation of Her2 is initiated by the formation of homodimers and heterodimers with
other members of the Her family with known ligands. This leads to a cascade of events that result in
tumor growth, cell motility and inhibition of apoptosis . Amplification of the Her2/neu gene and
over-expression of the protein occurs in about 20% of patients with breast cancer
Over-expression occurs as a result of amplification of the gene in 96.5% of cases.
Her2/neu as a Predictive and Prognostic Marker
Her2/neu over-expression and/or amplification have been established as a poor prognostic
marker in both node positive and node negative disease
To date testing at diagnosis is the
standard of practice as numerous studies have shown the importance of Her2/neu status in managing
patients with breast cancer. St. Gallen's guidelines in 2005  included the Her2 status of the tumor
as a factor in the risk classification of patients and subsequently in the choice of treatments.
Her2/neu over-expression is a predictor of response to therapy since clinical studies have shown that
these patients will benefit from anthracycline-based chemotherapy, possibly due to concomitant TopoII
Her2 over-expression may also identify patients who may not benefit from Tamoxifen
. More significantly is the use of the Her2/neu oncoprotein as a target for therapy. Trastuzumab
(Herceptin) is a humanized monoclonal antibody that targets the extracellular domain of the receptor.
Herceptin has shown efficacy in metastatic breast cancer in combination with chemotherapy  and as
a single agent . In 2005, the results of the HERA trial had shown improvement in disease free
survival and overall survival by the addition of Herceptin following chemotherapy in the adjuvant setting
. Studies demonstrated that Herceptin is only active against Her2 over-expressing tumors.
Review of testing methodologies and algorithm in clinical practice  showed that the most commonly
used methods are immunohistochemistry (IHC) to detect over-expression of the protein and in situ
hybridization (ISH) using either FISH or CISH to assess the presence of gene amplification. Both methods
have the advantage of being morphologically based using formalin fixed paraffin embedded tumor samples.
Because of the availability of IHC in most surgical pathology laboratories and lower cost, IHC is
commonly used to identify Her2 positive and negative tumors and equivocal results are retested using
ISH. This algorithm assumes excellent concordance between IHC and ISH test results. However, in
clinical practice there are studies that showed interlaboratory discrepancies which might be more
prevalent in low volume labs
Strict adherence to standardized methods, rigorous quality
controls and participation in external QA initiatives can produce significant improvement and a high
level of correlation between the IHC and ISH data
Available data does not demonstrate which
method, protein expression or in-situ hybridization methods, is superior in predicting therapeutic
response if the tumor's Her2/neu positive status is correctly made by either method. Thus, both
pathologists and oncologists are strongly advocating the use of standardized testing methodology and
participation in QA programs to properly identify the Her2/neu status of a patient's tumor. The aim is
to significantly reduce both false positive and negative results to <5%, i.e. achieve 95% concordance
rates between IHC and ISH test results.
The problem pathologists and oncologists are facing today in securing accurate and standardized
Her2/neu testing in breast cancer, is related to the following biological and methodology-related issues:
- Her2/neu oncoprotein over-expression and amplification of the gene is a continuous variable
and not a dichotomous status of positive and negative.
The cutoff points clinically used today are based on the clinical trial assay (CTA) data
which arbitrarily selected them .
In situ hybridization (ISH) methods are usually used in larger
labs by expert pathologists and although more complex and costly, appear to yield more consistent
- Intermediate ranges of over-expression of the Her2/neu protein may be seen in about 10-15%
of tumors and only one third of these are associated with amplification, mostly low level of
amplification of the gene. Borderline cases or low level amplification (1.8 – 2.2 scores) can also be
the source of discrepant results. However, these cases constitute about 3% of tumors.
- Polysomy of chromosome 17 may play a role in discrepant cases 
- Tumor heterogeneity and focal amplification of the Her2/neu gene can be seen in 1-2% of
cases and may contribute to discrepancy between IHC and ISH and interlaboratory results .
- Results of IHC testing could be adversely affected by pre-analytical variables such as
fixation. Also the subjective nature in interpretation of IHC, basically a qualitative rather than a
quantitative test, can result in interobserver variability.
- Lack of stringent use of standardized methodologies and non-compulsory
participation in external QA programs by testing labs.
Review of the Testing Algorithm and Pitfalls of IHC and ISH
The following points will be detailed in the presentation:
- Testing algorithms - definition of
positive, negative and borderline cases
- How to achieve accurate results using
- fixation (fixation type and duration)
, antigen retrieval, antibodies, controls,
- interpretation and scoring criteria 
- surrogate indicators for reflex testing with ISH
- Interpretation of ISH results
- FISH 
- CISH 
- Validation of CISH and FISH
- Quality assurance program
- models of QA programs in practice and results of some implemented QA programs will be discussed
Future Directions and Research Opportunities
- Clinical validation of cutoffs of positivity using response to Herceptin.
- Validation of the clinical benefits from Herceptin in borderline ranges of results, i.e.
IHC 2+ and cases with borderline FISH scores of 1.8 – 2.2.
- The role of new methodologies:
- assay of circulating serum Her2/neu and the correlation of the level of the extracellular domain
in the serum with disease status and treatment response 
- validation of image analysis in scoring IHC and ISH 
- new PCR platforms
- Development of sensitive and specific antibodies for IHC and probes for ISH
- The interaction of Her2/neu with other biological markers, for example,
cMYC , other members of the Her family and angiogenic factors.
- Mechanisms of Herceptin resistance, e.g. the role of C-terminal fragments 
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