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MEDICINE

case is just one example of why incalculable numbers of hours and by IHC and FISH using comparative genomic hybridization, also
healthcare dollars are continually spent on HER2 testing methods called chromosomal microarrays (CMAs).6-10 CMAs provide a
to “resolve” equivocal HER2 breast cancer into clearly actionable DNA-based approach to chromosome analysis with the capability
HER2-positive or HER2-negative categories. The collective effort of producing a high-resolution view of the HER2 gene on chro-
to create a binary, two-tier framework around HER2 status in breast mosome 17. The chromosome “ratio plot” allows simulated visual-
cancer is understandable given that oncology clinical practice guide- ization of the p arm, q arm, pericentromeric region, and HER2
lines have clearly actionable treatment directives only for unequiv- gene within the 17q12 amplicon. These high-resolution CMA im-
ocally positive or negative HER2 results. High-risk tumors with a ages of HER2 gene status on chromosome 17 in multiple types of
combination of low HER2 protein expression and nonamplified breast cancer have revealed the following interesting findings:
HER2 gene copy number fit neither of these categories. Yet tumors • CMA studies provide confirmation that ISH is a reliable method
with low HER2 protein expression represent a significant subset of for determining HER2 gene copy number independent of a ratio
breast cancer cases. Could these tumors be trying to announce their as long as formalin-fixed paraffin-embedded tissue handling is
biological reality by consistently showing 1 to 2+ protein and <6 within CAP/ASCO guidelines for formalin fixation times.
copy numbers after repeated rounds of testing? • CMAs have revealed that tumors with gains of entire copies of
Since the term “equivocal HER2” was introduced as part of the chromosome 17 (polysomy 17) occur in <10% of breast cancers
first College of American Pathologists/American Society of Clinical even though the HER2/cep 17 ratio used in dual- probe FISH
Oncology (CAP/ASCO) guidelines published in 2007, the term has is intended to correct for this biological phenomenon (cep 17 is
become synonymous with a third category of breast cancer.3 Fol- another area of chromosome 17 used as a denominator in ra-
lowing implementation of updated CAP/ASCO guidelines in 2013, tios). Instead of polysomy, many tumors contain segmental gains
the number of breast cancer cases falling into the equivocal category on chromosome 17, particularly on the long arm.11,12 A stan-
has increased, along with the number of additional tests that must dard definition of HER2 “amplification” by genomic copy num-
be performed to resolve equivocal results.4, 5 Within this equivocal ber analysis (including CMA) has not yet been established.
category, clinicians often end up with a collection of results from • CMA allows visualization of relative gains or losses of chromo-
repeated and alternative testing methods used to attempt to resolve some 17 regions used as the ratio denominator (cep17, TP53,
the equivocal HER2 status of the tumor. These test results often SMSCR, RARA), causing the ratio to skew towards false negative
disagree as to whether the tumor is HER2-positive, HER2-negative, or false positive.
or something in between. The discordant test results may arise from • Although CMA provides more detailed information about chro-
IHC, ISH, alternative chromosome 17 probes, RNA multigene ex- mosome 17 status in breast cancer than do conventional cytoge-
pression arrays, 21- gene recurrence score assays, DNA microarrays, netics or FISH, the complexity of the method and interpretation
and serum HER2 protein analysis, but only 2 of the aforementioned make it impractical for routine use by the clinical laboratory. Thus,
tests — IHC and FISH — are actually FDA approved for reporting IHC and FISH will remain, for the foreseeable future, the main-
HER2 status in breast cancer! stay of testing for HER2 status in breast cancer.

Given then that there are two excellent HER2 assays (IHC and The above observations from genomic pathology help explain
ISH) readily available in the clinical laboratory, the currently con- many of the primary problems with current HER2 testing, and they
fused state of HER2 testing in breast cancer is perplexing and raises suggest strategies that could potentially improve results reporting.
some questions: 1. Is it time to move away from dual-probe testing and the
• Are IHC and 2-probe ISH giving wrong answers 20% of the HER2/cep17 ratio to a single-probe approach? Beginning with
time, consistently, requiring alternative testing methods to re- the first Southern blots used to identify HER2 gene amplification
solve discrepancies? in breast carcinomas, HER2 gene testing has historically been re-
• Alternatively, could IHC and ISH be giving correct answers, but ported as a ratio. In the initial studies, HER2 gene DNA was
we misinterpret the data and thus miss the true HER2 biology compared with DNA of other genes such as ARG1 as a nonam-
of HER2 “equivocal” tumors? plified internal control.1 In the era of FISH, a ratio of HER2
gene copy number per nucleus to chromosome 17 centromere
Seeking answers to these questions, multiple genomic pathology copy number per nucleus is used as an internal control to “correct
groups have analyzed breast cancers that have been characterized for” polysomy 17. However, from CMA studies we know that

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