Practical Issues with Her2-neu Testing
Daniel W. Visscher, M.D.
Carol Reynolds, M.D.
How would you score this HER2-neu immunostain? What is the
likelihood that this neoplasm has HER2-neu amplification?
The score is 2+. The cells show prominent granular cytoplasmic staining, but
"moderate" membrane staining. About 20% of "2+" cases have gene amplification; this tumor was not
amplified in subsequent FISH analysis.
Her2-neu is a paradigm for recent pathogenetic, diagnostic and therapeutic
developments in breast cancer. The Her2-neu gene product is a transmembrane growth factor receptor,
encoded on chromosome 17q, which is expressed physiologically in fetal tissues but not in normal adult
breast. The molecule is functionally and structurally analogous to other tyrosine kinase receptors, such
as EGFR; these molecules normally initiate cellular signal transduction cascades after binding to their
cognate ligands (i.e. growth factor). Ligand binding is thought to "bridge" adjacent Her2-neu molecules,
causing dimerization of receptors. Conformational changes induced by dimerization leads to activation.
Highly elevated plasma membrane levels of Her2-neu result from gene amplification (see below). This
leads to spontaneous receptor activation, and hence constitutive signal transduction activity, due to
ligand independent dimerization/heterodimerization resulting from increased density of molecules. The
resulting sustained intracellular signaling has pleiotropic effects that facilitate neoplastic cell
In breast carcinomas, highly increased levels of plasma membrane protein generally reflect an acquired
chromosomal alteration called gene amplification, which occurs in 15-30% of unselected cases. Most of
the time (75%) this consists of tandem intrachromosomal DNA sequence repeats, typically numbering at
least 15-20 copies; these produce the so called homogeneously stained regions (HSR's) which correspond to
amplified DNA in metaphase karyotype spreads. Less frequently, gene amplification reflects either a
complex translocation of duplicated gene copies to other chromosomes or the presence of extrachromosomal
segments of gene encoding DNA (double minutes). Although cellular protein levels generally correlate
with gene copy number, some degree of dissociation is introduced by variable rates of transcription and
mRNA stability. In about 5% of overexpressed tumors, significantly elevated levels of Her2-neu protein
occurs without apparent gene amplification. (These cases represent one source of IHC-FISH "discrepancy",
Her2-neu amplification/overexpression in invasive breast carcinomas is associated with high tumor
grade, estrogen receptor negative status and more frequent nodal metastasis. Paradoxically perhaps,
Her-2 is frequently overexpressed in DCIS; at least 50% of comedo DCIS cases, in fact, have 3+ Her2-neu
staining. Amplification is also more frequent in tumors with extensive intraductal components. This is
thought to reflect the function of Her2 in facilitating a "spreading" (i.e. vs infiltrative) type of
growth, in keeping with observations that the molecule is present in aggregates at pseudopodia and
co-expressed with pathologic cell adhesion molecules.
The early Her2-neu literature hotly debated its prognostic significance. This controversy largely
became moot after the publication of studies demonstrating that amplification may be associated with
enhanced therapeutic responsiveness to anthracycline (Adriamycin) based chemotherapeutic regimens. (Some
data suggest that this reflects co-amplification with topoisomerase II-alpha, the target of
anthracyclenes, which is located very close to Her2 on chromosome 17q.) Other studies have shown that
Her2 overexpression antagonizes anti-estrogen therapy. Hence, determining the prognostic significance of
Her2-neu is materially confounded by variable treatments employed in the population being analyzed. The
transformation of Her2-neu from a prognostic to a predictive marker was completed with the introduction
of Trastuzumab (Herceptin), a humanized murine monoclonal antibody specific for Her2. This agent is
thought to augment the host immune response to neoplastic cells. It is presently employed in patients
with advanced metastatic disease, although trials to evaluate efficacy in an adjuvant setting are in
progress. The clinical response rate to Trastuzamab, when administered alone, is about 15-30%, and
60-80% when combined with one of the taxanes. Testing for Her2 status is clinically indicated for
patients with invasive breast carcinoma in order to determine eligibility for Herceptin therapy.
Oncologists are increasingly requesting that Her-2 analysis be performed on all invasive breast cancers
in order to predict anthracycline/anti-estrogen response.
Her2-neu testing has ignited two long simmering issues in anatomical pathology: quantification of
immunostaining intensity and genetic testing of tumor samples (i.e. at the DNA level). Presently, much
debate concerns the issue of whether Her2-neu status should be established using immunohistochemistry, to
evaluate protein expression, or interphase cytogenetics (fluorescence in situ hybridization, or FISH) to
assess gene amplification. As noted earlier, gene amplification is causally and thus predictably
associated with high levels of protein expression. In theory, therefore, the two technologies are
directed at different manifestations of the very same process. Why then, has the controversy arisen?
The answer, although partly technical, partly biological, and partly commercial, is probably best
approached in an historical context.
Investigators involved with the initial trials of Herceptin therapeutic response recognized that
breast carcinomas demonstrate a spectrum of immunoreactivity to monoclonal antibody reagents against the
Her2-neu protein. Those studies employed a four-part (0-3+) grading system, based primarily on stain
intensity (see below), to semiquantitate immunostaining. Importantly, they chose the threshold of 2+, or
"moderate" intensity (not 3+, or "stong") as "positive". Subsequent analysis of the data indicated that
therapeutic response was limited primarily, but not exclusively, to patients with more intense (i.e. 3+)
immunoreactivity. (Others have shown that patient survival is also related to the degree of
immunostaining.) In addition, studies such as the one by Pauletti et al., reported that gene
amplification status was superior to Her2-neu immunoreactivity in predicting disease outcome. The fact
that only a proportion (15-25% in most studies) of 2+ cases are amplified at the DNA level further seems
to imply that amplification status is most relevant. Since immunostaining, by definition, is assessed
subjectively and gene copy number is quantifiable by direct observation using hybridization technologies,
many have reasoned that FISH is most "accurate" and should thus be employed as the method of choice for
clinical Her2-neu testing.
A number of arguments have been put forward to challenge this conclusion. First, the study by
Pauletti has been criticized for suboptimal immunohistochemical methods. For example, only 70% of their
amplified cases were positive by immunohistochemistry. These data are at variance with the majority of
the literature, which are consistent in reporting presence of gene amplification in 90-100% of 3+
positive cases. Most studies also demonstrate that gene amplification is present in only 0-7% of 0-1+
staining tumors. Many would agree that the balance of the literature shows that expression status is a
tolerably accurate surrogate for gene copy number in breast carcinomas that are either 0-1+ or 3+ by
IHC. Collectively, this accounts for about 80% of breast carcinomas. Hence, supporters of IHC would
assert the main issue to resolve is the problem of cases having 2+ immunoreactivity. Unfortunately this
is not a trivial matter; an indeterminate, or 2+, result is obtained in 10-15% of unselected cases - a
frequency similar to 3+. Of course, all advocates of IHC presume optimal utilization of this
methodology, which is by no means a trivial consideration.
The problem of "2+" IHC cases is becoming more clearly understood. In the past, some FISH advocates
have suggested that the lack of gene amplification in most 2+ cases represent either falsely positive or
negative results that reflect intrinsic limitations of immunohistochemical methods, such as subjective
interpretation, variable methodology and fixation artifacts. For example, allthough interobserver
agreement for staining, overall, is about 85%, the kappa statistic for 2+ cases is much lower – on the
order of 0.4-0.6. Further, there are published data showing that "high volume" labs have better IHC-FISH
correlation that "low volume" labs, implying that technical or interpretation expertise are significant
factors in the IHC assay. Interestingly, there comparatively few interobserver, much less
interlaboratory, comparison studies in the cytogenetics literature. Our data suggest an agreement rate
for FISH on the order of 85% (in cases that have been referred due to 2-3+ staining). It should also be
noted that hybridization "fails" in 3-5% of cases.
We are of the opinion that technical issues are only one part of the problem with 2+ IHC cases, and
with Her2 testing overall. Gene amplification and its relationship to protein levels are biologically
complex. Average gene copy number in amplified tumors varies from 4 to 70 per nucleus and there is
significant intratumoral heterogeneity; the latter reflects a high level of genetic instability and is
manifested by markedly aneuploid karyotype spreads/ chromosome 17 polysomy in Her2 positive cases. We
have observed that most 2+ cases have a difficult to interpret heterogeneous staining pattern,
significantly complicating their interpretation. When confronted with such cases the issue becomes
whether sufficient portions of the lesion meet the "strong"/3+ threshold. (We have found, incidentally,
that many of these 2+ cases would be changed to 3+ if another block from the case were analyzed.) Image
analysis is one approach that has been shown to refine the interpretation of Her2-neu immunostains. A
number of sophisticated automated instruments are commercially available. It should be noted, however,
that quantitative analysis would not add significant information in 0+ or 3+ cases nor would they enhance
diagnostic accuracy in suboptimally stained sections.
Since cellular protein levels have been shown to correlate generally with gene copy number, some
authors have concluded that a 2+ staining result may accurately reflect marginal, or low level,
amplification status and thereby represent a fundamentally different subset of cases than 3+/highly
amplified tumors. Accordingly, one study reported that the gene copy number in 17/20 3+ cases was
>20, whereas only 5/14 2+ cases with amplification had > 15 copies. Our lab has made similar
observations (see Table in CD). We have also observed that amplification is limited to relatively small
subpopulations in about one third of 2+ cases that are amplified (see Table in CD). The clinical
significance of "low level" or focal amplification has yet to be defined in clinical studies. Presently
(see below) the threshold for Her-2 amplification using the FISH assay is very low; up to 25% of FISH
positive tumors exhibit marginal levels of gene amplification. It is important to note that, at
marginal levels of gene amplification, even FISH is subject to significant interlaboratory discordance.
With respect to Her2-neu expression, ELISA assays have demonstrated "low level" protein upregulation,
in the absence of gene amplification, in at least 40% of breast carcinomas. Little is known about the
biology and clinical significance of this finding, although it likely accounts for the majority of 1-2+
Practical issues with Her2-neu testing. We do not take an either/or position on the issue of
whether FISH or IHC is superior and the matter remains a topic of debate. Until the issue is resolved,
our opinion, to date at least, is that they are complementary. In our practice, tumors selected for
Her2-neu analysis are routinely "screened" using IHC, and the diagnostically "ambiguous" (2+) cases are
automatically reflexed for "definitive" evaluation using FISH. In view of the heterogeneity issues cited
above, it is our opinion that borderline cases at the 1-2+ and 2-3+ interfaces should also be referred
for FISH assay. (After all, the test is being employed to make a significant treatment decision.) This
algorithm reflects not only the considerations described above, but also the results of our own internal
comparison studies, which imply that the (genetic) status of Her2-neu can be reliably inferred using
immunohistology in unequivocally IHC positive/negative breast carcinomas. Cost and logistical issues are
also factors – FISH is at least three times more expensive than IHC. Most relevant to the readers of
this handout, of course, is that interphase cytogenetics requires equipment and technical expertise that
are not universally, or even widely, employed in the setting of anatomical pathology departments. We
would not be particularly surprised or dismayed if new developments in this evolving field were to impose
modification of the approach outlined above.
Apart from purely technical matters, such as antigen retrieval, a number of issues impact immunostain
assays. There are, for example, at least four commercially available antibodies, all of which appear
frequently in the Her2-neu literature. Comparison studies have generally shown a similar degree of
diagnostic specificity, sensitivity and predictive significance. However they differ with respect to FDA
endorsement, which is driven largely by marketing of internal/external control reagents and procedures
designed to enhance test performance and interpretation.
Although seemingly straightforward, we have observed that interpretation of staining can be
difficult, most often due to the following:
It is beyond our scope to review FISH technology in detail, and interested readers may consult a
number of sources for methodologic information. We should note that most cytogenetics labs issue a
report that states, not merely the number of Her2-neu copies per cell, but rather the ratio of Her2-neu
to chromosome 17 copy number in the tumor population. This ratio is obtained by direct visualization and
manual counting of neoplastic cell nuclei, typically 40-60, within intact tissue sections that have been
simultaneously ("dual") hybridized with two separate probes. One probe is sequence complementary to the
Her2-neu locus and the other to the chromosome 17 centromere region. The signals corresponding to target
hybridization may be detected and distinguished under fluorescence microscopy by use of uniquely-colored
signals (i.e. red vs green) which are developed as a component of the detection system for commercially
- Cytoplasmic staining is common, and may be quite strong, but should
not be considered specific. In true positive cases, there is intense, "crisp", linear (not granular)
circumferential plasma membrane immunoreactivity. Weak staining is observed in benign epithelium in up
to 25% of cases. Most authors would consider this to be "real" and not an artifact or reflection of
- Although gene status within the DCIS and invasive components of a
tumor are generally similar, evaluation of staining should be limited to the latter. Nonamplification
associated expression, frequently of 2+ intensity, is particularly common in DCIS. Also, experienced
observers have noted the occasional tumor with strong DCIS staining but weak/marginal staining in the
- Weakly (1-2+) immunoreactive cases usually exhibit considerable
staining heterogeneity, in contrast to the diffuse, strong pattern that characterizes the majority
(>90%) of highly amplified tumors. This does not discount the presence of heterogeneity with respect
to amplification, noted above, which likely characterizes about 30% of positive cases. "Intense" plasma
membrane staining, however, is sufficiently specific that it need only be observed in 10% of cells to
indicate amplification. In our experience, the distinction of "moderate" (2+) from "strong" (3+)
staining is a major source of interpretation variation. From a clinical standpoint, it is important to
avoid "overcalling" a moderately positive (i.e. "2+") stain. We strongly recommend performing a
positive control stain on a case with 3+ immunoreactivity and known positive amplification status.
- Some authors have noted that discrepancies between IHC and FISH are
more common when the specimen tested is a small core biopsy. Not only does the smaller sample magnify
the potential problem of heterogeneity, but "crushing" artifacts can make distinguishing cytoplasmic from
plasma membrane staining quite difficult. (A similar problem obscures sections with retraction
artifact.) The enhanced "edge effect" associated with diminished section size may further confound
- It is important to avoid drying of slides during the immunostain procedure. This results
in "mushy" cytoplasmic staining, even in positive tumors. Also, use of
distilled water is recommended for washing steps.
The need for evaluating chromosome 17 copy number reflects the frequent occurrence of chromosome
polysomy in amplified tumors; 7-8 copies per cell is common. In order to be amplified a cell must have
at least twice the number of Her2-neu signals to chromosome 17 centromere signals (hence a ratio of at
least 2.0). Some labs may concede the presence of equivocal or "borderline amplified" cases (i.e. if the
ratio is 1.7-2.0) or "duplication" (i.e. if the ratio is 2.0). Although the 2.0 threshold is more or
less uniformly accepted as the criterion for amplification, the clinical significance of varying degrees
of amplification (particularly low level amplification) has yet to be established in clinical trials. In
situ hybridization technology is successful is 90-95% of tests.
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