Molecular markers are studied for their potential to act
as prognostic or predictive factors. A prognostic factor influences the clinical
outcome independently of treatment, whereas a predictive factor correlates with
prognosis because it is linked to the response to a particular therapy. HER2 may
function as both a prognostic and a predictive factor; in addition, HER2 can be
the target of therapy.
The HER2/neu oncogene, also referred to as c-erbB-2/neu, encodes a protein
with a molecular weight of 185,000 daltons (p185). The gene product is a
transmembrane tyrosine kinase receptor belonging to a family of epidermal growth
factor receptors (EGFRs) that are structurally related to the human EGFR.[1,2]
The other members of this family are HER1 (also known as EGFR), HER3, and HER4.
This receptor family is known as the type 1-receptor tyrosine kinases.
Ligands for this family of receptors include the epidermal growth factor (EGF)
and neuregulins, also known as neu differentiation factors or heregulins. At
least six different ligands, the EGF-like ligands, activate the EGF receptor and
cause formation of homodimersan event believed to activate the intrinsic
tyrosine kinase, resulting in transautophosphorylation of tyrosine residues. EGF-like
ligands can also induce heterodimerization between other members of the HER
family, forming heterodimers such as HER1/HER2, HER1/HER3, and HER1/HER4
(Figures 1 and 2). The HER2 receptor is partially homologous to the EGFR.
However, to date, unlike for EGFR, HER3, and HER4, no ligand for HER2 has been
The second class of ligands for the HER receptors, collectively termed
neuregulins, bind directly to HER3 and HER4, but not to HER2 or the EGFR. It is
hypothesized that the main role of HER2 may be to dimerize with the other
members of the HER family of receptors. Interestingly, HER2 is the preferred
heterodimerization partner within the family and is frequently transactivated by
EGF-like ligands or neuregulins resulting from the formation
of heterodimerization with other members of the HER family. This
heterodimerization allows the participation of HER2 in signal transduction, even
in the absence of a cognate ligand.
The HER2 gene was first identified as a transforming oncogene in the DNA of
chemically induced neuroblastomas in the rat. HER2 is overexpressed or
amplified in approximately 20% to 25% of human breast cancers. The HER2 gene
product is composed of a cytoplasmic domain with tyrosine kinase activity, a
transmembrane domain, and an extracellular domain that may be shed from the
surface of breast cancer cells.
HER2 may be involved in the pathogenesis and clinical aggressiveness of
HER2-overexpressing tumors. Indeed, evidence supports a direct role for HER2
overexpression in the pathogenesis and poor clinical outcome of human tumors.
When the mutated gene is transfected into mouse fibroblast cells (NIH-3Y3), it
causes transformation, and the resulting cells are tumorigenic in nude mice.
Transgenic mice that overexpress the neu gene (the rodent homolog of the human
HER2 gene) develop breast cancer. Specific antibodies to the extracellular
domain of the human HER2/neu gene product inhibit the growth of experimental
tumors that overexpress the gene.
HER2 has been shown to be overexpressed in several human carcinomas
including, but not limited to, breast, ovarian, gastric, colon, and non-small-cell
lung cancer. Cellular proliferation is regulated by extracellular factors
that trigger signal transduction cascades from surface receptors through
cytoplasmic effectors and ultimately control progression through the cell cycle.
To date, the exact mechanisms by which oncogenic HER2 affects cell proliferation
and the cell-cycle regulatory components involved have not been identified.
as a Predictive Factor
Amplification of the HER2 proto-oncogene and overexpression of its protein
product in patients with breast cancer have been linked to a poor prognosis; ie,
a more aggressive clinical course and shortened survival.[12,13] Although
inconclusive, data have also suggested that HER2 overexpression may be useful as
a predictive factor, raising the possibility of pretreatment selection of
patients who might benefit from particular therapeutic strategies. Recent data
indicate that patients with lymph node-positive breast cancer whose tumors
overexpress HER2 may obtain additional benefit from adjuvant anthracycline-containing
chemotherapy (as opposed to non-anthracycline-containing regimens).[14,15]
The role of HER2 status in predicting response to adjuvant systemic therapy
and the method of defining HER2 status require further exploration before
definitive recommendations can be made on how best to utilize this
biomarker. Further investigations are also necessary to define the
responsiveness of HER2-positive tumors to hormonal therapy with selective
estrogen-receptor modulators (SERMs) and aromatase inhibitors.
Preclinical studies have suggested that estrogen-dependent cultured human
breast cancer cell lines are rendered hormone-independent after transfection
with multiple copies of the HER2/neu gene. Some studies have suggested that
patients whose tumors overexpress HER2/neu are less likely to respond to
tamoxifen and may have a worse outcome, compared to patients with normal HER2/neu
expression.[18,19] However, other studies have reported no worse outcome with
the use of adjuvant tamoxifen in patients with HER2-overexpressing
The American Society of Clinical Oncology (ASCO) recently updated its
recommendations for the use of tumor markers. In this document, ASCO
recommends the evaluation of HER2/neu status in all primary breast cancers, at
either the time of diagnosis or the time of recurrence. However, the data are
currently insufficient to recommend the routine use of HER2 overexpression to
identify patients with a higher risk of relapse. Complicating the evaluation of
the published data on HER2 as a prognostic factor is the lack of uniform use of
assays in assessing HER2 by immunohistochemistry or fluorescent in situ
The need for accurate detection of HER2 status is becoming more apparent, as
therapeutic decisions are influenced by this information in both the adjuvant
and advanced-stage setting.
A wide range of assay methods have been used to assess the HER2 status of
fresh and archival surgical specimens from breast carcinoma patients. Methods to
assess protein overexpression include immunohistochemistry, enzyme immunoassay,
and Western blot analysis; methods to evaluate gene amplification include FISH,
Southern blot analysis, and polymerase chain reaction (PCR); and methods to
assess messenger (m)RNA overexpression include Northern blot analysis and in
situ hybridization (Table 1).[22,23] In addition, circulating levels of the shed
extracellular domain of the HER2 receptor protein can be detected by serum
enzyme-linked immunosorbent assay (ELISA).
All of these assays have been used in research laboratories, but some have
not been routinely used in hospitals because they require specialized equipment
and/or the use of radioisotopes. On the other hand, a wide range of factors
affect both HER2 testing and determination of HER2 status, including
standardization of methods, standardization of antibodies (monoclonal/polyclonal)
used for immunohistochemistry, agreement on the adoption of common grading
criteria, scoring systems, and correlation of various assays.
Immunohistochemistry and FISH are the techniques routinely recommended for
determining HER2 status, and both have been approved by the US Food and Drug
Administration (FDA) for use in the selection of patients for therapy with the
monoclonal antibody trastuzumab (Herceptin). The other available techniques
should be used for research purposes only. However, many factors may compromise
successful immunohistochemical testing of HER2 status.
Form of FixationThe fixative should preserve antigenic integrity and
limit extraction, diffusion, or displacement of antigen during subsequent
processing. Formalin-fixed, paraffin wax-embedded tumor tissue samples are
appropriate for immunohistochemical assessment of HER2, whereas other methods of
tissue fixation can adversely affect reactivity. The rate of false-positive
cases rises to 50% with the use of an unsuitable fixative. Evidence suggests
that HER2 protein reactivity may deteriorate in fixed paraffin wax sections
after prolonged storage; the immunohistochemistry test should, therefore, be
performed on block sections that have been stored for no more than a few
Antigen RetrievalExcessive antigen retrieval can artifactually increase
immunoreactivity of cancer cells and bring about spurious membrane reactivity in
normal breast epithelial cells, as well as carcinoma cells.
AntibodySeveral antibodies are commercially available, including CB11
(mouse antihuman monoclonal antibody, Ventana, Tucson), TAB 250 (mouse antihuman
monoclonal antibody, Zymed, San Francisco), and HercepTest (rabbit antihuman
HER2/neu polyclonal antibody, DAKO, Carpinteria, Calif). No single antibody has
been consistently demonstrated to be superior in terms of sensitivity and
specificity (Figure 3). The polyclonal antiserum used in the FDA-approved
HercepTest, however, has been subjected to comprehensive standardization of
Scoring SystemHercepTest is the currently recommended scoring method. A
semiquantitative system based on the intensity of cell-membrane immunostaining
and the percentage of positive cells, it has a score range from 0 to 3+. Samples
scoring 3+ are regarded as unequivocally positive and 0/1+ as negative.
Borderline 1+/2+ and 2+ require confirmation with an alternative method,
When dealing with the analysis of an image, both brightness and spatial
resolution play crucial roles. Basically, the finer the resolution, the closer
we approach the original appearance of the image. The quality of resolution, in
turn, is significantly impaired by a so-called contouring phenomenon, which
mainly affects the perimeter evaluation of images, such as HER2 immunostain on
the cell membrane. Taken together, these optical phenomena are likely to
influence interpretation of immunohistochemistry. Interobserver variation in the
assessment of staining is considerable and can lead to misclassification of HER2
status. A simplified immunohistochemistry scoring method is advisable.
The accuracy, sensitivity, and reproducibility of HER2 immunohistochemical
assay can be substantially improved with the use of an image analyzer system to
quantify the immunohistochemical staining. The Automated Cellular Imaging System
(ACIS, ChromaVision, San Juan Capistrano, Calif) automatically scans the
immunohistochemically stained slide, and clearly distinguishes and quantifies
cell membrane staining from cytoplasmic staining using so-called color-space
transformation proprietary technology.
With the ACIS system, HercepTest is scored as a number between 0 and 4, with
a score < 2 indicating a negative result and > 2, a positive result. FISH
analysis is requested for scores ranging from 0.5 to 1.9. When compared with the
FISH standard, the ACIS immunohistochemical assay notably reduces intraobserver
and interobserver disagreement, thus improving the concordance rate and
sensitivity of the manual immunohistochemical assay.
False-Positive ResultsAnother pitfall of immunohistochemistry is the
possibility of false-positive results. In over 90% of cases, HER2 protein
overexpression is related to HER2/neu gene amplification, which results in
increased mRNA transcription and, ultimately, increased synthesis of the
glycoprotein receptor. When technical artifacts (eg, excessive antigen
retrieval) can be ruled out, HER2 overexpression in the absence of genomic
amplification could be the effect of a transcriptionally up-regulated
overexpression.[28,30] HER2 is a growth factor, and enhanced transcription in
the absence of gene amplification is a well-recognized mechanism of cellular
function through enhanced production of mRNA by phosphorylation of tyrosine
kinase acting on growth factors and other regulators of cell growth and
Transiently enhanced transcription unlinked with the corresponding oncogene
amplification has been immunohistochemically seen in colonic regenerating
epithelium and is a well-recognized mechanism in cellular homeostasis. This
does not seem to be the case for HER2 in breast cancer, however, and most
HercepTest-positive cases with a score of 2+ and a normal gene copy should be
regarded as true false-positives, unresponsive to trastuzumab. In contrast,
gene amplification without protein overexpression is an artifact and can only be
due to a failure of the immunohistochemistry assay; ie, posttranslational
modifications of the mRNA transcript may go undetected by inappropriate or
inadequate antibody detection methods.
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