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The goal of the HER-2 evaluation
of a tumor is to predict Herceptin responsiveness
or non-responsiveness 2 as to Herceptin-based chemotherapy. "Positivity" predicts (1) responsiveness to Herceptin & indicates (2) likely resistance to
therapies & (3) likely less benefit from non-anthracycline, non-taxane-containing chemo but better response to anthracycline
METHODS: Membrane product expression is asessed by IHC. Gene amplification is asessed by in-situ gene or oncogene hybridization with either a non-permanent fluorescent
tag (FISH) or permanenmt marker tags of chromogenic type (CISH) or silver deposition type (SISH
). Or, there is the reverse transcriptase quantitative PCR (RTq-PCR) test. These are all done on properly formalin fixed & paraffin embedded tumor tissue (FFPE).
Bullet Points :
FISH tumor tissue test: We have this done at
PhenoPath or Clarient labs & tends to be requested in 2+ indeterminate cases.
- IHC tumor tissue test: We perform this @ LML, & it is our first-line
check of the invasive tumor...true negativity means there is nothing for
Herceptin to go after.
- 10% neutral buffered formalin-fixed tumor tissue (from cores, lumpectomy,
or mastectomy) [Dr. Michael Lagios2 has always
used alcoholic formalin & says IHC works fine with it; Allen
Gown2says FISH works with it, too...but alcohol damages nuclear markers such as ER & TTF-1 if nuclei not yet formalin fixed]. Adequate…8
hours or more…fixation is vital2, 5! ...at least 6 and no more than 24 hours fixation8...though 2008 finds some authnorities defending 48 hours in order to account for fixation over a weekend.
- similar results are (should be) seen whether primary tumor or a metastasis4.
- the tissue block needs to contain normal breast & tumor
so that the interpreting pathologist can “normalize7” (mentally
subtract) the degree of staining in that patient’s benign
breast tissue against the tumor staining (tumor IHC minus normal
gland IHC = reported result) in order to grade the degree of over-expression & obtain IHC/FISH
concordance7...this happens in some kits and must not be confused with overall too-heavy marking. CAP/ASCO standards do not seem to consider this (so, importance is uncertain).
- IHC is directed at extracellular domain of the membrane receptor protein
coded by the HER-2/neu oncogene.
- degrees of IHC staining ...only membrane
staining counts (we use current CAP-ASCO criteria); IHC expression can be quite heterogeneous [L13-1583]… & FISH correlation is as follows:
- 0 =...no staining at all or up to <10% of cells have partial membrane
staining when normalized (correlates with normal FISH negative
for gene amplification).
- 1 + =...>10% of tumor cells with faint/barely perceptible
membrane-pattern of partially circumferential staining. Probably
FISH non-amplified. If you can only appreciate the membrane staining
at 40x...most likely 1+ and not 2+.
- 2 + =... weak to moderate circumferentially complete membrane
staining greater than the internal normal tissue in <30% of
tumor cells. If you can see the staining at 10x, could be
2-3+. Most likely (1) diploid chromosomes with only 3-6 FISH-positive
dots...normal is 2...which would give a ratio between 1.5 & 3...gene
amplification being a ratio greater than 2; or (2) aneuploid
chromosome 17 which turns out not to have a ratio above 2.
- 3 +...>30%8) with moderate to strong..."intense"...complete membrane staining.
If see distinct pattern at 4x, most likely 3+...slide should seem brownish to the naked eye. FISH amplified
in 95% if true 3+. FISH positive if >6 copies per nucleus8).
- concordance/discordance: IHC & FISH/CISH
are trying to predict/assure a Herceptin response if Herceptin is used (Dr.
Press2 notes a $50,000+ cost of just the drug
for a 52-week course; risk of Tx-induced cardiomyopathy increased
over 400% from 1% up to 4-6%6) & normalized IHC 3+ responds
and a 2+ with gene amplification (especially if high-level amp.)
responds like a 3+. By current convention you expect true product over-expression
to be associated with gene amplification.
- archetype: really overexpressed IHC, gene-amplified
by FISH/CISH, grade III, high Ki67, node positivity, and relative
chemoresistance to some agents, and half of such cases are low-degree
- really overexpressed IHC without remarkable Ki67 elevation
( discordant ?).
- 0 negative & 1+ are strongly predictive of negativity
for gene amplification; 2+ is indeterminate; 3+ is strongly
predictive of positivity for gene amplification and strongly
predictive of Herceptin responsiveness.
- 3+ IHC with FISH negative (or 3+ & ratio only 2.1 L07-4483) for gene amplification may be an
example of “single copy over-expression” of product
[LMC-00-6135; LMC-05-6300], an over-interpretation, or an insufficiently controlled technical problem such as too-intense antigen retrieval or wrong antibody used such as E-cadherin [L09-801].
- 2+ IHC, especially if the lab does not “normalize” and
mentally subtract out the degree of normal epithelial staining
on the slide, may not be true over-expression; a true normalized
2+ likely corresponds to low amplification of gene copies (3-6)
or to a non-amplified gene ratio in an aneuploid-increase of
- IHC not 2-3+ but gene is amplified according
to FISH or CISH or SISH results (possibly as many as 1-3% of cases9):
- indicates absence of full translation of the genetic information.
- amplification is reported, but it is really pseudoamplification
of the gene in aneuploid nuclei & was not detected because
lab did not use a ratio with another gene on same chromosome
such as CEP 17.
- amplified gene is producing a defective or aberrant oncoprotein
which the IHC antibody does not adequately recognize.
- testing on a post-chemotherapy tumor can show non-over-expressed
IHC while the gene status is actually amplified (chemo has
damaged oncoprotein production?) [LMC-01-4957] .
- a true status of product over-expression relates to increased
proliferation and is thought to happen rarely in nuclear grade
I (IDCs, classic lobular, or tubular ca.)2...warrants careful review.
- reasonable to FISH, CISH, or SISH test cases IHC <3 +, at least when tumor
ER/PR neg., and high proliferation by such as Ki672.
- Is a surrogate line of evidence to help define whether membrane product
is over-expressed…amplification implies product over-expression.
RTq-PCR: this is a quantitative test done from FFPE tissue and capable of correlating concordantly in p7% of cases with ISH10.
serum test: useful in patients who have an initial
test value = to or > 15 ng/ml;serum sample is
forwarded to a reference lab. Indications for serum test:
- results expressed as a ratio of average number of detected HER-2/neu “genes” [oncogenes] stained
per tumor nucleus to the number of chromosome 17 genes stained
per nuclei (this adjusts for variations in nuclear “ploidy”).
- if original tumor block is missing or recurrence is impractically
located for biopsy
- to complement the results of the IHC test
- to monitor response to hormonal therapy (cases with ER positivity & low-volume,
indolent metastatic disease)
- to monitor response to Herceptin/chemo
combination (ER neg. & symptomatic
Human epidermal growth factor receptor 2 gene ERBB2 (commonly referred to as HER-2...sometimes as HER-2/neu) amplification and/or membrane
correlates with aggressive traits likely for that breast cancer2, with HER-2 gene amplification in about 18-20% of all breast
Produced in the cytoplasm, protein goes to cell surface membrane and normally aggregates to become or induce clusters of about a thousand
receptors. If protein is neoplastically upregulated, those clusters of receptors enlarge and coalesce & lead to heavy IHC staining
8. The receptor is
a 185-kd protein cell-surface membrane receptor (in the family
of human epidermal growth factor receptors) with tyrosinase activity in the
intra-cellular domain (and the molecule having a transmembrane domain and an extracellular
domain). It is found normally in a number of epithelial cell types.
Through binding of specific growth factors, the receptor regulates
some aspects of cell growth and division. Increased receptors are thought to increase signalling for cell growth. This proto-oncogene is located
on the long arm of chromosome 17.
Proto-oncogenes encode important proteins utilized normally in normal
cell growth & differentiation, and they are genes with oncogenic
potential. When proto-oncogenes are altered by point mutation,
translocation or gene amplification, they thereby become oncogenes
and produce growth signals that may lead to cellular transformation
and the development of cancer. Herceptin attaches to the HER-2/neu
protein (neu) product (membrane receptors) and somehow, sometimes,
inhibits or cancels the dysfunctional effect of excess product or amplified
The mutated, amplified, or altered form of a proto-oncogene
is called an oncogene…an amplified HER-2 gene is an oncogenic
HER-2 genetic tumor status.
Breast cancer is diagnosed in about 180,000 American women
each year. The risk of breast cancer increases with age. For more than
a decade, circulating breast cancer antigens have been used in breast
cancer for monitoring therapy and evaluating possible recurrence. It
is conventionally considered that a breast cancer never changes its
HER-2 status. Since about 2003, we have seen in our practice the use of
the IHC tissue stain in a search for over-expressed HER-2/neu
oncogene-product protein as a prognostic indicator and a marker related
to choice of therapy (15-30% of breast cancers over-express HER-2 product).
This latter point has become particularly useful since the introduction
of Herceptin® as a chemotherapy agent which targets the HER-2/neu
receptor. And, in ER positive tumors,HER-2 positivity predicts responsiveness to anthracyclin based chemo and to
tamoxifen7. With immunohistochemistry (IHC) techniques, it has been found
that breast cancer patients who over-express HER-2/neu product in their
cancers have a poor prognosis with shorter disease-free and overall
survival than patients who do not over-express HER-2/neu.
Some suggest that elevations of shedded extracellular domain HER-2/neu
in serum1 may be used as evidence of early recurrence
(the serum material is part of the membrane product but not sure yet
of any correlations with intensity of IHC staining of tumor).
In addition, initially elevated levels of serum HER-2/neu correlate
with the presence of metastatic disease and suggest poor prognosis.
These levels may be valuable in predicting response to various forms
of therapy. Combining HER-2/neu serum measurements with other tumor
markers may improve the sensitivity of detection of recurrence.
A reference in 2002 indicated that Herceptin was only effective in
cases of IHC product over-expression; one in 2003 indicated effectiveness
only if gene status is amplified. Dr. Gown says that Herceptin response
has to do with product over-expression2… as
does NEJMed4…over-expression is the target
Synonyms: neu, c-erbB2, HER-2
- from an online Bayer report of 4 serum test reports at 12/01 San
Antonio breast conf., 31 Dec. 2001 [not online as of 7 July 2003].
- verbal or e-mail info from experts (such as Dr. Allen Gown, Dr.
- Genentech website & then
drop HER-2 into site search engine.
- articles & editorials, NEJMed 353(16):1652-1738, 20 October
- Neal Goldstein, et. al. AJCP 120:86-92, July 2003.
- 12/2005 San Antonio Breast Conf.
- Gown AM, et. al., "Extremely High Concordance Between Immunohistochemistry and FISH Testing for HER2/neu STATUS in Breast Cancer:
a Study of 6104 Cases, presented at 12/2006 San Antonio Breast Conference [showing the value of "normalization" in obtaining
IHC & FISH concordance.
- CAP/ASCO Guidelines ("New Guidelines Recommended Determining HER2 Status for All Invasive Breast Cancer") released 11-18 Dec
2006 & in article in CAP Today Dec. 2006.
- Dr. Jeff Ross, Albany Med. Ctr., Albany, NY @ Genetech sponsored lecture, Columbia, S. C. 5/13/2009.
- Baehner FI, et. al., JCO 28:4300-6, 2010 & reported by Allen Gown in spring 2011 vol. 14(2) issue of Phenomena by PhenoPath Lab.
(posted 2004; latest addition 6 June 2011)