measuring her-2 in brest cancer_pathology pattern reviews

8/3/2019 Measuring HER-2 in Brest Cancer_pathology Pattern Reviews

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Pathology Patterns Reviews

S26 Am J Clin Pathol 2002;117(Suppl 1):S26-S35 American Society for Clinical Pathology

A b s t r a c t

Measuring HER-2 is important for selecting

optimal therapy and predicting prognosis in breast

cancer patients. Current methods for evaluating HER-2

include measuring protein overexpression by

immunohistochemistry (IHC), measuring gene copy

number by fluorescent in situ hybridization (FISH), and

measuring shed antigen in the serum by enzyme-linked

immunosorbent assay (ELISA). This review compares

these 3 methods and analyzes the current literature

pertaining to this subject. In comparing IHC withFISH, the negative predictive value is excellent for

commonly used commercial antibodies but the positive

predictive value is highly variable. However, by

considering only strongly staining cases as positive by

IHC, the positive predictive value is markedly

improved. ELISA is useful in the follow-up care of

patients with breast cancer. An algorithm for using all 3

methods is presented.

The HER-2 (neu, or c-erbB-2) gene encodes a trans-

membrane receptor of the epidermal growth factor (tyrosine

kinase) receptor family, notably the only one without a

known ligand. About 20% to 30% of breast cancers show

increased activity of this membrane receptor, either by gene

amplification or by protein overexpression. HER-2 was first

described to be important as a prognostic marker of breast

cancers in 1987,1 and subsequently measurement of HER-2

has gained prominence in the ancillary laboratory testing of

breast cancer, second only to estrogen and progesterone

receptor testing. Patients with HER-2 gene amplification,protein overexpression, or both have decreased overall

survival and a higher probability of disease recurrence.2

Perhaps even more important than prognosis, measurement

of HER-2 became crucial in predicting response to therapy

when trastuzumab (Herceptin, Genentech, San Francisco,

CA), a humanized monoclonal antibody that targets the

HER-2 receptor, became available in 1998. Other therapies

commonly used in breast cancer also may be related to HER-

2 levels: HER-2 may predict response to anthracycline-

containing chemotherapy regimens, and HER-2 may predict

response to hormonal therapy.3,4

In addition to breast cancer,HER-2 gene amplification and protein overexpression may

have a role in a variety of other carcinomas, including colon,

salivary gland, and gynecologic carcinomas.

The best method for measuring HER-2 is controversial.

The existing methods include the following: (1) measuring

the gene copy number by Southern blot analysis, polymerase

chain reaction (PCR), fluorescent in situ hybridization

(FISH), or chromogenic in situ hybridization (CISH); (2)

measuring messenger RNA by Northern blot analysis or

PCR; and (3) measuring protein expression by Western blot

or immunohistochemical analysis. Shed antigen may be

Measuring HER-2 in Breast Cancer

Immunohistochemistry, FISH, or ELISA?

I-Tien Yeh, MD

Key Words: Breast cancer; HER-2; Immunohistochemistry; Fluorescent in situ hybridization; Enzyme-linked immunosorbent assay


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Am J Clin Pathol 2002;117(Suppl 1):S26-S35 S27 American Society for Clinical Pathology

measured in the serum by enzyme-linked immunosorbent

assay (ELISA). Evaluation of HER-2 can be performed on

paraffin-embedded tissue sections when using immunohisto-

chemical and FISH methods. A tissue approach permits

assessment of tumor morphologic features and correlation,

permits the use of archival material, and avoids the dilutional

artifacts of most of the other approaches.

Of the aforementioned methods, 3 have gained US Food

and Drug Administration (FDA) approval for patient care

purposes: immunohistochemical analysis, FISH, and ELISA.

As of this writing, there are 2 approved immunohistochem-

ical methods: the HercepTest (DAKO, Carpinteria, CA), a kit

using a polyclonal antibody to the internal domain of theHER-2 receptor, and CB11 (Ventana Medical Systems,

Tucson, AZ), a monoclonal antibody to the internal domain

of the HER-2 receptor. There also are 2 FDA-approved FISH

kits, INFORM (Ventana Medical Systems, Tucson, AZ),

which uses a single probe for the HER-2 gene, and Pathvy-

sion (Vysis, Downers Grove, IL), which uses 2 probes, 1 for

the HER-2 gene and 1 for the centromeric region of chromo-

some 17. Only 1 FDA-approved ELISA assay is available:

HER-2/neu serum assay ECD (Oncogene Science, Bayer,

Cambridge, MA). Table 1 enumerates some basic compar-

isons of these methods. Sites of action of the differentmethods on the cell are pictured in Figure 1.

Immunohistochemical Analysis

Immunohistochemical analysis detects the actual HER-2

receptor on the cell membrane through the use of antibodies

against the HER-2 receptor Image 1. This receptor also is

the target of trastuzumab therapy, and it would seem to

follow that that actual detection of the HER-2 receptor would

predict response to trastuzumab therapy. However, there area number of variables that affect the results of HER-2 testing

by immunohistochemical analysis, and the consequent

inconsistencies in results probably contribute to lack of

correlation between trastuzumab treatment and immunohis-

tochemical findings. These variables include fixation,

storage, antigen retrieval, reagent optimization, the specific

antibody and its domain, controls, the scoring system, and

interobserver variability in interpretation.

Prolonged fixation will cross-link antigenic sites and

make them unavailable for immunohistochemical analysis.5,6

Requests for testing of archival cases are sometimes made

Table 1Comparison of Immunohistochemical Analysis, FISH, and ELISA

Immunohistochemical Analysis FISH ELISA

Specimen type Tissue Tissue SerumTechnical complexity Moderate High LowTarget Protein P185 DNA Protein extracellular domain, P105Interpretation Subjective Quantitative Quantitative

Clinical applications Trastuzumab therapy, prognosis Trastuzumab therapy, prognosis Follow-up monitoring

ELISA, enzyme-linked immunosorbent assay; FISH, fluorescent in situ hybridization.

Figure 1 HER-2 receptor. The HER-2 receptor is a

transmembrane receptor, denoted by P185, with an external

domain and an internal domain. Immunohistochemical

analysis (IHC) uses an antibody against either the internal or

external domain. The cleaved portion of the HER-2 receptor

is detected by enzyme-linked immunosorbent assay (ELISA),

whereas fluorescent in situ hybridization (FISH) analyzes the

gene copy number on chromosome 17. CISH, chromogenic in

situ hybridization; ECD, extracellular domain. For proprietary

information, see the text.


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Yeh / MEASURING HER-2 IN BREAST CANCER

for patients with recurrent or metastatic disease, but with

prolonged storage, antigens may degrade, leading to false-

negative assay results. Antigen-retrieval methods have

largely solved these problems, but there still is variability in

methods and, thus, variability in the retrieval of antigens. In

fact, false-positive assay results are possible, since HER-2 is

a normal gene, and if antigen retrieval is successful enough,

normal expression will be seen. The difference between

normal expression (tens of thousands of receptors per cell)

and overexpression (millions of receptors per cell) is

substantial. Jacobs et al7 tried to take this factor into account

in their scoring system, which subtracts out the normal

staining, but normal tissue is not always available in a small

needle biopsy specimen for comparison. Individual laborato-

ries optimize immunoassays for their own conditions (eg,

temperature, humidity, manual vs automated), so antibody

and other reagent concentrations may vary.

Numerous antibodies against the HER-2 receptor are

available commercially, including both polyclonal andmonoclonal antibodies. In general, monoclonal antibodies

are considered more specific. Of the 2 FDA-approved anti-

bodies, the HercepTest uses a polyclonal antibody, and CB11

is a monoclonal antibody. The antibody used in the

HercepTest, A0485, is also available as a stand-alone product

and is used by some laboratories in lieu of the kit. The anti-

genic domain of both FDA-approved antibodies is for the

internal domain of the HER-2 receptor. Some of the

commercially available antibodies are against the external

domain of HER-2. Probably the most widely used external

domain antibody is TAB250 (Zymed, South San Francisco,CA). There is some concern that the external domain of

HER-2 may be cleaved (this is the portion measured by the

serum ELISA assay), and, thus, antibodies against the

external domain may not be as sensitive as antibodies against

the internal domain.8 However, this concern is not borne out

in comparison of antibodies (see FISH vs Immunohisto-

chemical Analysis).

DAKO, in the HercepTest, recommends using cell line

controls that are known to have negative, low, and high levels

of HER-2 expression. While in theory this is an excellent

idea, in reality, a control really should have undergone thesame processing steps as the tissue in question, and, therefore,

Jacobs et al7 proposed using a method of analysis in which

the level of staining of the background benign breast tissue is

recorded and subtracted from the staining level of the tumor.

This proposal is based on normal tissues always having a

normal HER-2 gene copy number and normal expression.

Stark et al9 demonstrated rare cases of increased HER-2 gene

copy number by PCR in archival benign breast biopsy speci-

mens of patients in whom breast carcinoma subsequently

developed, although in that study, no overexpression was seen

in benign cells by immunohistochemical analysis.


The scoring system in widest use is based on the

scoring system recommended by the HercepTest: 0, no

staining; 1+, weak partial membranous staining in more

than 10% of cells; 2+, weak to moderate complete

membranous staining in more than 10% of cells; and 3+,

strong complete membranous staining in more than 10%

of cells. Note that granular cytoplasmic staining is nonspe-

cific (some antibodies result in cytoplasmic staining that

may be misinterpreted by inexperienced observers). Anegative assay is reported with 0 and 1+ staining, whereas

2+ and 3+ are reported as positive. This scoring system

has been strongly critiqued in the last few years because in

2+ cases, gene amplification could be demonstrated in

only about a quarter of the cases in the initial trastuzumab

trials using the clinical trial assay.10 For trastuzumab

response, cases with immunohistochemical results inter-

preted as 2+ responded to treatment only 4% of the time to

treatment, whereas 3+ cases responded 17% of the time

(trastuzumab product insert). In addition, only a minority

of 2+ cases showed gene amplification. Negative and posi-tive cases had good correlation. These data have been

interpreted to indicate that a 2+ level of staining should not

be interpreted as positive, but rather regarded as a border-

line value, and that confirmatory testing by FISH should

be performed. Other scoring systems, using 0 to 211 and 0

to 4+7 also have been used, as well as one using an addi-

tive score of 0 to 8, with staining intensity and proportion

of cells stained separately recorded.12

Interobserver variability has been a well-documented

problem with pathologic interpretation and, indeed, has been

documented in reading immunohistochemical results. In the

Image 1 Invasive breast cancer showing strong, complete

membranous staining by immunohistochemical analysis (400).


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study by Thomson et al,13 3 observers, including an experi-

enced breast pathologist, a general surgical pathologist, and a

pathology resident, independently interpreted slides in ablinded manner. Negative staining and strong staining (0, 3+)

showed the greatest degree of agreement in scoring, whereas

intermediate degrees of staining (1+ and 2+) showed signifi-

cant variations in scoring. Hoang et al14 also compared inter-

observer reproducibility and found interobserver agreement

in 85% of cases among 4 observers, with agreement in nega-

tive and strong positive cases and variability in weak positive

cases. This disparity in scoring by observers may well be a

good part of the reason for reported differences in results

when the same antibody is used in different laboratories

Table 2, Table 3, Table 4, and Table 5.13-29

Fluorescent In Situ Hybridization

FISH quantifies the number of HER-2 gene copies thatare located on chromosome 17 Image 2. Normal cells

contain 2 copies of the HER-2 gene, but when a cell is under-

going division, the cell may contain 4 copies of the gene.

Gene-amplified cells contain more than 4 copies or have a

HER-2/chromosome 17 gene copies ratio greater than 2.

The most compelling reason to use FISH analysis is

that comparison of the clinical trial assay with FISH,

which was used during the trastuzumab trials, showed that

prediction of response was superior using FISH.10 These

data were presented to the FDA and led to the recent

approval (December 2001) of the Pathvysion FISH assay

Table 2HercepTest vs FISH*

Sensitivity Specificity PPV NPV Efficiency

No. of FISHAuthors Cases Method 3+ Only 3+ Only 3+ Only 3+ Only 3+ Only

Bnkfalvi et al15 40 Ventana 100 100 77 100 50 100 100 100 81 100Birner et al16 207 Vysis 97 97 76 97 45 89 99 99 80 97

Hoang et al14 100 Vysis 100 100 91 97 71 89 100 100 93 98Jacobs et al17 48 Ventana 42 74 42 74Lebeau et al18 79 Vysis 100 90 79 100 65 100 100 96 85 97Pauletti et al19 80 Vysis 78 80 73 84 79 Thomson et al13 127 Vysis 59-77 91-93 89 93-97 (0);

50-70 (1+)Tubbs et al27 144 Vysis 83 81 76 93 46 75 95 95 77 90

FISH, fluorescent in situ hybridization; NPV, negative predictive value; PPV, positive predictive value; 3+, strong complete membranous staining in more than 10% of cells.* For the methods used to calculate sensitivity, specificity, PPV, NPV, and efficiency and for proprietary information, see the text. Skewed population, cases negative by FISH only, using data presented in paper, without subtraction of benign staining. Limited analysis on subgroup of 900 cases; 5 cases with low levels of gene amplification excluded from analysis. Three observers, using a modified statistical method outlined in reference.

Table 3

A0485 vs FISH*



Birner et al16 207 Vysis 100 100 69 90 40 72 100 100 73 92Jacobs et al7 90 Ventana 78 96 86 93 91 Jimenez et al21 41 Ventana 100 100 87 100 83 100 100 100 92 100

and VysisLebeau et al18 79 Vysis 100 90 79 100 65 100 100 96 85 97Onody et al22 100 Ventana 87 91 62 97 90 Ridolfi et al23 116 Ventana 97 95 70 100 59 100 98 98 78 99

and VysisThomson et al13 127 Vysis 63-84 95-98 93-100 93-97 (0);

53-87 (1+)

Wang et al24 52 Ventana 93 91 68 89 54 77 96 96 75 9052 Vysis 93 91 66 89 50 77 96 96 73 90

Wang et al20|| 189 Vysis 99 98 66 94 65 92 99 99 79 96

FISH, fluorescence in situ hybridization; NPV, negative predictive value; PPV, positive predictive value; 3+, strong complete membranous staining in more than 10% of cells.* For the methods used to calculate sensitivity, specificity, PPV, NPV, and efficiency and for proprietary information, see the text. Data for 2 FISH methods not separately specified. Three observers, using a modified statistical method outlined in the article. Two FISH methods performed on all cases.|| Manual method only.


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for the selection of patients for trastuzumab therapy. In

addition, studies of HER-2 measured by FISH are more

consistently predictive of prognosis than are immunohisto-chemical studies.30

Slamon31 argues for the use of FISH as the primary

testing method for these reasons: (1) FISH data will separate

curves of patient prognosis, even at low levels of gene ampli-

fication; (2) when immunohistochemical analysis is done as

the primary assay, then FISH is superimposed, there is a

significant change in the outcome data, but the opposite is

not true. This implies that immunohistochemical analysis is

missing some cases (false-negative results), although the

theoretical ideal immunohistochemical analysis would be

more sensitive than FISH.31

FISH is less subject to the problems of fixation, storage,

antigen preservation, and interobserver variability than is

immunohistochemical analysis. DNA is relatively stable andnot subject to problems of preservation and storage.

Formalin fixation, used in the vast majority of pathology

laboratories for breast tissue, has no significant detrimental

effect on the preservation of DNA. Interobserver variability

is not usually much of a problem in a quantitative test, but

the fluorescent signals are not always clearly defined;

clumping of signals occurs, and some cases are undecipher-

able by FISH for this reason. Cases may be indeterminate by

FISH assay because of heterogeneity in the gene number

among cells.21 It sometimes is difficult to get an exact count

of gene copy number because the examined tissue section is

Table 4CB11 vs FISH*



Bnkfalvi et al15 40 Ventana 86 83 100 100 100 100 97 97 97 97Birner et al16 207 Vysis 80 76 96 98 80 88 96 96 93 95

Couturier et al25 100 Ventana 92 89 99 99 92 89 99 99 98 99Gancberg et al26 160 Vysis 43 100 100 84 87 Jimenez et al21 41 Ventana

and Vysis 100 100 93 100 89 100 100 100 96 100Lebeau et al18 85 Vysis 91 85 96 100 91 100 96 97 95 97Thomson et al13 127 Vysis 63-65 97-98 95-96 86-93 (0);

30-33 (1+)Tubbs et al27 144 Vysis 73 68 86 93 57 85 93 93 83 91

FISH, fluorescence in situ hybridization; NPV, negative predictive value; PPV, positive predictive value; 3+, strong complete membranous staining in more than 10% of cells.* For the methods used to calculate sensitivity, specificity, PPV, NPV, and efficiency and for proprietary information, see the text. If


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only 5 m thick, whereas the tumor nucleus may be several

times this size, thereby necessarily missing some signals.

Counting multiple cells (40 for INFORM and 60 for Pathvy-

sion) and using the presence of 2 chromosome 17

centromeric signals (Pathvysion) helps ensure a more accu-

rate count. Measuring chromosome 17 and giving a ratio of

gene copy number of HER-2 to chromosome 17 excludes

cases with chromosome 17 polysomy from being scored aspositive, although the data are not completely clear

whether cases with polysomy chromosome 17 respond to

trastuzumab treatment.

Compared with immunohistochemical analysis, FISH

is more expensive, more time-consuming, and technically

more difficult to perform. FISH reagent costs are typically

at least 3 times the costs of immunohistochemical analysis.

Technical assay performance takes approximately 16 hours

for FISH compared with 3 hours for immunohistochemical

analysis, and technicians need to be specially trained for

FISH. Technologists may be delegated the time-consuming task of counting cells, but the differentiation

of tumor cells vs normal cells and in situ vs invasive

carcinoma cells needs to be performed by a trained

pathologist.

Additional negative aspects of FISH testing include

missing some overexpressed cases, the need for special

equipment, and the lack of permanent slides for review.

FISH detects only gene amplification and will not detect

protein overexpression, which occurs in 3% to 10% of

breast cancer cases.32 The additional equipment required

for FISH (fluorescent microscope) is often available in

standard hospital laboratories for other purposes, such as

for evaluating kidney biopsy specimens, but most patholo-

gists do not routinely perform fluorescent microscopy.

Special filters are required, as well as photography equip-

ment, if permanent records are desired. Fluorescent signals

will quench, and although slides may be viewed more than

once, they cannot be retained for years, as immunostained

slides can be. The use of photography may overcome thepermanent record problem, but of course photos permit the

viewing of only small areas of the original slide. On a

high-power image, one may not be sure whether the area

being viewed is part of an in situ or an invasive carcinoma.

This is a crucial point in determining a positive assay, as in

situ areas more often are positive for HER-2 than are inva-

sive areas of tumor, but it is the invasive area that should

be analyzed for determination of HER-2 status.

FISH vs Immunohistochemical Analysis

The College of American Pathologists issued a

consensus statement, as a result of a 1999 conference,

regarding the measurement of HER-2 as a prognostic

marker.33 Recommendations include specifying the method

and reagent used. For cases that are indeterminate by

immunohistochemical analysis, another method is recom-

mended to confirm the results, such as FISH. At that time,

specific reagents or methods for HER-2 testing were not

recommended because of lack of standardization and

comparability data. Since that time, a number of studies

A B

Image 2 A, Breast cancer, fluorescent in situ hybridization (FISH) for the HER-2 gene (orange dots). This tumor is gene

amplified for HER-2. B, Breast cancer, FISH with probe for the centromeric region of chromosome 17 (green dots). Note thatsome nuclei overlap and cells in division appear to have more than 2 copies.


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have been published directly comparing FISH and immuno-

histochemical analysis.7,13-29,32 Tables 2 through 5 show a

summary of recent literature detailing the results of

comparison of FISH with the HercepTest and with 3

commonly used antibodies, A0485, CB11, and TAB250.

For purposes of this review, data presented in the arti-

cles were analyzed and the following parameters were

calculated, where possible (TP, true positive; FP, false posi-

tive; TN, true negative; and FN, false negative): Sensitivity

= TP/(TP + FN); Specificity = TN/(FP + TN); Positive

Predictive Value = TP/(TP + FP); Negative Predictive Value

= TN/(TN + FN); and efficiency = (TP + FP)/(TP + FP +

TN + FN).34 There are variations in study populations,

methods, and scoring, even when analyzing the same anti-

bodies, which hinders the comparison of studies. The

analyses were performed on invasive breast cancer cases, if

specified, using standard scoring of 0 and 1+ as negative

and 2+ and 3+ as positive for each antibody, with FISH

used as the gold standard. The analyses then wererepeated by excluding the 2+ cases (or weakly positive

cases, if an alternative scoring method was used) and using

the scores 0 and 1+ as negative and considering only the 3+

cases as positive (Tables 2-5). Note that some of the articles

did not present data so that the second analysis could be

performed from published material. In reviewing these

data, the following observations are made: (1) There is

substantial variation among laboratories in reported sensi-

tivity, specificity, positive predictive value, and efficiency,

but the negative predictive value is excellent across all anti-

bodies. (2) The positive predictive value is increased byexcluding 2+ cases. (3) There is no consistent pattern of an

antibody having the best sensitivity, specificity, or other

parameter, although the monoclonal antibodies seem to

have better positive predictive values than the polyclonal,

especially when considering both 2+ and 3+ cases as posi-

tive; results seem to depend on the laboratory performing

the assay.

An automated reading system for immunohistochemical

analysis has been tested and may show increased sensitivity

with improved correlation with FISH.20 However, this

method adds substantially to the cost of the procedure; sensi-tivity and specificity are improved more by simply excluding

the 2+ cases, as shown in the present analysis.

It seems clear that immunohistochemical results that are

intermediate (2+) should not be regarded as positive, but

rather as borderline, and that these specimens should

undergo further testing. The 3+ cases by immunohistochem-

ical analysis are excellent predictors of gene amplification,

and negative (0 and 1+) immunohistochemical results predict

negative gene amplification very well. A few false-negative

results will occur using either immunohistochemical analysis

or FISH as the primary testing method.


Enzyme-Linked Immunosorbent Assay

The external domain of the HER-2 receptor is the ligand-

binding portion, also known as the extracellular domain

(ECD). Proteolytic processes cause the external domain to be

shed into the serum, possibly mediated by matrix metallopro-

teinases,35 and this shed ECD has been shown to be present in

breast cancer patients. ELISA is a monoclonal assay that can

be performed on either cell lysates from tissues or on fluids.

Because immunohistochemical analysis and FISH permit

histologic correlation on tissues, which is not possible on cell

lysates, the marketing of ELISA has concentrated on its use

in serum. ELISA can be performed on serum at any time

during the disease and has the advantage of being a quantita-

tive test. However, serum ELISA levels tend to vary with

tumor burden, time, and factors such as the phase of

menstrual cycle. HER-2 ECD levels may be detected in

healthy patients, and studies comparing healthy patients and

those with breast cancer are ongoing, with optimal levels forthe cutoff value still being defined. ELISA is available on an

automated platform (Bayer Diagnostics, Tarrytown, NY), so

that reproducibility should be excellent.

Circulating HER-2 antigen levels correspond to expres-

sion shown by immunohistochemical analysis and tumor

bulk,36-38 but relatively little ECD is shed during early-stage

disease. Only 25% to 50% of patients with HER-2positive

breast cancer by tissue assays will have detectable serum

HER-2 ECD. The clinical sensitivity of the assay is nearly 0

in stage I tumors and increases to about 40% for stage IV

tumors.39,40 For this reason, the serum assay may be usefulonly in cases of metastatic disease. Approximately 17% to

20% of patients with breast cancer whose tumors initially

test negatively for HER-2 may experience recurrence, with

HER-2positive serum. Possible reasons for this may be

tumor heterogeneity with selection for an HER-2positive

clone with disease progression, a false-negative initial assay

result, mutations after the initial assay, and up-regulation of

HER-2 expression by estrogen deprivation.

For disease monitoring, surprisingly, trastuzumab does

not seem to interfere with the ELISA assay results, although

this drug is a monoclonal antibody against HER-2. Nor doesa high level of HER-2 ECD inhibit response to trastuzumab

therapy. Trastuzumab seems to down-regulate HER-2 and

prevent cleavage.

Immunohistochemical Analysis, FISH,

and ELISA

For pathologists, immunohistochemical testing remains

the favored initial testing method in typical hospital laborato-

ries because most such laboratories are set up to perform


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Use of one assay method does not exclude measurement

by alternative methods. A triage system of using immunohis-

tochemical analysis as a screening method, FISH as a confir-

matory test,23,27,41 and ELISA for disease monitoring has

been proposed42 Figure 2. This triage system uses the rela-

tively cost-effective immunohistochemical method for

detecting high expressers on tissue and uses the more expen-

sive FISH method for a limited number of borderline cases.

ELISA ECD is most likely to be clinically useful in the early

detection of recurrent disease in HER-2positive tumors and,

possibly, to monitor results of trastuzumab therapy.

Future Directions

CISH is a new method of analyzing HER-2 that combines

the best characteristics of both immunohistochemical analysis

and FISH, that is, being able to identify gene copy number on

a permanent slide that can be visualized by a standard lightmicroscope Image 3. CISH assays depend on a technique

called subtractive hybridization,43,44 which uses a DNA probe,

visualized by a peroxidase reaction. Compared with FISH, this

method is easier to perform, easier to visualize, and results in a

stable prepared slide that does not require special microscopes

or filters to view. Compared with immunohistochemical

analysis, one can actually identify gene amplification and at

least semiquantitate the gene copy number, and reading the

gene copy number will not depend on factors such as the

degree of antigen retrieval. These positive aspects make this

technique one that may become widely used. However, thereare too few data on the clinical usefulness and pathologic

correlations to make a recommendation.45,46

The need to measure HER-2 continues to gain impor-

tance in the management of patients with breast carcinoma,

as well as patients with a variety of other carcinomas. Selec-

tion of optimal methods to measure HER-2 likely will

depend on availability, ease of use, cost, and correlation with

clinical characteristics.

From the Department of Pathology, University of Texas Health

Science Center at San Antonio.

Address reprint requests to Dr Yeh: Dept of Pathology,

University of Texas Health Science Center at San Antonio, 7703

Floyd Curl Dr, San Antonio, TX 78229.

Acknowledgments: I thank Tonya Keys for help with

manuscript preparation, technologists Oredius Pressley and

Guadalupe Castillo for help in the laboratory, and Jaishree

Jagirdar, MD, for reading and commenting on the manuscript.

References

1. Slamon DJ, Clark GM, Wong SG, et al. Human breastcancer: correlation of relapse and survival with amplification

of the HER-2/neu oncogene. Science. 1987;235:177-182.


immunohistochemical analysis, but not necessarily FISH. In

addition, the costs for immunohistochemical analysis arerelatively low, and it is less time-consuming to perform and

interpret. FISH has become favored by many oncologists

because of the relatively high false-positive rate (primarily in

low-positive, 2+ cases) and occasional false-negative results

seen with immunohistochemical analysis. The particular

antibody used may not be as important as the laboratory that

is performing the assay. In some hands, one antibody

performs better than others, and it should be up to the indi-

vidual laboratory to determine the best method for its own

conditions. At this time, serum ELISA seems to be mainly

useful for monitoring advanced-stage disease.

Figure 2 Proposed triage of HER-2 testing procedures. ELISA,

enzyme-linked immunosorbent assay; FISH, fluorescent in

situ hybridization; IHC, immunohistochemical analysis.

Image 3 Chromogenic in situ hybridization for HER-2 in

breast cancer (brown dots).


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