shnitt and jacobs ajcp 2000

8/3/2019 Shnitt and Jacobs AJCP 2000

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AJCP / COMMENTARY

806 Am J Clin Pathol 2001;116:806-810 American Society of Clinical Pathologists

Current Status of HER2 Testing

Caught Between a Rock and a Hard Place

Stuart J. Schnitt, MD, and Timothy W. Jacobs, MD

The analysis of breast cancer specimens for alterations in

the HER2/neu (c-erb-B2) gene or its protein product has

become common practice in surgical pathology laboratories.

Although some clinicians use the information derived from

these assays for assessing patient prognosis and evaluating

the likelihood of response to various chemotherapeutic agents

and to tamoxifen, the major clinical role of assessment of

HER2 status is to help determine patient suitability for treat-

ment with trastuzumab (Herceptin), a monoclonal antibody

targeted to the HER2 protein.1,2

Although there are a variety of methods available to

assess HER2 status in clinical breast cancer specimens,

assessment of protein overexpression using immunohisto-

chemical analysis and evaluation of gene amplification

using fluorescence in situ hybridization (FISH) are the

methods most commonly used in clinical practice today.3,4

There are currently 4 commercially available, US Food

and Drug Administrationapproved assays for deter-

mining HER2 status in breast cancer samples, although

these have not all been approved for the same purpose

Table 1. Among these 4 assays, 2 are immunohisto-

chemical assays (HercepTest, DAKO, Carpinteria, CA; and

Pathway, Ventana Medical Systems, Tucson, AZ) and 2 are

FISH assays (PathVysion, Vysis, Downers Grove, IL; and

Inform, Ventana). All of these assays can be performed

using automated instrumentation. In addition, there are

numerous commercially available anti-HER2 primary anti-

bodies and immunohistochemical detection systems for

HER2 testing for which Food and Drug Administration

approval has not been sought or received.

While the clinical value of assessing HER2 status in

human breast cancers to help determine suitability for

trastuzumab therapy is indisputable, the manner in which

the test should be performed and the results evaluated and

reported is a matter of heated debate and controversy

involving several groups with somewhat different (and

sometimes conflicting) agendas including pathologists,

medical oncologists, basic scientists, commercial vendors,

the lay press, and patients and their families. The College

of American Pathologists recently issued recommenda-

tions regarding HER2 testing for patients with breast

cancer.5 However, these recommendations largely reflect

the current lack of consensus with regard to how best to

evaluate HER2 status.

Table 1US Food and Drug AdministrationApproved Assays for HER2

Test Type Indication

HercepTest (DAKO, Carpinteria, CA) Immunohistochemical Suitability for trastuzumab (Herceptin)Pathway (Ventana Medical Systems, Tucson, AZ) Immunohistochemical Suitability for trastuzumabPathVysion (Vysis, Downers Grove, IL) Fluorescence in situ hybridization Assessing prognosis; predicting response

to chemotherapyInform (Oncor/Ventana) Fluorescence in situ hybridization Assessing prognosis


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Immunohistochemical analysis offers advantages over

FISH in that it is relatively inexpensive and fast, uses a light

microscope, and can be performed in most pathology labora-

tories. In addition, the results have been linked to prognosis

and response to treatment.

Furthermore, it seems more logical to determine HER2

protein expression (by immunohistochemical analysis) than

the level of gene amplification (by FISH) when treatmentssuch as trastuzumab are specifically targeted toward the

HER2 protein on the cell surface. Immunohistochemical

analysis should, therefore, be the more biologically relevant

assay, particularly in cases in which there is discordance

between gene copy number and level of protein expression.

However, the numerous commercially available anti-HER2

antibodies vary widely in sensitivity and specificity,

staining results are subject to variations in tissue fixation

and processing, there is no standard scoring system, there is

no uniformly accepted threshold for positivity, and the

results are not quantitative. FISH offers several advantages

over immunohistochemical analysis in that the options for

reagents are limited (there are only 2 commercially avail-

able reagent kits available at this time), the threshold for

positivity has been standardized, the results are quantita-

tive, and there are internal positive controls. Moreover,

DNA, the target for FISH, seems less affected by variations

in tissue fixation and processing than does protein, the

target for immunohistochemical analysis. However, the

capability for performing FISH is limited to a much smaller

number of pathology laboratories than is immunohisto-

chemical analysis, and FISH is more expensive, is techni-

cally more cumbersome, requires longer procedure and

interpretation times than does immunohistochemical

analysis, and requires a fluorescence microscope for inter-

pretation. In addition, although published data have

supported a link between FISH results and prognosis, data

linking FISH assay results and response to therapy

(including response to trastuzumab) are more limited.6

In most studies, the concordance rates between

immunohistochemical and FISH results are in the 80% to

95% range, with the highest levels of concordance among

cases that are either completely negative or strongly positive

by immunohistochemical analysis.3,7 Much lower levels of

agreement between immunohistochemical analysis and FISHresults are seen for cases in which the immunohistochemical

result is less than strongly positive.8-10

So, given that immunohistochemical analysis and FISH

each have their advantages and disadvantages, what is the

practicing pathologist to do in order to best serve the clinical

needs of patients with breast cancer and the physicians caring

for them? Unfortunately, there is no definitive answer to this

question, and, in a way, pathologists are caught between the

proverbial rock and hard place. How, then do we move

forward? As we see it, there are a number of options, with

each having its pros and cons:

Abandon immunohistochemical analysis and use FISH

as the exclusive method for evaluating HER2 status of breast

cancers. A number of investigators have argued that because

of the inherent limitations of immunohistochemical analysis

for evaluating HER2 protein overexpression on fixed,

paraffin-embedded tissue sections, FISH should be consid-ered the method of choice for determining HER2 status in

clinical breast cancer samples and could be used as a surro-

gate for HER2 protein expression.11 While this argument

certainly has merit, there currently are numerous practical

barriers to the routine use of FISH for determining HER2

status in breast cancers. In particular, as noted, the procedure

is more complicated, time consuming, and expensive than

immunohistochemical analysis, requires a fluorescence

microscope, and requires considerably more pathologist

interpretation time than does immunohistochemical

analysis.3 It is possible that the development of automated

methods for performing the assay and screening the slides

may make FISH more practical for the routine determination

of HER2 status.

Use immunohistochemical analysis as a screening step

followed by FISH in selected cases. Many authors recently

have advocated the use of a combined approach for

assessing HER2 status.9,10 In this scenario, immunohisto-

chemical analysis is used as the initial screening step for all

cases, and, following evaluation of the immunohistochem-

ical stains, a subset of cases is subjected to FISH, which is

considered the gold standard. The most commonly

proposed algorithm is to screen cases with immunohisto-

chemical analysis and then perform FISH on cases in which

the immunohistochemical stains show only weak or

moderate membrane staining (2+ in the HercepTest

scoring system), since many such cases seem to represent

immunohistochemical false-positive results. While this

represents a reasonable suggestion, a number of studies

have clearly shown that some immunohistochemically nega-

tive cases show gene amplification and some strongly posi-

tive cases (3+ in the HercepTest scoring system) lack

gene amplification.11 Moreover, preliminary data suggest

that among cases that are strongly positive (3+) by

immunohistochemical analysis, only the cases that alsoshow gene amplification by FISH respond to trastuzumab

treatment.6 Thus, limiting FISH testing to cases scored as

2+ by immunohistochemical analysis will not eliminate

all false-positive and false-negative immunohistochemical

results, and precisely which subset of cases should be

subjected to FISH following immunohistochemical analysis

remains a matter of debate. Furthermore, it is not at all clear

which immunohistochemical assay should be used as the

screening test in such a scenario.


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Develop methods to standardize immunohistochemical

analysis and/or render it more objective and quantitative.

One of the major problems with immunohistochemical

analysis is the lack of standardization with regard to primary

antibodies, detection systems, epitope/antigen retrieval, inter-

pretation, and reporting of results.12 The DAKO HercepTest

represented an important step toward standardizing immuno-

histochemical staining for HER2 by introducing predilutedreagents in kit form and a scoring system. However, some

studies have reported that this test has lower than desirable

specificity, whereas others have found that it has lower than

desirable sensitivity.8,10,11,13-15 Nevertheless, standardization

of immunohistochemical methods, with appropriate quality

control, remains an important goal.

Another major criticism of immunohistochemical

analysis has been that interpretation of the results is subjec-

tive and prone to interobserver variability, particularly at

intermediate levels of protein expression (ie, 2+ by the

HercepTest scoring system).13 Recently, a number of inves-

tigators have attempted to overcome this limitation of

immunohistochemical analysis by using computer-assisted

image analysis to quantitate HER2 immunohistochemical

results. For example, in the October 2001 issue of the

Journal, Wang et al16 retrospectively studied 189 breast

cancer cases using immunohistochemical analysis and

FISH and quantitated the immunohistochemical analysis

staining using the Automated Cellular Imaging System

(ACIS; ChromaVision Medical Systems, San Juan Capis-

trano, CA). The results of this study indicated that

immunohistochemical analysis quantitated with ACIS

showed a higher level of concordance with FISH than

immunohistochemical stains interpreted using visual

inspection (91% vs 86%) and that ACIS provided higher

sensitivity (but slightly lower specificity) than visually

interpreted immunohistochemical stains. Similar results

using other image analysis software have recently been

reported by others.17 However, Wang et al16 also appropri-

ately noted a number of limitations to the ACIS method,

which also apply to other computer-assisted image analysis

methods, including the cost of purchasing and maintaining

the hardware and computer software required for the quan-

titation, unresolved technical issues, and lack of data about

clinical validation of quantitative immunohistochemicalanalysis for HER2 protein determination. Perhaps most

important, unless the immunohistochemical analysis proce-

dure being subjected to image analysis has been optimized

by calibrating it against an appropriate standard,18 quantifi-

cation may simply provide an objective way to report an

incorrect result rather than a more reliable or accurate

result. Thus, while quantitation of immunohistochemical

results using computer-assisted image analysis could

provide certain important advantages over qualitatively or

semiquantitatively interpreted immunohistochemical stains,

there are a number of barriers and limitations to the wide-

spread implementation of this type of technology.

Develop methods to assess HER2 gene copy numbers

that can be evaluated using routine light microscopy. A

method that would combine the advantages of both immuno-

histochemical analysis and FISH might well be the most reli-

able and practical means to routinely assess HER2 status (ie,a DNA-based method that could be readily interpreted using

routine light microscopy). One such method is chromogenic

in situ hybridization (CISH). This approach permits the iden-

tification and quantitation of HER2 gene copies using light

microscopy since it uses a chromogenic rather than a fluores-

cent detection system. One study has indicated a high level

of concordance between CISH and FISH in enumerating

HER2 gene copy numbers.19 However, additional studies,

including those assessing the clinical value of CISH, are still

needed. Until such data are available, CISH should be

considered an investigational technique.

Develop tests that better predict the response of patients

to HER2-targeted therapy. Even if there were a method that

had 100% sensitivity, specificity, and accuracy in the deter-

mination of HER2 status of breast cancers, available data

suggest that this would still not permit the identification of

all patients who might or might not respond to HER2-

targeted antibody treatment with trastuzumab.2 Therefore,

there is great interest in developing a test that better predicts

response to such treatment. In particular, it is possible that

evaluation of the activated (phosphorylated) form of HER2,20

assessment of the level of expression of other members of

the epidermal growth factor receptor family in conjunction

with assessment of HER2, or assessment of downstream

effector molecules may provide more accurate information

than simply measuring HER2 status. However, additional

studies are needed to assess the clinical value of these alter-

native testing approaches.

Abandon tissue-based HER2 testing altogether. Some

investigators have studied the use of assays for circulating

HER2, in particular the measurement of the serum level of

the HER2 extracellular domain (ECD) in determining

HER2 status.21,22 Although the early results of such studies

showed promise, several potential problems exist in using a

serum assay for HER2 ECD. First, the assay has low sensi-tivity compared with immunohistochemical analysis or

FISH on corresponding tissue samples.22 Detection of ECD

is dependent on several factors, such as the level of antigen

production and release by the patients tumor and the half-

life of the protein in the serum. In addition, use of this assay

is subject to problems similar to those currently experienced

with immunohistochemical analysis and FISH, such as stan-

dardization of method and determination of appropriate

assay cutoff levels.


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Await results of studies directly comparing different

assays for HER2 with regard to their relationship to clin-

ical outcome. Perhaps more important than anything else at

this time, the various available assays for determining

HER2 status need to be directly and rigorously compared

within the setting of prospective clinical trials to determine

their relative merit and also to determine the appropriate

threshold for HER2 positivity. It should be noted, however,that the threshold for HER2 positivity may vary, depending

on the clinical question being asked. For example, the level

of HER2 protein expression required to declare a case

HER2-positive may be different when evaluating response

to treatment with trastuzumab than when determining sensi-

tivity to anthracyclines. Similarly, determining gene copy

number (eg, by FISH) may be more appropriate in some

clinical settings than others. Some recently published23 and

ongoing studies are attempting to address these issues.

In the meantime, in the absence of definitive answers,

it seems most prudent for pathologists to closely follow the

developments in this field and to work with their clinical

colleagues to formulate an approach to HER2 testing that is

most suitable at their institution. Ideally, before it is intro-

duced into clinical laboratory practice, any immunohisto-

chemical assay should be validated against another assay

such as FISH or a polymerase chain reactionbased assay.14

Based on extensive discussions with our clinical

colleagues, the approach we have settled on at our institu-

tion is to initially test all cases by immunohistochemical

analysis using a method we have validated against FISH.3,24

For cases that are strongly positive or clearly negative by

immunohistochemical analysis, no further testing is

performed. For cases that are equivocal or weakly positive

by immunohistochemical analysis (corresponding to 1+

or 2+ by HercepTest scoring) we perform FISH. We view

this as an interim solution until the issue of how best to

assess the HER2 status of breast cancers becomes more

clearly defined.

From the Department of Pathology, Beth Israel Deaconess

Medical Center and Harvard Medical School, Boston, MA.

Address reprint requests to Dr Schnitt: Dept of Pathology,

Beth Israel Deaconess Medical Center, 330 Brookline Ave,

Boston, MA 02215.Acknowledgments: We thank Anne Vincent-Salomon, MD,

and Jerome Couturier, MD, Insitut Curie, Paris, France, for their

helpful suggestions and comments.

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N Engl J Med. 2001;344:783-792.

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4. Hanna W, Kahn HJ, Trudeau M. Evaluation of HER-2/neu(erbB-2) status in breast cancer: from bench to bedside.Mod Pathol. 1999;12:827-834.

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17. Lehr HA, Jacobs TW, Yaziji H, et al. Quantitative evaluationof HER-2/neu status in breast cancer by fluorescence in situhybridization and by immunohistochemistry with imageanalysis.Am J Clin Pathol. 2001;115:814-822.

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