RAPID PUBLICATION
`Specificity of HercepTest in Determining HER-2/neu Status
`of Breast Cancers Using the United States Food and Drug
`Administration–Approved Scoring System
`
`By Timothy W. Jacobs, Allen M. Gown, Hadi Yaziji, Melissa J. Barnes, and Stuart J. Schnitt
`
`Purpose: To evaluate the specificity of the HercepTest
`for Immunoenzymatic Staining (Dako Corp, Carpinteria,
`CA) for determining HER-2/neu protein expression in
`breast cancer.
`Materials and Methods: Forty-eight invasive breast
`cancers previously found to be HER-2/neu–negative by
`two different immunohistochemical (IHC) assays and
`not amplified for the HER-2/neugene by fluorescence in
`situ hybridization were studied using the HercepTest kit.
`HercepTest was performed according to the manufactur-
`er’s guidelines, and the results were scored on a 0 to 31
`scale using the United States Food and Drug Administra-
`tion (FDA)–approved grading system. In this system,
`cases scored as 21 or 31 are considered HER-2/neu–
`positive.
`Results: Among these 48 cases, the IHC score using
`the FDA-approved scoring system was 0 in four cases
`(8.3%), 11 in 16 (33.3%), 21 in 21 (43.8%), and 31 in
`seven (14.6%). Therefore, 58.4% of these cases were
`
`categorized as HER-2/neu–positive, and the specificity
`of the HercepTest kit for HER-2/neu expression was
`41.6%. However, with the use of a modified scoring
`system that took into account the level of staining of
`nonneoplastic epithelium, the specificity increased to
`93.2%.
`Conclusion: Our results indicate that the HercepTest
`kit, when used in accordance with the manufacturer’s
`guidelines and the FDA-approved scoring system, re-
`sults in a large proportion of breast cancers being
`categorized as positive for HER-2/neu protein expres-
`sion and that many of these seem to be false-positives.
`Consideration of the level of staining of nonneoplastic
`epithelium resulted in improved specificity. The current
`FDA-approved scoring system for HercepTest results
`should be reevaluated before its widespread use in
`clinical practice.
`J Clin Oncol 17:1983-1987. r 1999 by American
`SocietyofClinicalOncology.
`
`THE HER-2/neu (c-erbB-2) oncogene encodes a 185-
`
`kda transmembrane protein (p185), which is overex-
`pressed in 20% to 30% of invasive breast carcinomas.1,2
`Since 1987, when Slamon et al1 first reported a significant
`relationship between amplification of the HER-2/neu onco-
`gene and poor clinical outcome in breast cancer patients,
`numerous studies have examined the utility of HER-2/neu as
`a prognostic factor. Recent evidence also supports a role for
`HER-2/neu status of breast cancers as predictive of their
`sensitivity or resistance to various forms of systemic therapy.
`Most recently, HER-2/neu protein expression has been used
`to select patients for treatment with trastuzumab (Herceptin,
`Genentech, Inc, South San Francisco, CA), a monoclonal
`antibody to the HER-2/neu protein. Initial clinical trials have
`indicated that this therapy may be useful in prolonging the
`survival of patients with advanced, metastatic breast carci-
`noma.3-5 Several studies have also indicated that tumors that
`overexpress HER-2/neu may show resistance to certain
`forms of chemotherapy (such as cyclophosphamide/metho-
`trexate)6-11 and sensitivity to others (such as doxorubi-
`cin).12-15 Furthermore, some clinical studies have suggested
`that HER-2/neu overexpression is predictive of resistance to
`tamoxifen.11,16-19
`Therefore, analysis of the HER-2/neu status of breast
`cancer specimens is assuming increasing clinical relevance.
`
`Immunohistochemistry (IHC) is commonly used for evaluat-
`ing HER-2/neu protein expression on formalin-fixed, paraffin-
`embedded samples of breast cancer.20-22 However, given that
`various assay protocols, HER-2/neu antibodies, and scoring
`systems are currently in use, variability in HER-2/neu IHC
`results has become a matter of legitimate concern.23-27 A
`standardized IHC kit for the evaluation of HER-2/neu
`protein expression (HercepTest for Immunoenzymatic Stain-
`ing, Dako Corp, Carpinteria, CA) has recently been ap-
`proved by the United States Food and Drug Administration
`(FDA). Of note, this release coincided with the FDA’s
`approval of trastuzumab.28 As a result of these develop-
`ments, there is now great interest among both clinicians and
`pathologists in evaluating the ability of the HercepTest assay
`to accurately determine the HER-2/neu status of breast
`
`From the Department of Pathology, Beth Israel Deaconess Medical
`Center and Harvard Medical School, Boston, MA; and PhenoPath
`Laboratories and IRIS, Seattle, WA.
`Submitted February 8, 1999; accepted April 9, 1999.
`Address reprint requests to Stuart J. Schnitt, MD, Department of
`Pathology, Beth Israel Deaconess Medical Center–East Campus,
`330 Brookline Ave, Boston, MA 02215; email sschnitt@caregroup.
`harvard.edu.
`r 1999 by American Society of Clinical Oncology.
`0732-183X/99/1707-1983
`
`JournalofClinicalOncology, Vol 17, No 7 (July), 1999: pp 1983-1987
`
`1983
`
`Downloaded from jco.ascopubs.org on December 1, 2014. For personal use only. No other uses without permission.
`Copyright © 1999 American Society of Clinical Oncology. All rights reserved.
`
`IMMUNOGEN 2192, pg. 1
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`1984
`
`cancers. The specificity of the assay is of particular concern,
`because low specificity, manifested as a large number of
`false-positive results, could result in inappropriate use of
`trastuzumab.
`The purpose of this study was to evaluate the HercepTest
`kit in a series of breast cancers previously shown to be
`negative for HER-2/neu protein expression by two other
`IHC assays and nonamplified for the HER-2/neu gene by
`fluorescence in situ hybridization (FISH).
`
`MATERIALS AND METHODS
`
`Study Design
`
`We previously studied 100 consecutive cases of invasive breast
`cancer for HER-2/neu protein overexpression using two IHC methods
`and for HER-2/neu gene amplification by FISH.29,30 Of note, the two
`prior IHC assays employed different methodology and scoring systems
`but used the same primary antibody (Dako rabbit antihuman polyclonal
`antibody to c-erbB-2 oncoprotein, code number A0485; Dako Corp),29,30
`which is a concentrate of the same anti–HER-2/neu antibody provided
`in prediluted form in the HercepTest kit. In these prior analyses, 22.9%
`of the cases showed HER-2/neu protein overexpression by one of the
`IHC methods, 23.7% showed HER-2/neu overexpression by the second
`IHC method, and 25.8% showed HER-2/neu gene amplification by
`FISH. Among these 100 cases, there was sufficient tissue remaining in
`the paraffin block for further IHC analysis in 48 cases that lacked
`HER-2/neu overexpression by both IHC assays and lacked HER-2/neu
`gene amplification by FISH. These 48 cases constitute the population
`for this study. All cases had been accessioned at Beth Israel Deaconess
`Medical Center (BIDMC), Boston, MA, between July 24, 1997, and
`February 18, 1998. The tissue from these cases was fixed initially in
`alcoholic formalin (Anatech, Ltd, Battle Creek, MI) followed by
`fixation in 10% neutral buffered formalin. For each case, 4-µm thick
`tissue sections were cut from a representative paraffin block and applied
`to positively charged slides.
`In the first of the prior IHC assays, performed at PhenoPath
`Laboratories (PPL), Seattle, WA, tissue sections were subjected to
`heat-induced epitope retrieval (HIER) by immersion in citrate buffer
`(pH 6.0) preheated to greater than 90°C and heating in a Black &
`Decker vegetable steamer (Black & Decker Corp, Towson, MD) for 20
`minutes before incubation with the anti–HER-2/neu antibody on a Dako
`Autostainer. Primary antibody was localized using the LSAB1 Detec-
`tion System (labeled streptavidin biotin immunoperoxidase; Dako
`Corp) according to the manufacturer’s instructions using the Dako
`Autostainer. Membrane staining intensity and pattern were evaluated
`using a 0 to 41 scale (0, completely negative; 11, faint membranous
`positivity; 21, moderate membranous positivity; 31, strong, circumfer-
`ential membranous positivity; and 41, extremely strong, circumferen-
`tial membranous positivity). For a score of 21 to 41, membrane
`staining in the majority of the tumor cells was required to be present.
`Cytoplasmic immunostaining was noted but not incorporated into the
`final scoring. For each case, infiltrating carcinoma and adjacent normal
`epithelium (if available) were separately scored. A final subtracted score
`of the tumor minus normal epithelium was used to correct for variability
`in background staining of normal epithelium (which should not
`overexpress the HER-2/neu protein). Either a final subtracted score of $
`2 or tumor cell staining of 31 or greater was required to categorize a
`case as HER-2/neu–positive.
`
`JACOBS ET AL
`
`In the second of the previous IHC assays, performed at BIDMC,
`tissue sections were subjected to HIER by heating in a microwave oven
`in citrate buffer (pH 5 6) for a total of 10 minutes before immunostain-
`ing using the Ventana 320 automated immunostainer (Ventana Medical
`Systems, Tucson, AZ). The primary antibody to the HER-2/neu
`oncoprotein was used at a 1:500 dilution, and diaminobenzidine (DAB,
`Sigma Chemicals, St. Louis, MO) was used as the chromogen. HER-2/neu
`staining was considered positive when the tumor cells showed intense
`circumferential cell membrane staining, easily identified with a 103
`objective. In all of these cases, staining was observed in the majority (.
`50%) of the tumor cells. Tumors in which there was cytoplasmic
`staining without distinct cell membrane staining were scored as negative.
`All 48 cases had also been previously analyzed for HER-2/neu gene
`amplification using the Oncor/Ventana INFORM HER-2/neu Gene
`Detection System (Ventana Medical Systems; formerly sold by Oncor,
`Inc, Gaithersburg, MD) at BIDMC in a laboratory certified by Oncor as
`proficient in the procedure. The methodology and interpretation were in
`accordance with the guide accompanying the kit31 as previously
`described.29 Briefly, tissue sections were digested with proteinase,
`denatured, and hybridized with Oncor biotinylated HER-2/neu DNA
`probe. Oncor Fluorescein-Labeled Avidin Detection Reagent and Oncor
`Anti-Avidin Antibody were used for probe detection. Nuclei were
`counterstained with 48-68-diamidino-28-phenylindole (DAPI)/Antifade.
`Slides were examined using a fluorescence microscope. Twenty ran-
`domly selected invasive tumor cell nuclei in each of two separate,
`distinct microscopic areas were evaluated for HER-2/neu gene copy
`number (ie, a total of 40 nuclei per case). Cases were scored as
`amplified by FISH when the mean number of fluorescent signals per
`nucleus was greater than four.
`
`HercepTest IHC Assay
`
`In this study, HER-2/neu protein expression was evaluated using the
`HercepTest for Immunoenzymatic Staining at PPL according to the
`protocol described in the manufacturer’s guide accompanying the kit.
`Tissue sections were deparaffinized in two 5-minute changes of xylene
`and were rehydrated through alcohols to distilled water. Subsequently,
`sections were subjected to HIER by immersing the slides in Dako Epitope
`Retrieval Solution (0.01 mol/L citrate buffer; pH 5 6) preheated to
`95°C, and then heated in waterbath at 95°C for a total of 40 minutes,
`followed by a 20-minute cooldown period at room temperature. Slides were
`incubated with the primary rabbit polyclonal antibody to the HER-2/neu
`oncoprotein (as supplied prediluted in the HercepTest kit) on a Dako
`Autostainer for 30 minutes at room temperature. Antibody was local-
`ized by incubating slides with the Dako Visualization Reagent (dextran
`polymer conjugated with horseradish peroxidase and goat antirabbit
`immunoglobulins) for 30 minutes using the Dako Autostainer. Diamino-
`benzidine (DAB) was used as the chromogen, and the sections were
`counterstained with hematoxylin. Positive controls were included in
`each staining run and consisted of freshly cut breast cancer cases known
`to express HER-2/neu and a control slide consisting of three pelleted,
`formalin-fixed, paraffin-embedded human breast cell lines with staining
`intensity scores of 0, 11, and 31 (supplied in the HercepTest kit).
`Negative controls consisted of substituting normal rabbit serum (Dako
`Negative Control Reagent) for the HER-2/neu primary antibody. Only
`membrane staining intensity and pattern were evaluated using the 0 to
`31 scale as illustrated in the HercepTest kit scoring guidelines. As defined in
`the HercepTest kit guide, scores of 0 or 11 were considered negative
`for HER-2/neu overexpression, 21 was weak positive, and 31 was
`strong positive. To qualify for 21 and 31 scoring (ie, positive), complete
`membrane staining of more than 10% of tumor cells had to be observed.
`
`Downloaded from jco.ascopubs.org on December 1, 2014. For personal use only. No other uses without permission.
`Copyright © 1999 American Society of Clinical Oncology. All rights reserved.
`
`IMMUNOGEN 2192, pg. 2
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`HERCEPTEST SPECIFICITY
`
`We also used a modification of this scoring system that took into
`consideration the level of staining of nonneoplastic epithelium present
`on the same slide as the cancer. In this system, nonneoplastic epithelium
`was also graded on a 0 to 31 scale using the same criteria used for
`assessment of tumor cell staining. Cases were considered HER-2/neu
`positive only when the difference between the tumor cell staining score
`and the nonneoplastic epithelial cell staining score was $ 2.
`
`Calculation of HercepTest Assay Specificity
`
`Specificity of the HercepTest was defined as the number of true-
`negative cases (ie, cases that were negative for HER-2/neu protein
`expression by HercepTest
`that were also negative for HER-2/neu
`protein expression by both prior IHC assays and negative for HER-2/
`neu gene amplification by the FISH assay) divided by the total number
`cases that were HER-2/neu–negative by both prior IHC assays and by
`the FISH assay (ie, true-negatives and false-positives by HercepTest).
`Specificity was expressed as a percentage.
`
`RESULTS
`Patient Data and Histologic Features of Carcinomas
`
`The median age of the patients was 66 years (range, 36 to
`89 years). Thirty-two of the 48 carcinomas (66.7%) were of
`infiltrating ductal
`type, seven (14.6%) were infiltrating
`lobular, four (8.3%) were invasive cancers with both ductal
`and lobular features, three (6.3%) were mucinous (colloid)
`carcinomas, and two (4.2%) were tubular carcinomas. The
`median size of the tumors was 15 mm (range, 6 to 90 mm).
`Histologic grading was performed using the Elston and
`Ellis32 modification of the Bloom-Richardson grading sys-
`tem. Sixteen of the 48 carcinomas (33.3%) were grade 1, 18
`(37.5%) were grade 2, and 14 (29.2%) were grade 3. Twenty
`of the 48 patients (41.7%) were axillary lymph node-
`negative and 13 (27.1%) were node-positive. Fifteen pa-
`tients did not undergo axillary lymph node dissection.
`Forty-one of the 48 cases (85.4%) were estrogen receptor
`(ER)-positive and six (12.5%) were ER-negative. ER status
`was not determined in one case (Table 1).
`
`HER-2/neu Status
`
`All 48 cases were negative for HER-2/neu protein expres-
`sion by previous IHC assays at both PPL and BIDMC, and
`none were amplified for the HER-2/neu gene by FISH.29,30
`However, using the HercepTest IHC kit and the FDA-
`approved scoring system, 28 of these cases (58.4%) were
`interpreted as positive (score of 21 or 31), and 20 (41.6%)
`were interpreted as negative (score of 0 or 11) (Table 2).
`Therefore, if the results of the three previous HER-2/neu
`assays performed on these cases are considered true-
`negative results, then under these circumstances, the specific-
`ity of the HercepTest kit for HER-2/neu protein expression
`was 41.6%.
`
`1985
`
`Table 1. Clinical Data and Pathologic Features of Cases Analyzed for
`HER-2/neuProtein Overexpression by HercepTest (n 5 48)
`
`Characteristic
`
`No.
`
`%
`
`Age, years
`Median
`Range
`Histologic type
`Infiltrating ductal
`Infiltrating lobular
`Mixed ductal and lobular
`Mucinous (colloid)
`Tubular
`Size, mm
`Median
`Range
`Histologic grade
`1
`2
`3
`Axillary nodal status
`Negative
`Positive
`No axillary dissection
`ER status
`Positive
`Negative
`Not determined
`
`66
`36-89
`
`15
`6-90
`
`66.7
`8.3
`8.3
`6.3
`4.2
`
`33.3
`37.5
`29.2
`
`41.7
`27.1
`31.2
`
`85.4
`12.5
`2.1
`
`32
`7
`4
`3
`2
`
`16
`18
`14
`
`20
`13
`15
`
`41
`6
`1
`
`In 44 of these cases, nonneoplastic epithelium was present
`on the same tissue sections as the cancer. The HercepTest
`score in the benign epithelium was 0 in five cases (11.4%),
`11 in 15 (34.1%), 21 in 21 (47.7%), and 31 in three
`(6.8%). The difference between the tumor cell score and the
`nonneoplastic epithelial cell score was $ 2 in only three
`cases, and these three cases were considered HER-2/neu–
`positive. Therefore, when the level of staining of nonneoplas-
`tic epithelium was taken into consideration, the specificity of
`the HercepTest increased to 93.2%.
`
`DISCUSSION
`In this study, the HercepTest kit, when used in accordance
`with the manufacturer’s guidelines and FDA-approved scor-
`ing system, categorized as HER-2/neu–positive almost 60%
`
`Table 2. HER-2/neuStatus by IHC Using the HercepTest Kit
`
`Dako IHC Score*
`
`Dako IHC Interpretation*
`
`0
`11
`21
`31
`
`Negative
`Negative
`Weak positive
`Strong positive
`
`Cases
`
`%
`
`8.3
`33.3
`43.8
`14.6
`
`No.
`
`4
`16
`21
`7
`
`NOTE. HercepTest was performed on 48 cases that were all negative for
`HER-2/neu protein expression by two other IHC assays and nonamplified for
`the HER-2/neugene by FISH.
`*According to the manufacturer’s FDA-approved guidelines.
`
`Downloaded from jco.ascopubs.org on December 1, 2014. For personal use only. No other uses without permission.
`Copyright © 1999 American Society of Clinical Oncology. All rights reserved.
`
`IMMUNOGEN 2192, pg. 3
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`1986
`
`of invasive breast cancers that were previously shown to
`lack both HER-2/neu protein expression and HER-2/neu
`gene amplification in prior assays. These findings are subject
`to a number of different interpretations. First, it could be
`argued that the results using the HercepTest kit accurately
`reflect the HER-2/neu protein expression status of these
`cases and that our prior results, in which these cases were
`categorized as HER-2/neu–negative, were incorrect. How-
`ever, this interpretation seems unlikely for several reasons.
`First,
`the HER-2/neu positivity rate by IHC using the
`HercepTest kit in this selected series of cases was substan-
`tially higher than the 20% to 30% rate of positivity noted in
`unselected series of breast cancers reported in other stud-
`ies.1,2 Second, all of the cases in this study that were scored
`as positive using the HercepTest kit lacked HER-2/neu gene
`amplification as determined by a FISH assay. Although prior
`studies have clearly shown that breast cancers may exhibit
`HER-2/neu protein expression in the absence of gene
`amplification, this phenomenon has been observed in only
`3% to 7% of cases.33-35 Third, the HercepTest assay was
`performed and the results were scored strictly in accordance
`with the manufacturer’s recommendations and FDA-
`approved scoring system. Therefore, neither technical nor
`interpretive deviations from the proscribed method are likely
`to explain these results.
`An alternative explanation for our findings is that the
`HercepTest assay, when used according to the manufactur-
`er’s FDA-approved guidelines, has low specificity for the
`detection of HER-2/neu protein expression. This interpreta-
`tion is in agreement with the recent findings of Roche and
`Ingle.36 These investigators noted a HER-2/neu positivity
`rate of 54% using the HercepTest kit in 59 cases that were
`found to be HER-2/neu–negative using another IHC assay. It
`could be argued that this comparison is not entirely valid,
`because these authors compared results of the HercepTest
`assay to an assay that uses a different antibody (monoclonal
`antibody CB11). However, our prior negative IHC results on
`the cases evaluated in the current study were obtained using
`the same primary anti–HER-2/neu antibody supplied in the
`HercepTest kit. Therefore, it seems unlikely that the ob-
`served discrepancies in IHC results between the HercepTest
`and other IHC assays is related to the nature of the primary
`antibody alone. It
`is possible that other methodologic
`
`JACOBS ET AL
`
`aspects of the assay contributed to the low specificity
`observed in the study of Roche and Ingle36 and in our study.
`However, our results strongly suggest that the low specific-
`ity is in large part related to the use of the recommended
`scoring system, because a dramatic improvement in specific-
`ity was noted when the level of staining of nonneoplastic
`epithelium was taken into account in our modified scoring
`system.
`Previous studies have highlighted a number of potential
`problems in the use of IHC assays for HER-2/neu, including
`variability in tissue fixation and processing, variable sensitiv-
`ity and specificity of commercially available antibodies, and
`differences in scoring criteria.23-25 In our experience, varia-
`tions in the type of fixative, length of tissue fixation, and
`details of tissue processing can result in differences in the
`intensity of specific staining for HER-2/neu in tumor cells as
`well as in variable levels of staining of nonneoplastic
`epithelium. In particular, fixatives that contain alcohol
`(including alcoholic formalin) can result
`in prominent
`staining of nonneoplastic epithelium in some cases. Any
`scoring system must, therefore, take into account the immu-
`table fact that different fixation and processing protocols will
`be used in different laboratories. Our results suggest that
`consideration of the level of staining of nonneoplastic
`epithelium helps to ‘‘normalize’’ the level of HER-2/neu
`staining by serving as an internal control and may help to
`compensate for interlaboratory differences in tissue fixation
`and processing. However, one potential limitation of this
`approach is the lack of nonneoplastic epithelium in associa-
`tion with some primary tumors and in metastatic lesions.
`The development of standardized methods for HER-2/neu
`IHC is clearly an important goal. However, our results
`suggest that the HercepTest kit, the first such proposed
`standardized assay, has low specificity for HER-2/neu
`protein expression when used in accordance with the
`manufacturer’s guidelines and FDA-approved scoring sys-
`tem. Pathologists who perform assays for HER-2/neu and
`clinicians who use this information in formulating therapeu-
`tic recommendations need to be aware of these issues. In
`particular, the current FDA-approved scoring system for
`HercepTest should be re-evaluated before widespread use of
`the scoring system in clinical practice.
`
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`Copyright © 1999 American Society of Clinical Oncology. All rights reserved.
`
`IMMUNOGEN 2192, pg. 4
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`HERCEPTEST SPECIFICITY
`
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