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`Case 1:18-cv-01363-CFC Document 82-8 Filed 03/22/19 Page 2 of 10 PageID #:
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`RAPID PUBLICATION
`Comparison of Fluorescence In Situ Hybridization
`and Immunohistochemistry for the Evaluation
`of HER-2/neu in Breast Cancer
`
`By Timothy W. Jacobs, Allen M. Gown, Hadi Yaziji, Melissa J. Barnes, and Stuart J. Schnitt
`
`Purpose: To compare fluorescence in situ hybridiza-
`tion (FISH) and immunohistochemistry (IHC) in the deter-
`mination of HER-2/neustatus of breast cancers.
`Materials and Methods: FISH and IHC for HER-2/neu
`were performed on formalin-fixed paraffin sections of
`100 consecutive invasive breast cancers. FISH was per-
`formed at Beth Israel Deaconess Medical Center, Boston,
`MA, using the Oncor/Ventana INFORM kit (Ventana
`Medical Systems, Tucson, AZ; formerly sold by Oncor,
`Inc, Gaithersburg, MD) in a laboratory certified as profi-
`cient in this procedure. IHC was performed at Pheno-
`Path Laboratories, Seattle, WA, using a polyclonal anti-
`body to the HER-2/neu protein. FISH and IHC were
`analyzed in a blinded fashion, and the results were then
`compared. Procedure and interpretation times and re-
`agent costs for FISH and IHC were also compared.
`Results: HER-2/neuwas amplified by FISH in 26% of
`cases, and 23% were HER-2/neu–positive by IHC. FISH
`
`IN 1987, SLAMON et al1 first reported a significant
`
`relationship between amplification of the HER-2/neu
`(c-erbB-2) oncogene and adverse clinical outcome in pa-
`tients with breast cancer. Although subsequent studies have
`largely confirmed this association in patients with node-
`positive disease,2-22 whether or not HER-2/neu gene amplifi-
`cation or overexpression is an independent prognostic factor
`in patients with node-negative breast cancer remains a
`matter of controversy.6-11,14-16,19,22-37 More recently, there
`has been considerable interest
`in the potential role of
`HER-2/neu gene amplification and overexpression as a
`predictor of response or resistance to various therapeutic
`modalities in patients with breast cancer. In particular, the
`results of recent clinical trials have indicated that treatment
`with a monoclonal antibody to the HER-2/neu protein
`(trastuzumab [Herceptin, Genentech, Inc, South San Fran-
`
`From the Department of Pathology, Beth Israel Deaconess Medical
`Center and Harvard Medical School, Boston, MA; and PhenoPath
`Laboratories and IRIS, Seattle, WA.
`Submitted January 15, 1999; accepted April 7, 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/99/1707-1974
`
`and IHC were both assessable in 90 cases. Concordance
`between FISH and IHC results was seen in 82 of these
`cases (91%, P F .001). The FISH procedure required
`more technologist time and more interpretation time
`per case for the pathologist than IHC. Reagent costs
`were substantially higher for FISH than for IHC.
`Conclusion: There is a high level of correlation be-
`tween FISH and IHC in the evaluation of HER-2/neu
`status of breast cancers using formalin-fixed paraffin-
`embedded specimens. Although the choice of which
`assay to use should be left for individual laboratories to
`make based on technical and economic considerations,
`our results may make it difficult to justify the routine use
`of FISH for determination of HER-2/neustatus in breast
`cancer.
`J Clin Oncol 17:1974-1982. r 1999 by American
`SocietyofClinicalOncology.
`
`cisco, CA]) may be useful in prolonging the survival of
`patients with metastatic disease.38-40 Furthermore, some
`studies have indicated that tumors that overexpress HER-2/
`neu may show resistance to certain forms of cytotoxic
`therapy14,21,28,41-43 and sensitivity to others.44-47 Finally,
`some recent experimental and clinical studies have sug-
`gested that HER-2/neu overexpression is associated with
`resistance to tamoxifen.43,48-51 As a result of this informa-
`tion, there is a growing clinical demand for HER-2/neu
`analysis of current and archived breast cancer specimens.
`There are a variety of methods available to determine the
`HER-2/neu status of breast cancers. These include assays to
`evaluate gene amplification, including Southern blot,1,2 slot
`blot,52,53 and dot blot analyses,19,54 polymerase chain reac-
`tion,55,56 in situ hybridization,57 and fluorescent
`in situ
`hybridization (FISH)53,58,59; assays to determine mRNA
`overexpression, such as Northern blot analysis,2 slot blot
`analysis,52 and in situ hybridization52; and methods to assess
`protein overexpression, including Western blot analysis,2
`immunoassays,60 and immunohistochemistry (IHC).2,61-64
`Many of these methods are beyond the scope of most
`pathology laboratories for technical reasons. Furthermore,
`most of these assays require prospective collection of fresh
`tissue and are not applicable to archival material. Therefore,
`the most viable choices for HER-2/neu analysis in both
`routine clinical practice and in clinical research studies are
`
`1974
`
`JournalofClinicalOncology,Vol 17, No 7 (July), 1999: pp 1974-1982
`
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`Copyright © 2018 American Society of Clinical Oncology. All rights reserved.
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`Case 1:18-cv-01363-CFC Document 82-8 Filed 03/22/19 Page 3 of 10 PageID #:
`9881
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`FISH AND IHC FOR HER-2/neu IN BREAST CANCER
`
`1975
`
`FISH or IHC, performed on sections cut from formalin-
`fixed, paraffin-embedded specimens. Although each of these
`methods has its advantages and disadvantages, direct com-
`parisons of these two assays have been few and are limited
`by small numbers of cases.65-69 The purpose of this study,
`therefore, was to compare FISH and IHC in a single cohort
`of invasive breast cancers.
`
`MATERIALS AND METHODS
`
`Study Design
`
`The study population consisted of 100 consecutive cases of invasive
`breast cancer accessioned at Beth Israel Deaconess Medical Center,
`Boston, MA, between July 24, 1997, and February 18, 1998. These
`specimens had been fixed initially in alcoholic formalin (Anatech, Ltd,
`Battle Creek, MI) followed by fixation in 10% neutral-buffered
`formalin. Only cases with sufficient invasive carcinoma for multiple
`assays were included in the study. For each case, 4-µm thick tissue
`sections were cut from a representative paraffin block and applied to
`positively charged slides. FISH for HER-2/neu gene amplification was
`performed using the Oncor/Ventana INFORM Gene Detection System
`(Ventana Medical Systems, Tucson, AZ; formerly sold by Oncor, Inc,
`Gaithersburg, MD) at Beth Israel Deaconess Medical Center in a
`laboratory certified by Oncor as proficient in the procedure. IHC for
`HER-2/neu protein expression was performed at PhenoPath Laborato-
`ries, Seattle, WA, using a polyclonal antibody to the HER-2/neu protein.
`Interpretation of FISH and IHC were each performed by investigators
`blinded to the results of the other assay. The procedure (technologist)
`and interpretation (pathologist) time and reagent costs were determined
`for FISH and IHC.
`
`FISH for HER-2/neu Gene Amplification
`
`FISH for HER-2/neu was performed using the Oncor/Ventana
`INFORM HER-2/neu Gene Detection System as described in the guide
`accompanying the kit.70 Tissue sections (on slides) were baked over-
`night at 65°C, deparaffinized in three 5-minute changes of xylene,
`transferred through two 2-minute changes of 100% ethanol, and
`allowed to air-dry. Slides were immersed for 15 minutes in 30% Oncor
`Pretreatment Solution (Oncor, Inc, Gaithersburg, MD) at 43°C, briefly
`washed in 23 sodium chloride/sodium citrate (SSC) at room tempera-
`ture, dehydrated through 100% ethanol, and air-dried. Slides were then
`incubated for 60 minutes in Oncor Protein Digesting Enzyme Working
`Solution at 37°C. Slides were briefly washed in 23 SSC at room
`temperature, dehydrated through 100% ethanol, and allowed to air dry.
`Tissue sections were denatured by immersing slides in Oncor Denatur-
`ation Solution for 8 minutes at 75°C, followed by rinsing through 100
`ethanol at 220°C, and air-drying. Oncor biotinylated HER-2/neu DNA
`probe was prewarmed for 5 minutes at 37°C before application. Slides
`were incubated with probe overnight (12 to 16 hours) at 37°C in a
`humidified chamber. The amount of probe used (range, 10 to 20 µL) was
`adjusted according to the size of the tissue section to be covered. After
`hybridization, slides were immersed in Oncor Post-Hybridization Wash
`Solution at 43°C for 15 minutes, which was followed by washing in 23
`SSC at 37°C for 10 minutes with agitation. The wash was repeated with
`fresh 23 SSC. Slides were then immersed in 13 phosphate-buffered
`detergent (PBD) at 18° to 25oC. After application of Oncor Blocking
`Reagent One for 5 minutes at room temperature, Oncor Fluorescein-
`Labeled Avidin Detection Reagent was applied to the slides for a
`20-minute incubation in a humidified chamber at room temperature.
`
`Slides were then subjected to three 2-minute washes in 13 PBD at room
`temperature. After incubation with Oncor Blocking Reagent Two for 5
`minutes at room temperature, Oncor Anti-Avidin Antibody was applied
`to the slides (for signal amplification) and incubated for 20 minutes at
`room temperature in a humidified chamber. Slides were subjected to
`three 2-minute washes in 13 PBD at room temperature and incubated
`with Oncor Blocking Reagent One for 5 minutes in a humidified
`chamber at room temperature. Oncor Fluorescein-Labeled Avidin
`Detection Reagent was then applied to the slides, with incubation for 20
`minutes at room temperature followed by three 2-minute washes in 13
`PBD at room temperature. Nuclei were counterstained with 48-68-
`diamidino-28-phenylindole (DAPI)/Antifade. Slides were stored in the
`dark at 270°C for up to 5 days before analysis. Positive controls were
`included in each staining run and consisted of freshly cut paraffin
`sections of cases known to be amplified for the HER-2/neu gene by
`FISH.
`Slides were evaluated for HER-2/neu gene copy number using a Zeiss
`Axioscope fluorescence microscope (Zeiss, Inc, Thornwood, NY) by
`one observer (T.W.J.). Each slide was examined using DAPI, dual-band
`pass, and triple-band pass filters. Slides were scanned at low power
`(103 objective) using the DAPI filter to identify areas with optimal
`tissue digestion and nonoverlapping nuclei. Twenty randomly selected
`invasive tumor nuclei in each of two separate, distinct microscopic
`areas were evaluated (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. Cases with $ 20 mean signals
`per nucleus were scored as 20. Borderline cases (ie, 3.5 to 4.5 mean
`signals per nucleus) were scored by a second observer (S.J.S.). For each
`case, the corresponding hematoxylin and eosin slide was reviewed
`before FISH interpretation. In cases where invasive carcinoma could
`not be reliably identified by DAPI during the interpretation of FISH, the
`corresponding hematoxylin- and eosin-stained slide was re-reviewed
`concurrently. For each case, the following factors were also noted:
`adequacy of digestion, intensity of specific fluorescent signal, nonspe-
`cific fluorescent background, and tissue histology and quality (including
`degree of overlapping of tumor nuclei). If any of these factors precluded
`reliable interpretation of a case (as outlined in the Oncor Procedure and
`Interpretation Guide70),
`then the case was evaluated by a second
`observer (S.J.S.) and the assay was repeated. A case was categorized as
`uninformative (uninterpretable) if FISH could not be reliably inter-
`preted after repeating the assay a second time.
`
`IHC for HER-2/neu Protein Expression
`
`Four-µm sections from each case were deparaffinized and rehydrated
`in graded alcohols. The slides were subjected to heat-induced epitope
`retrieval by immersing them in 0.01 mol/L citrate buffer (pH 6.0)
`preheated to greater than 90°C and then heated in a Black & Decker
`vegetable steamer (Black & Decker Corp, Towson, MD) for 20 minutes,
`followed by a 20-minute cooldown period at room temperature. Slides
`were then incubated with an anti–HER-2/neu polyclonal antibody
`(Dako Corp, Carpinteria, CA; 1:1,000 dilution) on a Dako Autostainer
`(Dako Corp) for 30 minutes at room temperature. The primary antibody
`used in this study is the same anti–HER-2/neu antibody currently
`available in the Dako HercepTest kit (Dako Corp). However, in the
`HercepTest kit, the antibody is provided in a prediluted form. For each
`case, one slide was incubated with phosphate-buffered saline (PBS)
`instead of the primary antibody as a negative control. For positive
`controls, a composite slide composed of formalin-fixed cell pellets of
`the following four cell lines (obtained from Dr. Nora Disis, Fred
`Hutchinson Cancer Research Center, Seattle, WA) was used: MCF-7 (a
`cell line negative for HER-2/neu overexpression) and three human
`
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`Copyright © 2018 American Society of Clinical Oncology. All rights reserved.
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`

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`Case 1:18-cv-01363-CFC Document 82-8 Filed 03/22/19 Page 4 of 10 PageID #:
`9882
`
`1976
`
`carcinoma cell lines showing increasing levels of overexpression of
`HER-2/neu (BT-20 [low overexpressor], SKBR-3 [intermediate overex-
`pressor], and SKOV-3 [high overexpressor]). Antibody was localized
`using the LSAB1 Detection System (labeled streptavidin biotin
`immunoperoxidase; Dako Corp) according to the manufacturer’s instruc-
`tions using the Dako Autostainer (Dako Corp) and counterstained with
`hematoxylin.
`All slides were scored by one observer (A.M.G.). Only 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, circumferential membranous posi-
`tivity; and 41, extremely strong, circumferential membranous positiv-
`ity). 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 $ 21 or tumor cell staining of 31 or greater was
`required for the case to be considered positive.
`
`Procedure Time, Interpretation Time, and Reagent Costs
`
`The procedure time for each run of the FISH procedure was recorded
`by the technologist who performed the assay, and the average time per
`run was calculated. Similar data were recorded for the IHC procedure.
`The time required for interpretation of each FISH and IHC slide was
`recorded by the interpreting pathologist, and the average interpretation
`time per case was calculated for each procedure. The reagent cost per
`case was calculated by determining the actual retail cost of the FISH
`reagents and IHC reagents required to stain the study cases and dividing
`these total costs by the number of study cases evaluated by each assay.
`
`Statistical Methods
`Statistical significance was set at P , .05. Data were analyzed by the
`Mann-Whitney rank sum test, x2 test, and Fisher’s exact test, where
`appropriate. Computations were performed with the SigmaStat for
`Windows software (Version 2.03, SPSS, Inc, Chicago, IL).
`
`RESULTS
`Patient Data and Histologic Features of Carcinomas
`
`The median age of the patients was 53.5 years (range, 27
`to 89 years). Seventy-two carcinomas were of infiltrating
`ductal type, 11 were infiltrating lobular, nine were invasive
`cancers with both ductal and lobular features, three were
`mucinous (colloid) carcinomas, three were tubular carcino-
`mas, one was an invasive micropapillary carcinoma, and one
`was a metaplastic carcinoma. The median size of the tumors
`was 15 mm (range, 6 to 102 mm). Histologic grading was
`performed using the Elston and Ellis71 modification of the
`Bloom-Richardson grading system. Twenty-seven carcino-
`mas were grade 1, 37 were grade 2, and 35 were grade 3. The
`metaplastic carcinoma was not graded, because there are no
`universally accepted criteria for the grading of such lesions.
`
`JACOBS ET AL
`
`FISH for HER-2/neu
`
`Ninety-three of the 100 cases were interpretable for
`HER-2/neu gene amplification by FISH. In 24 of these 93
`cases (25.8%), HER-2/neu was interpreted as amplified (ie,
`mean fluorescent signals per nucleus . four). The average
`number of signals per nucleus in the amplified cases ranged
`from 4.2 to 20.0 (median, 12.0). In nonamplified cases, the
`mean number of fluorescent signals per nucleus ranged from
`1.3 to 3.6 (median, 1.8). Two cases were considered
`borderline, demonstrating 4.2 and 3.6 mean signals each per
`nucleus. However, in the subsequent analyses, the former
`case was considered amplified and the latter nonamplified.
`For eight of the 100 cases, the FISH assay was repeated a
`second time because the initial slides were unassessable.
`Only one of these cases was interpretable after the second
`assay. Of the seven cases considered uninterpretable after
`two assays, all had low-level specific fluorescent signal. Five
`also had high nonspecific fluorescent background staining,
`five were underdigested, and four had overlapping tumor
`nuclei. Re-review of hematoxylin- and eosin-stained slides
`concurrent with the FISH evaluation was necessary in 41
`cases to reliably identify invasive tumor cells. There were
`several different reasons that such re-review was necessary.
`In 11 of these 41 cases (27%), invasive carcinoma could not
`be reliably distinguished from carcinoma-in-situ on the
`DAPI stain alone. Similarly, well differentiated (grade 1)
`invasive carcinoma could not be accurately distinguished
`from benign glands in seven of 41 cases (17%) on the DAPI
`stain. Seven of 41 cases (17%) were infiltrating lobular
`carcinomas, six had small tumor cell groups or glands, and
`one each was mucinous carcinoma, metaplastic carcinoma,
`or partially overdigested. All seven cases determined to be
`uninterpretable after a second assay required concurrent
`review of the hematoxylin- and eosin-stained sections. A
`second observer was required for the evaluation of the FISH
`slides in 28 cases,
`including all eight cases that were
`reassayed as well as the two borderline cases. Of the seven
`amplified cases that required a second observer, five had
`fewer than seven mean signals per nucleus. Eight of 24 cases
`scored as amplified had fewer than 10 mean signals per
`nucleus (range, 4.2 to 8.7 signals). In six of these cases, the
`fluorescent signal was dispersed (as opposed to clustered).
`
`IHC for HER-2/neu
`
`Ninety-six of the 100 cases were assessable for HER-2/
`neu, with 22 of these 96 cases (22.9%) interpreted as
`positive. All four unassessable cases had tumor cell mem-
`brane staining of 11 or 21 but no normal breast ducts or
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`Case 1:18-cv-01363-CFC Document 82-8 Filed 03/22/19 Page 5 of 10 PageID #:
`9883
`
`FISH AND IHC FOR HER-2/neu IN BREAST CANCER
`
`Table 1. Comparison of HER-2/neuStatus of 90 Invasive Carcinomas
`by FISH and IHC
`
`FISH Amplified
`
`FISH Not Amplified
`
`IHC-positive
`IHC-negative
`
`18
`5
`
`3
`64
`
`lobules on the slide. The IHC assay was repeated a second
`time in three cases. These cases had insufficient tumor
`present on the slides in the initial assay.
`
`Comparison of HER-2/neu FISH and IHC
`
`The results of both the FISH and IHC assays were
`assessable in 90 cases. Results of these two assays were
`concordant in 82 of these cases (91.1%; P , .001) (Table 1).
`Of 23 cases amplified by FISH, five were negative by IHC,
`and of 67 cases not amplified by FISH, three were positive
`by IHC (Table 1; Figs 1 and 2). Four of five cases that were
`negative by IHC and amplified by FISH had fewer than 10
`mean signals per nucleus (range, 4.2 to 8.4 signals), with one
`case having 10.9 mean signals per nucleus (Fig 1). The three
`cases that were nonamplified by FISH and positive by IHC
`had between 1.7 and 2.1 mean fluorescent signals per
`nucleus. Of the four cases that were unassessable by IHC,
`one was also uninterpretable by FISH, one was amplified,
`and two were nonamplified by FISH. Of the seven cases that
`were uninterpretable by FISH, one was also unassessable by
`IHC, one was positive, and five were negative by IHC.
`
`1977
`
`The FISH procedure required more technologist time than
`did IHC (10.4 hours over 2 days v 6 hours on 1 day).
`Pathologist interpretation time per case was longer for FISH
`than for IHC (mean 8.9 minutes v 2.5 minutes). Reagent
`costs were substantially higher for FISH than for IHC (Table
`2). A total of nine Oncor/Ventana INFORM kits were
`required for 108 FISH assays (100 cases, eight of which
`were repeated a second time) at a cost of $2,000 per kit (total
`cost of $18,000). Therefore, the average cost per case for
`FISH was $166.67 ($18,000 divided by 108). In contrast, the
`total cost of the IHC reagents required for 103 assays (100
`cases, three of which were repeated a second time) was $608
`(primary antibody, $208; detection system, $350; diamino-
`benzidine [chromagen], $50). The average cost per case for
`IHC was, therefore, $5.90 ($608 divided by 103).
`
`DISCUSSION
`The results of this study indicate that there is a high level
`of correlation between FISH and IHC in the evaluation of
`HER-2/neu status of invasive breast cancers using formalin-
`fixed, paraffin-embedded tissue. The levels of HER-2/neu
`gene amplification (26%) and overexpression (23%) found
`in our study are in keeping with the published range of 20%
`to 30%.1,72 In addition, the level of concordance of FISH and
`IHC (91%) is similar to that seen in prior studies comparing
`assays of HER-2/neu gene amplification with those of
`HER-2/neu protein expression.2,53,55,58,59,73
`
`Fig 1. Average number of HER-
`2/neu signals per nucleus by FISH
`for invasive breast carcinoma cases
`interpreted as HER-2/neu–negative
`by IHC (n 5 90). Cases with more
`than four signals per nucleus by
`FISH were considered amplified.
`
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`
`

`

`Case 1:18-cv-01363-CFC Document 82-8 Filed 03/22/19 Page 6 of 10 PageID #:
`9884
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`1978
`
`JACOBS ET AL
`
`Fig 2. Average number of HER-
`2/neu signals per nucleus by FISH
`for invasive breast carcinoma cases
`interpreted as HER-2/neu–positive
`by IHC (n 5 90). Cases with more
`than four signals per nucleus by
`FISH were considered amplified.
`
`When FISH and IHC results were compared, five cases
`interpreted as negative by IHC were scored as amplified by
`FISH. All but one of these cases had a mean of fewer than 10
`fluorescent signals per nucleus (range, 4.2 to 8.4 signals per
`nucleus) by FISH. In a study by Ciocca et al,73 the
`HER-2/neu gene was amplified (as measured by Southern
`blot analysis) without overexpression of protein (as mea-
`sured by Western blot analysis) in 9% of cases. However,
`these authors did not distinguish invasive from in situ
`carcinoma. Of cases that showed HER-2/neu gene amplifica-
`tion by Southern blot, Slamon et al2 found discordance (ie,
`no overexpression) on Western blot in 6%, Northern blot in
`2%, and IHC in 1%. However, at least one assay of gene
`expression was concordant with the Southern blot results in
`these cases. At the cellular level, Szollosi et al74 found good
`
`Table 2. Comparison of Procedure and Interpretation Time and Reagent
`Costs for FISH and IHC
`
`Procedure (technologist) time per assay, hours
`Mean
`Range
`Interpretation (pathologist) time per case, minutes
`Mean
`Range
`Cost per case, United States dollars
`
`FISH
`
`IHC
`
`10.4
`9.2-11.3
`
`8.9
`2.1-26.5
`166.67
`
`6.0*
`
`2.5
`0.5-5.0
`5.90
`
`*The IHC assays were performed in three runs, with no appreciable variation
`in time between runs (3.5 hours of hands-on technologist time and 2.5 hours of
`autostainer time for each run).
`
`overall correlation between HER-2/neu oncoprotein expres-
`sion and gene copy number. However, on a cell-by-cell
`basis, heterogeneity was found between these parameters. It
`is possible that in cases with low-level gene amplification,
`gene transcription and posttranscriptional and translational
`events could be abnormal or downregulated, leading to low
`HER-2/neu oncoprotein expression or abnormal protein or
`epitope production. Alternatively, the low gene copy number
`(, 10 mean signals/nucleus) could represent aneuploidy for
`chromosome 17 rather than gene amplification per se. In a
`study correlating HER-2/neu gene expression and DNA
`ploidy, cases that were negative for HER-2/neu oncoprotein
`by IHC had DNA indices that ranged from diploid to up to
`three times normal.75 Use of a reference probe, such as to
`chromosome 17 alpha-satellite DNA or D21S16 on 17q,53
`could resolve the ploidy status of such cases. However, the
`Oncor/Ventana INFORM kit does not include such a probe,
`which precluded testing for this in our study.
`Three cases interpreted as HER-2/neu–positive by IHC
`were scored as nonamplified by FISH. This could represent
`single-copy overexpression of the HER-2/neu gene at the
`mRNA transcription level and/or beyond, with resultant
`overexpression of protein. Alternatively, it may be due to
`gene amplification that is below the detection level of the
`FISH assay. These scenarios were alluded to in the study of
`Slamon et al,2 in which low HER-2/neu DNA levels were
`found with overexpression of mRNA and protein levels in
`
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`
`

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`Case 1:18-cv-01363-CFC Document 82-8 Filed 03/22/19 Page 7 of 10 PageID #:
`9885
`
`FISH AND IHC FOR HER-2/neu IN BREAST CANCER
`
`10% of cases. Similar findings of absent HER-2/neu gene
`amplification with protein expression have been reported in
`3% to 7% of cases in other studies.53,58,59 Regardless of the
`molecular mechanisms involved, the findings of discordant
`gene and gene product results may have therapeutic implica-
`tions, because the gene product (ie, HER-2/neu oncoprotein)
`is now being used as the target for treatment modalities, such
`as the anti-HER-2 monoclonal antibody, trastuzumab (Her-
`ceptin).38-40,76 Therefore, IHC may well be the more biologi-
`cally relevant assay on the basis of these considerations.
`A number of previous studies have suggested that IHC for
`HER-2/neu lacks sensitivity and specificity and that this
`assay is subject to considerable interobserver variability in
`the interpretation of the results.63,77-79 The largest and
`perhaps the most widely cited study in this regard is that of
`Press et al.63 These investigators performed HER-2/neu IHC
`on formalin-fixed paraffin-embedded tissue, comparing the
`sensitivity and specificity of 28 different antibodies. The
`results were compared with known Southern blot, Northern
`blot, Western blot, and frozen section IHC for the cases. The
`sensitivity of the various antibodies studied ranged from 6%
`to 82%, and with one antibody, the rate of tumor positivity
`was as low as 2%. However, there are several important
`limitations to the IHC assays used in that study. First,
`epitope retrieval methods were not used for 27 of the 28
`antibodies evaluated. It is now clear that epitope retrieval is
`important for obtaining optimal staining of formalin-fixed
`paraffin-embedded tissue with at least some of the commer-
`cially available HER-2/neu antibodies.80 Second, the peroxi-
`dase/antiperoxidase method was used as the detection sys-
`tem. This method has substantially lower sensitivity than the
`avidin-biotin complex systems that are currently in wide-
`spread use. Finally, only a very small tissue sample was
`evaluated for each case, because the authors used multitu-
`mor tissue blocks. This could have resulted in cases that
`exhibited regional variation in HER-2/neu staining being
`erroneously categorized as negative. Therefore, it is difficult
`to extrapolate the results of the study of Press et al63 to
`studies of HER-2/neu IHC that use current reagents and
`methodology.
`In this study, the FISH assay required more technologist
`time and more interpretation time by the pathologist than did
`IHC. Moreover, FISH requires the use of a fluorescence
`microscope and often required re-review of the hematoxy-
`
`1979
`
`lin- and eosin-stained sections to definitely identify areas of
`invasive carcinoma. IHC interpretation is performed using a
`standard light microscope and permits simultaneous evalua-
`tion of immunostaining and morphology on the same slide.
`The FISH slides must be stored at 220°C or lower and are
`subject to quenching of the fluorescent signal, whereas
`IHC-stained slides can be stored in standard slide files and
`the reaction product is permanent. Finally, the cost of the
`FISH procedure is substantially greater than that of the IHC
`assay.
`In summary, we have documented a high level of
`concordance between FISH and IHC in the evaluation of
`HER-2/neu status on the same cases of invasive breast
`carcinoma. The technical and interpretation times and re-
`agent costs per case are substantially lower for IHC com-
`pared with FISH. However, there are a number of important
`caveats in the interpretation of our results. First, our findings
`are based on the analysis of consecutive breast cancer cases
`that were fixed and processed in a relatively uniform manner
`in a single pathology department. It remains to be deter-
`mined if similar results can be obtained in laboratories that
`analyze specimens that are subject to more variability in
`tissue fixation and processing, such as reference laboratories
`that receive specimens from a variety of different institu-
`tions. It is also not known which of these procedures (IHC or
`FISH) is subject to greater interlaboratory concordance with
`regard to both performance of the assay and interpretation of
`the results, and studies are needed to address this important
`issue. Finally, it is imperative that these two assays be
`compared directly in the same study population to determine
`which, if either of these, has greater clinical relevance with
`regard to predicting prognosis and response to therapy.
`Although the choice of which assay to use for evaluating the
`HER-2/neu status of breast cancers should be left for
`individual
`institutions to make based on technical and
`economic considerations, our results may make it difficult to
`justify the routine use of FISH for the determination of
`HER-2/neu status in breast cancer.
`
`ACKNOWLEDGMENT
`We thank Oncor Corp, Gaithersburg, MD, and Dako Corp, Carpinte-
`ria, CA, for generously supplying the reagents for the FISH and IHC
`assays, respectively.
`
`REFERENCES
`1. Slamon DJ, Clark GM, Wong SG, et al: Human breast cancer:
`3. Tandon AK, Clark GM, Chamness GC, et al: HER-2/neu onco-
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`2. Slamon DJ, Godolphin W, Jones LA, et al: Studies of the
`4. Thor AD, Schwartz LH, Koerner FC, et al: Analysis of c-erbB-2
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`Downloaded from ascopubs.org by Reprints Desk on December 4, 2018 from 072.037.250.187
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`Copyright © 2018 American Society of Clinical Oncology. All rights reserved.
`
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