RESEARCH ARTICLE
A New Immunohistochemical ER/PR Image Analysis System A Multisite Performance Study Aziza Nassar, MD,* Cynthia Cohen, MD,w Sally S. Agersborg, MD, PhD,z Weidong Zhou, MD,z Kathleen A. Lynch, MD,z Eugene R. Heyman, PhD,y Allen Olson, PhD,y Holger Lange, PhD,y and Momin T. Siddiqui, MDw
Background: Aperio provides a new image analysis (IA) solution for immunohistochemistry (IHC) as part of its digital pathology system. To be used in a clinical setting, substantial equivalence to scoring by manual microscopy (MM) needs to be shown. A multisite study was conducted to assess the performance of Aperio’s IHC IA solution for estrogen receptor (ER) and progesterone receptor (PR). Design: A total of 260 formalin-fixed, paraffin-embedded breast tissue specimens were assayed at 2 clinical sites for ER and PR. The ability to score ER/PR slides in terms of (1) percentage of positive nuclei with cutoffs of 1%, 5%, and 10% and (2) average staining intensity as 0, 1+, 2+, and 3+ score was assessed. At each site, 3 pathologists performed a blinded read of the glass slides using their microscopes. The glass slides were then scanned, and after a wash-out period and randomization of the slides, the pathologists viewed the images on a computer monitor and outlined a representative set of tumor regions to be analyzed by IA. Each of the methods: MM and IA were evaluated separately and comparatively. Results: Comparable or higher percent agreements were obtained for IA compared with MM (ERFpercent of positive nuclei with cutoffs: MM: 91.3% to 98.8%/IA: 93.8% to 98.8%/IA vs. MM: 92.5% to 97.5%, and intensity score: MM: 55.0% to 86.3%/IA: 88.8% to 90.0%/IA vs. MM: 63.8% to 86.3%; PRFpercent of positive nuclei with cutoffs: MM: 83.8% to 99.0%/IA: 85.0% to 99.0%/IA vs. MM: 81.3% to 99.0%, and intensity score: MM: 58.8% to 88.0%/IA: 68.8% to 88.0%/IA vs. MM: 58.8% to 84.0%). Conclusions: The study results show that Aperio’s digital IHC IA solution for ER/PR is substantially equivalent to scoring by MM.
Received for publication September 8, 2010; accepted September 23, 2010. From the *Department of Pathology, Mayo Clinic, Rochester, MN; wDepartment of Pathology and Laboratory Medicine, Emory University Hospital, Atlanta, GA; zDepartment of Hematology/ Oncology, Quest Diagnostics Nichols Institute, San Juan Capistrano; and yAperio Technologies Inc, Vista, CA. Reprints: Aziza Nassar, MD, Department of Pathology, Mayo Clinic, Rochester, MN (e-mail:
[email protected]). Copyright r 2011 by Lippincott Williams & Wilkins
Appl Immunohistochem Mol Morphol
Key Words: estrogen receptor, progesterone receptor, image analysis, digital pathology, breast cancer, hormone receptors (Appl Immunohistochem Mol Morphol 2011;19:195–202)
H
ormonal receptors are considered both prognostic and predictive markers in breast cancer. In patients with early-stage estrogen receptor (ER)-positive breast cancer, adjuvant endocrine therapy given for 5 years after primary surgery delays local and distant relapse, and prolongs overall survival.1 In addition, endocrine therapy substantially reduces the incidence of contralateral breast cancer in patients with primary breast cancer by about 50%.1 Approximately 60% to 70% of breast cancers are hormone-dependent (positive for ER and/or PR).2 In the seminal study by Harvey et al,3 a 9-point, semiquantitative Allred score of intensity and percent of positive cells (ranging from 0 to 8) was performed on a series of almost 2000 patients and results were correlated with response to adjuvant endocrine therapy.3 An Allred score of 3 or more (ie, the definition of ER positivity) corresponds to as few as 1% weakly staining tumor cells, which is sufficient to predict a significant benefit from endocrine therapy.3 Mann and colleagues4 have demonstrated that as many as 9% of women may have false-negative ER immunohistochemical (IHC) studies if their resection, rather than core needle biopsy, is used.4 A false-negative rate of 24% was found, when paraffin blocks of cancer known to express ER were distributed to various laboratories for staining and interpretation.4 Goldstein et al5 have demonstrated that at least 6 to 8 hours of formalin fixation time for breast biopsies is required to obtain reliable ER determination.5 Rhodes et al6 in an analysis of data from 66 laboratories participating in a United Kingdom external quality assurance program, found that ER IHC staining results were highly affected by the efficiency of the antigen retrieval step, and that this was, in fact, the single most important factor contributing to interlaboratory variability.6 The College of American Pathologists and American Society of Clinical Oncology recently issued recommendations and guidelines for IHC staining of ER and
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PR in breast cancer.1 Image analysis (IA) was thought to be a desirable method of identifying percentage of tumor cells that are immunoreactive, and designed to improve interobserver and intraobserver reproducibility.1 To be widely accepted for clinical use, digital pathology systems need to be cleared/approved by the Food and Drug Administration (FDA), and performance studies must show this new technology to the clinical community. This study evaluates the performance of Aperio Technologies Inc’s Digital Pathology Platform (Vista, CA) using FDA-approved antibodies for IHC quantitation with IA, in 2 different clinical centers, compared with manual microscopy (MM).
MATERIALS AND METHODS Two Clinical Laboratory Improvement Amendment-qualified clinical sites participated in the study. Before their participation in the study, each clinical site obtained exemption status from an Institutional Review Board. Each laboratory has conducted the study separately using their own antibody clones, and the results are analyzed separately for each laboratory. Eighty formalin-fixed paraffin-embedded breast tissue specimens from clinical site 1 were used for the ER study. The specimens were selected based on their clinical scores on file to provide equal distribution of ER slides in the percentage of positive nuclei ranges 0%, 1% to 4%, 5% to 9%, 10% to 49%, and 50% to 100%. All specimens for the ER study were IHC stained using Dako in-vitro diagnostic FDA-cleared Monoclonal Mouse Anti-Human Estrogen Receptor a (Clone 1D5) (Dako, Carpinteria, CA). Both clinical sites participated in the PR study. A total set of 180 formalin-fixed, paraffin-embedded breast tissue specimens from both clinical sites were used for the PR study; 80 slides from clinical site 1 and 100 slides from clinical site 2. The specimens at clinical site 1 were selected based on their clinical scores on file to provide equal distribution of PR slides in the percentage of positive nuclei ranges 0%, 1% to 4%, 5% to 9%, 10% to 49%, and 50% to 100%. The specimens at clinical site 2 were routine specimens from their clinical operation, representing the true target population of cases in a typical clinical setting. All specimens for the PR study were immunohistochemically stained at the clinical sites using Dako in-vitro diagnostic FDA-cleared Monoclonal Mouse Anti-Human Progesterone Receptor (Clone PgR 636) (Dako, Carpinteria, CA). The distribution of the ER and PR reactivity in the different clinical sites are presented in Tables 1 and 2. The study was performed primarily at the participating clinical sites and all parts except the scanning of glass slides were performed at their facilities using their typical workflow. The glass slides were prepared in the sites’ clinical laboratories and read by board-certified staff pathologists. For the scanning of glass slides, ScanScope XT instruments were operated in a simulated clinical setting at Aperio (designed to be representative of a
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TABLE 1. Percentage of Positive Nuclei Distribution for Estrogen Receptor Data Set from Clinical Site 1 Percentage
Clinical Site 1
0 1-5 5-10 10-50 50-100 Total
23 4 3 5 45 80
typical laboratory environment). All ScanScope XT instruments used in the study were production units and were delivered, installed, and maintained in accordance with the approved procedures, per Aperio’s Quality Systems Procedures, and as described in product documentation and labeling. Three board-certified pathologists at each clinical site performed a blinded manual review of each ER and PR-stained glass slide using a conventional light microscope. The pathologist reported the percentage of positive nuclei (0%, 1%, yy. 100%) and average intensity score of 0, 1+, 2+, and 3+ for each of the reviewed glass slides. All glass slides from the 2 laboratories were then sent to Aperio Headquarters, and were scanned with a 20 objective using a different ScanScope XT instrument for each clinical site. For ER/PR tissue scoring applications, the ScanScope Aperio digital microscopy locates tissues on a slide and generates a scanned image of the entire tissue. Other details of the hardware and software on the ScanScope Aperio digital microscopy system are available online (http://www.aperio.com). After a wash-out period of over 1 week and subsequent randomization of the slides, the same 3 pathologists at each clinical site outlined a representative set of tumor regions for each digital slide to be analyzed by IA using the ScanScope Systems’ remote editing capability (Fig. 1). The pathologist views this image on a computer monitor and selects multiple tissue regions of appropriately stained invasive carcinoma (Figs. 2, 3). The pathologists’ annotations of tumor region outlines were blinded from each other. The IA reported the ER/PR score for each of the 3 pathologists for each of the slides. The IA itself was run in batch mode blinded from the pathologists to avoid influencing them in their choice of the tumor regions.
TABLE 2. Percentage of Positive Nuclei Distribution for Progesterone Receptor Data Set from Clinical Sites 1 and 2 Percentage 0 1-5 5-10 10-50 50-100 Total
Clinical Site 1
Clinical Site 2
Total
16 3 8 14 39 80
33 6 3 11 47 100
49 9 11 25 86 180
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Microscope
New IHC ER/PR IA
Pathologist #1, #2, #3
Manual Microscopy
Slide Score Glass Slides
Wash-out
ScanScope®
ImageScope
Pathologist #1, #2, #3
Glass Slides
Image Analysis Slide Score
Tumor Regions Annotations
Image Analysis
FIGURE 1. Study illustration of the study design for manual microscopy and image analysis.
For each clinical site, IA algorithms were configured initially using an automatic training procedure with a representative training data set of 20 of each ER and PR slides, and the scores from the same 3 pathologists.
The IA algorithm reported the percentage of positive nuclei (0%, y 100%) and average intensity score of 0, 1+, 2+, and 3+ for each of the digital slides (Fig. 1).
FIGURE 2. Captured image of an estrogen receptor-immunostained slide on a computer monitor. r
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FIGURE 3. Annotations of an estrogen receptor-immunostained tumor on a computer monitor.
The ScanScope Aperio system generates a series of scores for these annotated areas selected by pathologists including both individual region scores and the average score of selected regions. In ER/PR IHC evaluation, the score is the average brown pixel intensity using a color threshold designed to measure the brown color associated with the 3, 30 -diaminobenzidine tetrahydrochloride stain. The specific algorithm selects those pixels within the selected tissue regions which meet the brown 3, 30 diaminobenzidine tetrahydrochloride color threshold. This color selection chooses pixels that are associated with the nuclear domain. The ScanScope Aperio digital microscope calculates the ER/PR IHC average intensity value on a scale from 0 to 255. Owing to the more quantitative data of ScanScope Aperio digital microscopy, this score can be reported in tenths of integer units (eg, 1.3 and 2.1).
Immunohistochemistry Five-micron sections of formalin-fixed paraffinembedded tissue were studied. Sections are deparaffinized in xylene and grades of alcohol, then rehydrated in water. Antigen retrieval is in citrate buffer (pH 6) using an electric pressure cooker for 3 minutes at 1201C (12 to 15 pounds per square inch) and cooling for 10 minutes before immunostaining. All immunostains were performed on an automated DAKO immunostainer (DakoCytomation Autostainer, Glostrup, Denmark). Immunostaining was performed with a polymer-based
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detection system (Envision-plus, DakoCytomation, Carpinteria, CA) using ER (monoclonal 1D5; 1:50, Dako) and PR (monoclonal PgR636; 1:400; Dako). Tissues are exposed to 3% hydrogen peroxide for 5 minutes, primary antibody for 30 minutes, labeled polymer, horseradish peroxidase for 30 minutes, diaminobenzidine as chromogen for 5 minutes, and DAKO automation hematoxylin as a counterstain for 5 minutes. These incubations are performed at room temperature; between incubations, sections are washed with Trisbuffered saline buffer. Coverslipping is performed using the Tissue-Tek SCA (Sakura Finetek, Inc, Torrance, CA). Known positive breast cancer controls, and negative controls with primary antibody replaced by buffer, were included in each run. Positivity was indicated by the presence of dark brown nuclear staining for ER and PR. The intensity of the staining for ER and PR (graded from 0 to 3+) and the percentage of positive cells were evaluated.
Statistical Analysis The statistical analyses are presented for ER and PR for each of the scores: percentage of positive nuclei and intensity. The statistical analyses are presented across all slides for MM and IA, and comparatively between the 2 methods for the 2 clinical sites each with 3 pathologists using percent agreement along with an exact 95% confidence interval. r
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TABLE 3. Interpathologist PA for MM, IA, and MM Versus IA Using Allred Scoring System for Estrogen Receptor Immunohistochemical Analysis in Clinical Site 1 Pathologist 1 Versus 2 MM IA MM versus IA
Pathologist 1 Versus 3
Pathologist 2 Versus 3
PA (%)
PA 95% CI
PA (%)
PA 95% CI
PA (%)
PA 95% CI
92.5 97.5 92.5
(84.4-97.2) (91.3-99.7) (84.4-97.2)
91.3 98.8 95.0
(82.8-96.4) (93.2-99.9) (87.7-98.6)
98.8 98.8 95.0
(93.2-99.9) (93.2-99.9) (87.7-98.6)
CI indicates confidence interval; IA, image analysis; MM, manual microscopy; PA, percent agreement.
RESULTS The statistical analyses are presented across all slides for each of the methods: MM and IA, and comparatively between the 2 methods for ER (clinical site 1 only) and PR (both clinical sites) for the interpretation of the Allred score (Tables 3, 4). An Allred score Z3 is considered positive and an Allred score r2 is considered negative. Although we set up cut-off thresholds for percentage of ER-positive nuclei, in reality, most cases were either positive in more than 50% of tumor nuclei or were completely negative (Table 1).
Interpathologist and Intrapathologist Agreement for ER Analysis Using intensity scores, interpathologist agreement for the performed (blinded) IA (88.8% to 90.0%) was comparable with or better than the interpathologist agreement for MM (55.0% to 86.3%) for ER IHC. The agreement between the pathologists MM versus performed (blinded) IA (63.8% to 86.3%) was comparable with the interpathologist agreement for MM (55.0% to 86.3%) for ER IHC using intensity scores. Tables 5 to 7 show percent agreement with an exact 95% confidence interval for the clinically relevant dichotomous outcome obtained by applying a cut-off threshold at 1%, 5%, and 10%, to the percentage of ERpositive nuclei for clinical site 1. The interpathologist agreement for MM (91.3% to 98.8%) (Table 5) was comparable with the interpathologist agreement for the performed (blinded) IA (93.8% to 98.8%) (Table 6). The agreement between the same pathologist’s MM versus performed (blinded) IA (92.5% to 97.5%) (Table 7) was
comparable with the interpathologist agreement for MM (91.3% to 98.8%) (Table 5).
Interpathologist and Intrapathologist Agreement for PR Analysis Using intensity scores for PR IHC analysis, interpathologist agreement for the performed (blinded) IA (68.8% to 88.0%) was comparable with the interpathologist agreement for MM (58.8% to 88.0%). Agreement between the same pathologist’s MM and performed (blinded) IA (58.8% to 84%) was comparable with the interpathologist agreement for MM (58.8% to 88.0%). For PR IHC analysis with cut-off thresholds, interpathologist agreement at both clinical sites for MM (83.8% to 99.0%) (Table 8) was comparable with the interpathologist agreement for the performed (blinded) IA (85.0% to 99.0%) (Table 9). Agreement between the same pathologist’s MM versus performed (blinded) IA (81.3% to 99.0%) (Table 10) was comparable with the interpathologist agreement for MM (83.8% to 99.0%) (Table 8) for PR IHC analysis.
DISCUSSION A major advent in the standardization of IHC quantitation is the automated IA system. Computerbased programs have been developed to eliminate the inherent variability and subjectivity of IHC interpretation. One such program is the Automated Cellular Imaging System (ACIS III; DAKO), which combines automated microscopy with color-based IA software. The ACIS III is currently FDA-approved in the quantitative analysis of human epidermal growth factor receptor 2
TABLE 4. Interpathologist PA for MM, IA, and MM Versus IA Using Allred Scoring System for Progesterone Receptor Immunohistochemical Analysis in Clinical Sites 1 and 2 Pathologist 1 Versus 2 Clinical Clinical Clinical Clinical Clinical Clinical
site site site site site site
1 2 1 2 1 2
MM MM IA IA MM versus IA MM versus IA
Pathologist 1 Versus 3
Pathologist 2 Versus 3
PA (%)
PA 95% CI
PA (%)
PA 95% CI
PA (%)
PA 95% CI
87.5 97.0 88.8 92.0 88.8 94.0
(78.2-93.8) (91.5-99.4) (79.7-94.7) (84.8-96.5) (79.7-94.7) (87.4-97.8)
85.0 98.0 85.0 97.0 90.0 95.0
(75.3-92.0) (93.0-99.8) (75.3-92.0) (91.5-99.4) (81.2-95.6) (88.7-98.4)
82.5 97.0 91.3 89.0 83.8 89.0
(72.4-90.1) (91.5-99.4) (82.8-96.4) (81.2-94.4) (73.8-91.9) (81.2-94.4)
CI indicates confidence interval; IA, image analysis; MM, manual microscopy; PA, percent agreement.
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TABLE 5. Interpathologist Agreement for Manual Microscopy Using 1%, 5%, and 10% Cut-off Threshold, Respectively, for Estrogen Receptor Immunohistochemical Analysis in Clinical Site 1 Pathologist 1 Versus 2 1% Cut-off 5% Cut-off 10% Cut-off
Pathologist 1 Versus 3
Pathologist 2 Versus 3
PA (%)
PA 95% CI
PA
PA 95% CI
PA
PA 95% CI
92.5 96.3 93.8
(84.4-97.2) (89.4-99.2) (86.0-97.9)
91.3 95.0 95.0
(82.8-96.4) (87.7-98.6) (87.7-98.6)
98.8 98.8 96.3
(93.2-99.9) (93.2-99.9) (89.4-99.2)
CI indicates confidence interval; PA, percent agreement.
TABLE 6. Interpathologist Agreement for Image Analysis Using 1%, 5%, and 10% Cut-off Threshold, Respectively, for Estrogen Receptor Immunohistochemical Analysis in Clinical Site 1 Pathologist 1 Versus 2 1% Cut-off 5% Cut-off 10% Cut-off
Pathologist 1 Versus 3
Pathologist 2 Versus 3
PA (%)
PA 95% CI
PA (%)
PA 95% CI
PA (%)
PA 95% CI
97.5 93.8 95.0
(91.3-99.7) (86.0-97.7) (87.7-98.6)
98.8 93.8 96.3
(93.2-99.9) (86.0-97.7) (89.4-99.2)
98.8 97.5 98.8
(93.2-99.9) (91.3-99.7) (93.2-99.9)
CI indicates confidence interval; PA, percent agreement.
TABLE 7. Same Pathologist’s Agreement for Manual Microscopy Versus Image Analysis Using 1%, 5%, and 10% Cut-off Threshold, Respectively, for Estrogen Receptor Immunohistochemical Analysis in Clinical Site 1 Pathologist 1 1% Cut-off 5% Cut-off 10% Cut-off
Pathologist 2
Pathologist 3
PA (%)
PA 95% CI
PA (%)
PA 95% CI
PA (%)
PA 95% CI
92.5 93.8 95.0
(84.4-97.2) (86.0-97.7) (87.7-98.6)
95.0 96.3 93.8
(87.7-98.6) (89.4-99.2) (86.0-97.9)
95.0 97.5 96.3
(87.7-98.6) (91.3-99.7) (89.4-99.2)
CI indicates confidence interval; PA, percent agreement.
Table 8. Interpathologist Agreement for Manual Microscopy Using 1%, 5%, and 10% Cut-off Threshold, Respectively, for Progesterone Receptor Immunohistochemical Analysis in Both Clinical Sites (1 and 2) Pathologist 1 Versus 2 Clinical Clinical Clinical Clinical Clinical Clinical
site site site site site site
1 2 1 2 1 2
at at at at at at
1% cut-off 1% cut-off 5% cut-off 5% cut-off 10% cut-off 5% cut-off
Pathologist 1 Versus 3
Pathologist 2 Versus 3
PA (%)
PA 95% CI
PA (%)
PA 95% CI
PA (%)
PA 95% CI
87.5 97.0 88.8 98.0 88.8 97.0
(78.2-93.8) (91.5-99.4) (79.7-94.7) (93.0-99.8) (79.7-94.7) (91.5-99.4)
85.0 97.0 85.0 99.0 92.5 99.0
(75.3-92.0) (91.5-99.4) (75.3-92.0) (94.6-99.9) (84.4-97.2) (94.6-99.9)
87.5 94.0 83.8 97.0 88.8 96.0
(78.2-93.8) (87.4-97.8) (73.8-91.1) (91.5-99.4) (79.7-94.7) (90.1-98.9)
CI indicates confidence interval; PA, percent agreement.
TABLE 9. Interpathologist Agreement for Image Analysis Using 1%, 5%, and 10% Cut-off Threshold, Respectively, for Progesterone Receptor Immunohistochemical Analysis in Both Clinical Sites (1 and 2) Pathologist 1 Versus 2 Clinical Clinical Clinical Clinical Clinical Clinical
site site site site site site
1 2 1 2 1 2
at at at at at at
1% cut-off 1% cut-off 5% cut-off 5% cut-off 10% cut-off 10% cut-off
Pathologist 1 Versus 3
Pathologist 2 Versus 3
PA (%)
PA 95% CI
PA (%)
PA 95% CI
PA (%)
PA 95% CI
88.8 92.0 88.8 95.0 90.0 95.0
(79.7-94.7) (84.8-96.5) (79.7-94.7) (88.7-98.4) (81.2-95.6) (88.7-98.4)
85.0 97.0 88.8 97.0 86.3 99.0
(75.3-92.0) (91.5-99.4) (79.7-94.7) (91.5-99.4) (76.7-92.9) (94.6-99.9)
91.3 89.0 92.5 94.0 88.8 96.0
(82.8-96.4) (81.2-94.4) (84.4-97.2) (87.4-97.8) (79.7-94.7) (90.1-98.9)
CI indicates confidence interval; PA, percent agreement.
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TABLE 10. Same Pathologist’s Agreement for Manual Microscopy Versus Image Analysis Using 1%, 5%, and 10% Cut-off Threshold, Respectively, for Progesterone Receptor Immunohistochemical Analysis in Both Clinical Sites (1 and 2) Pathologist 1 Clinical Clinical Clinical Clinical Clinical Clinical
site site site site site site
1 2 1 2 1 2
at at at at at at
1% cut-off 1% cut-off 5% cut-off 5% cut-off 10% cut-off 10% cut-off
Pathologist 2
Pathologist 3
PA (%)
PA 95% CI
PA (%)
PA 95% CI
PA (%)
PA 95% CI
88.8 95.0 83.8 99.0 88.8 96.0
(79.7-94.7) (88.7-98.4) (73.8-91.1) (94.6-99.9) (79.7-94.7) (90.1-98.9)
90.0 94.0 81.3 92.0 85.0 94.0
(81.2-95.6) (87.4-97.8) (71.0-89.1) (84.8-96.5) (75.3-92.0) (87.4-97.8)
86.3 89.0 90.0 97.0 90.0 98.0
(76.7-92.9) (81.2-94.4) (81.2-95.6) (91.5-99.4) (81.2-95.6) (93.0-99.8)
CI indicates confidence interval; PA, percent agreement.
(HER2), ER, and PR IHC. It also includes software for the analysis of tissue microarrays.7 ChromoVision Medical Systems (Chicago, IL) that first developed the technology, performed clinical tests using tissue microarray to prove substantial equivalence to the FDA-approved method.7 In several studies, ACIS has been shown to have high concordance with manual quantitation methods especially for immunostains such as ER, PR, and HER2.8–18 Although ChromoVision was considered the pioneer and the first-generation digital imaging technology, Aperio has taken the technology to a second level. They have integrated IA using cell-based versus pixel-based analysis into a digital pathology workflow that actually saves the pathologist time as he/ she can use IA with the click of a button while reading the slide on a computer monitor. Imaging analysis was introduced to reduce interobserver variability and improve prediction of response to therapy. Another advantage of IA seems to be the potential for increased standardization. ACIS was approved by the FDA in 1999 and has been used in breast cancer hormone receptor expression testing since 2000.7 As ER, PR, and HER2 expression in breast carcinoma are important predictive and prognostic factors for assessing response to therapy and patient outcome, accurate results, optimal sensitivity, and standardization are necessary. The use of computer-aided microscopy has been suggested as a way to improve interobserver reproducibility in IHC evaluation.19–22 Computer-aided microscopy involves the digitization of stained tissue and the automated evaluation of IHC with IA. In the case of ER/ PR, the College of American Pathologists/American Society of Clinical Oncology guidelines recognize IA as an effective tool for achieving consistent interpretation of IHC staining, provided that a pathologist confirms the result.1 Computer-aided quantitative assessment of IHC staining has potentially several benefits. It can provide a true continuous and reproducible assessment of staining. The human eye has difficulty distinguishing subtle differences in staining intensity using a continuous scale.22 Consequently, scoring systems tend to be nominal (ie, 0, 1+, 2+, and 3+). Studies have shown that accurate distinction between nominal categories is difficult and r
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often arbitrary, and this difficulty contributes to a significant lack of reproducibility.23 Automated systems can consistently preselect stained areas and extract a score from them or point out the same “hot spot” areas to different observers; the selection of different areas to be assessed by different observers has been identified as a source of interobserver variability.24 Automated systems may be used for screening strongly positive or strongly negative slides so that the ever-increasing reading load of a pathologist will be relieved of obvious cases. In addition to increased reproducibility, automated systems have the potential to increase prognostic accuracy by revealing differences in biomarker expression that are not discernable to the pathologist because of an inability to distinguish between fine levels of expression.22 As already performed for HER2,25 guidelines recently have been developed for handling, processing, and staining breast carcinoma for receptors. Of necessity, these guidelines include a standardized cut-off value to define internationally accepted interpretation of ER.1,26 The consensus group agreed on a minimum of 1% of ERpositive cells as the most reasonable cutoff based on correlation between level of ER expression and response to endocrine therapy.1,26 The guidelines recommend that ER interpretation should include evaluation of both percentage of positive tumor cell nuclei and intensity of staining reaction.1,26 Furthermore, quantitative IA is encouraged for samples with low percentages of nuclear staining or in cases with multiple observers in the same institution. IA is considered a valuable way to quantify intensity and assure day-to-day consistency of control tissue reactivity.1 This study assessed the performance of IA, using the Aperio ScanScope imaging system and algorithms. It showed comparative equivalence to MM in the scoring of ER and PR IHC assays in breast carcinoma tissues. Aperio technology is a promising digital analysis system providing a more sensitive and less subjective image cytometric quantitation of receptor expression than MM. REFERENCES 1. Hammond ME, Hayes DF, Dowsett M, et al. American Society of Clinical Oncology/College of American Pathologists guideline
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2. 3.
4. 5.
6.
7.
8. 9.
10.
11.
12.
13.
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recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer. J Clin Oncol. 2010; 28:2784–2795. Johnston SR. New strategies in estrogen receptor-positive breast cancer. Clin Cancer Res. 2010;16:1979–1987. Harvey JM, Clark GM, Osborne CK, et al. Estrogen receptor status by immunohistochemistry is superior to the ligand-binding assay for predicting response to adjuvant endocrine therapy in breast cancer. J Clin Oncol. 1999;17:1474–1481. Mann GB, Fahey VD, Feleppa F, et al. Reliance on hormone receptor assays of surgical specimens may compromise outcome in patients with breast cancer. J Clin Oncol. 2005;23:5148–5154. Goldstein NS, Ferkowicz M, Odiish E, et al. Minimum formalin fixation time for consistent estrogen receptor immunohistochemical staining of invasive breast carcinoma. Am J Clin Pathol. 2003;120: 86–92. Rhodes A, Jasani B, Barnes DM, et al. Reliability of immunohistochemical demonstration of estrogen receptors in routine practice: interlaboratory variance in the sensitivity of detection and evaluation of scoring systems. J Clin Pathol. 2000;53:125–130. US Food and Drug Administration. 510(k) Summary of Substantial Equivalence, ChromaVision Medical Systems, Inc. (Automated Cellular Imaging System). http://www.fda.goc/cdrh/pdf3/k032113. pdf. [Accessed May 19, 2009]. Bloom K, Harrington D. Enhanced accuracy and reliability of HER-2/neu immunohistochemical scoring using digital microscopy. Am J Clin Pathol. 2004;121:620–630. Ciampa A, Xu B, Ayata G, et al. HER-2 status in breast cancer: correlation of gene amplification by FISH with immunohistochemistry expression using advanced cellular imaging system. Appl Immunohistochem Mol Morphol. 2006;14:132–137. Tawfik OW, Kimler BF, Davis M, et al. Comparison of immunohistochemistry by automated cellular imaging system (ACIS) versus fluorescence in-situ hybridization in the evaluation of HER-2/neu expression in primary breast carcinoma. Histopathology. 2006;48:258–267. Bejar J, Sabo E, Misselevich I, et al. Comparative study of computer-assisted image analysis and light-microscopically determined estrogen receptor status of breast carcinomas. Arch Pathol Lab Med. 1998;122:346–352. Cavaliere A, Bucciarelli E, Sidoni A, et al. Estrogen and progesterone receptors in breast cancer: comparison between enzyme immunoassay and computer-assisted image analysis of immunocytochemical assay. Cytometry. 1996;26:204–208. Kohlberger PD, Breitenecker F, Kaider A, et al. Modified true-color computer-assisted image analysis versus subjective scoring of
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14.
15.
16.
17. 18.
19. 20. 21. 22. 23. 24.
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estrogen receptor expression in breast cancer: a comparison. Anticancer Res. 1999;19:2189–2194. Ballouk F, Boguniewicz A, Ross JS, et al. Image analysis quantification in hormone receptor assay of mucinous carcinoma of the breast. Comparison with biochemical analysis. Anal Quant Cytol Histol. 1995;17:151–156. Colley M, Kommoss F, Bibbo M, et al. Assessment of hormone receptors in breast carcinoma by immunohistochemistry and image analysis. II. Estrogen receptors. Anal Quant Cytol Histol. 1989; 11:307–314. Diaz LK, Sahin A, Sneige N. Interobserver agreement for estrogen receptor immunohistochemical analysis in breast cancer: a comparison of manual and computer-assisted scoring methods. Ann Diag Pathol. 2004;8:23–27. Bacus S, Flowers JL, Press MF, et al. The evaluation of estrogen receptor in primary breast carcinoma by computer-assisted image analysis. Am J Clin Pathol. 1988;90:233–239. Turbin DA, Leung S, Cheang MC, et al. Automated quantitative analysis of estrogen receptor expression in breast carcinoma does not differ from expert pathologist scoring: a tissue microarray study of 3484 cases. Breast Cancer Res Treat. 2008;110:417–426. Seidal T, Balaton AJ, Battifora H. Interpretation and quantification of immunostains. Am J Surg Pathol. 2001;25:1204–1207. Braunschweig T, Chung JY, Hewitt SM. Perspectives in tissue microarrays. Comb Chem High Throughput Screen. 2004; 7:575–585. Braunschweig T, Chung JY, Hewitt SM. Tissue microarrays: bridging the gap between research and the clinic. Expert Rev Proteomics. 2005;2:325–326. Camp RL, Chung GG, Rimm DL. Automated subcellular localization and quantification of protein expression in tissue microarray. Nat Med. 2002;8:1323–1327. Paik S, Bryant J, Tan-Chiu E, et al. Real-world performance of Her2 testing-national surgical adjuvant breast and bowel project experience. J Natl Cancer Inst. 2002;94:852–854. Van Diest PJ, van Dam P, Henzen-Logmans SC, et al. A scoring system for immunohistochemical staining: consensus report of the task force for basic research of the EORTC-GCCG. Clin Pathol. 1997;50:801–804. Wolff AC, Hammond EH, Schwartz JN, et al. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. Clin Oncol. 2007;25:118–145. Yaziji H, Taylor CR, Goldstein NS, et al. Consensus recommendations on estrogen receptor testing in breast cancer by immunohistochemistry. Appl Immunohistochem Mol Morphol. 2008;16:513–520.
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