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Evaluation of HER2/neu Status by Immunohistochemistry Using Computer-Based Image Analysis and Correlation With Gene Amplification by Fluorescence In Situ Hybridization Assay: A 10-Year Experience and Impact of Test Standardization on Concordance Rate.

The HER2 is a member of the HER family of transmembrane receptors with tyrosine kinase activity encoded by the HER2/neu gene. (1) Approximately 15% to 25% of invasive breast cancers overexpress HER2 and this is associated with aggressive behavior and poor outcome. (2,3) Several molecular targets directed against the HER2 receptor have been developed that have improved the outcome for these patients. (4) The overexpression of HER2 is most commonly assessed by immunohistochemistry (IHC). Despite the ease and ready availability of IHC, the lack of consistency in HER2/neu IHC testing/scoring and poor concordance with the FISH assay have cast doubt on the reliability of IHC tests. (5-8)

In an effort to improve the accuracy of HER2 testing with better concordance between the 2 assays, the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) proposed several methodologic and interpretation recommendations for HER2 test standardization. (9) One of the suggestions was that image analysis (IA) could be an effective tool for objective interpretation, and that validation and calibration of IA instrumentation is essential before implementation in the laboratory. Studies (10-19) have shown IA to be more objective and reproducible with higher correlation with the fluorescence in situ hybridization (FISH) assay. However, most of these studies included small series of cases and were not reflective of routine clinical practice. According to a recent CAP survey, approximately 33% of laboratories in the United States used IA for HER2/neu IHC assessment. (20)

In this retrospective study, we analyzed HER2/neu IHC results that were performed in our laboratory from 2002 to 2011 and quantified by IA. The IHC results were correlated with the FISH assay. We also determined the impact of standardization of test performance in accordance with the ASCO/CAP guidelines on HER2 concordance rate. We correlated HER2 status with race/ethnicity, pathologic parameters, and expression of estrogen receptor (ER), progesterone receptor (PR), and Ki-67.


The database included consecutive cases of primary and metastatic breast cancers that were diagnosed at Parkland Health and Hospital System, and the University Hospital of the University of Texas (UT) Southwestern Medical Center, Dallas, Texas. The study was a retrospective analysis of HER2/neu test IHC results from 2002 to 2011 that were scored by IA and confirmed by FISH. Only cases that were concurrently tested by IHC and FISH were selected for this study. Assessment of HER2 was performed in parallel with assessment of ER, PR, and Ki-67 as part of routine workup of patients for therapy planning and treatment. All tests were performed in the Department of Pathology, UT Southwestern Medical Center. In our practice, several pathologists with different years of experience are involved in the day-to-day sign-out of breast prognostic markers, including HER2. To maintain consistency in reporting and minimize interobserver variability among the pathologists, IA was used for scoring of HER2/neu, ER, PR, and Ki-67.

Immunohistochemical Staining and IA Scoring

Parallel sections from the same tumor block were used for IHC and FISH testing. Immunostaining was performed by using standardized automated techniques according to manufacturer's protocol. The Automated Cellular Imaging System (ACIS, Clarient, Inc, San Juan Capistrano, California) was used for scoring of HER2/ neu IHC from 2002 to November 2009, and the Ventana Imaging System (VIAS, Ventana Medical Systems, Tucson, Arizona) from December 2009 onward. The change from ACIS to the VIAS was due to a switch from Dako (Carpinteria, California) to Ventana antibodies and test procedures. Both systems were validated in the Clinical Laboratory Improvement Amendments of 1988 (CLIA)certified laboratory before clinical use. Antibodies used with the ACIS were ER (1:200; 1D5), PR (1:1000; PgR), HER2/neu (1:400; A0285), Ki-67 (1:500; MIB-1), and p53 (1:100; DO-7) (Dako). For the VIAS, the prediluted antibodies ER (SP1), PR (1E2), HER2/neu (4B5), Ki-67 (30-9), and p53 (DO-7) (Ventana) were used according to manufacturer's protocol.

The ACIS system consists of a robotic bright-field microscope that digitizes the slide stained by IHC; digital images are analyzed by proprietary software. For HER2/neu evaluation, the ACIS recognizes 256 levels of staining intensity that are converted to fractional scores. A continuum of staining intensity levels is scored in decimal increments. Six to 10 areas of the invasive tumor with the highest staining intensity (hot spots) were selected according to manufacturer's recommendation and the average score was calculated and taken as the final score. The manufacturer's default IA score of greater than 2.0 was reported as positive (3+), 1.4 to 1.9 as borderline (2+), and less than 1.4 as negative (0 to 1+). In 2008 the manufacturer's threshold for IHC 3+ score was raised to 2.5 or greater; scores ranging from 1.4 to less than 2.5 were scored as borderline; and less than 1.4, as negative.

The VIAS imaging device has a special optimized microscope, a touch screen control, and image analysis software. Ten non-overlapping images of the invasive tumor that are most representative of the 0 to 3+ score were selected by the operator for digital image acquisition and analysis. The VIAS algorithm generated a continuous scaled score for HER2/neu and the average score of the selected images was taken as the final score. The manufacturer's cutoff scores for negative (0 and 1+), 2+, and 3+ were 0 to 1.5, 1.51 to 2.50, and 2.51 to 3.5, respectively. However, owing to false-positive 3+ IHC results with the manufacturer's cutoff range, when compared to the FISH assay, it was decided to raise the threshold for 3+ to 3.5, and those between 2.51 and 3.49 were downgraded to 2+. Results for ER, PR, and Ki-67 were reported as percentage positive staining nuclei.

HER2/neu FISH Analysis

The FISH assay was performed by using the Vysis PathVysion kit (Abbott-Vysis, Abbott Park, Illinois) according to the manufacturer's protocol. Before 2007 a ratio of HER2/neu to CEP17 of 2.0 or greater was interpreted as positive for HER2/neu gene amplification and less than 2.0 as negative. From 2008, the ASCO/CAP scoring criteria were used: ratio less than 1.8 or HER2/neu signals less than 4 were reported as nonamplified; ratio of 1.8 to 2.2 or 4 to 6 HER2/ neu signals as borderline amplification; and signals greater than 6 or a ratio of greater than 2.2 as amplified.

Statistical Analysis

The sensitivity, specificity, and positive, negative, and overall concordance rate for HER2/neu IHC were calculated by using the FISH assay as the gold standard and excluding the IHC 2+ and equivocal FISH cases. The IHC 3+/ FISH+ result was considered true positive. The relationship between race/ethnicity and HER2 status was analyzed by using the [chi square] test and unadjusted post hoc multiple comparisons. The [chi square] test was used for comparing categorical variables and analysis of variance for differences in the mean HER2/neu amplification ratios in the different IHC categories. The Wilcoxon rank sum test was used as a nonparametric test for differences in continuous measures between the IHC 3+/FISH+ and IHC 3+/FISH--groups. Comparison of HER2 rates by year was analyzed by simple linear regression. All reported P values are 2-sided and performed at the .05 level. Multiple comparisons were not adjusted. All analyses were performed by using SAS 9.3 (SAS Inc, Cary, North Carolina).


There were 3093 HER2/neu IHC and concurrent FISH test results for 2264 patients from 2002 to 2011. In 1567 of 2264 patients (69.2%), a test was performed only 1 time and 697 patients (30.7%) had test results that were performed on more than 1 occasion. Of the 2264 patients, 2018 (89.1%) were diagnosed with primary breast cancer and 246 (10.9%) with metastatic breast cancer. The median age of the 2264 patients was 55.4 years (range, 21.1-99.7 years). Of the 2018 patients diagnosed with primary invasive breast cancer, 861 (42.7%) were white, 643 (31.9%) were African American, 350 (17.3%) were Hispanic, and 164 (8.1%) were of other/ unknown descent (Table 1). Clinical stage was available for 1772 of 2018 patients (87.8%); 1284 (72.5%) had stage 1 and 2 disease and 488 (27.5%) had stage 3 and 4 disease. Nodal status was available for 749 of 2018 patients (37.1%): 501 (66.9%) had N0 status; 177 (23.6%), N1; 38 (5.1%), N2; and 33 (4.4%), N3. There were significant differences in rates of HER2 positivity among different racial/ethnicity groups (P = .001). Differences in rates between white and Hispanic individuals was significant (P = .003) as well as between Hispanic and African American individuals (P = .002). The overall rate of HER2 positivity was 21.7% (492 of 2264) among all the patients tested and 22.3% (690 of 3093) for all tumors tested.

For the 3093 tumors, FISH test result was positive in 69.3% (594 of 857) of IHC 3+ cases and nonamplified in 27.3% (234 of 857) (Table 2). Conversely, of the 690 FISH+ cases, 594 (86.1%), 72 (10.4%), and 24 (3.5%) were IHC 3+, 2+, and negative, respectively. In the 2306 FISH-- group, 1174 (50.9%), 898 (38.9%), and 234 (10.1%) were IHC negative (0, 1+), 2+, and 3+, respectively. Conversely, 95.9% (1174 of 1224) of the HER2/neu IHC-negative (0,1+) cases were also negative by FISH.

Excluding the IHC 2+ and borderline FISH test results, the overall concordance between IHC and FISH assay was 87.3% (1768 of 2026). The positive and negative concordance rates were 71.7% (594 of 828) and 98.0% (1174 of 1198), respectively. When analyzed by year, there was an improvement in overall concordance rates (>90%) from 2008 onward with corresponding increase in specificity (Figure). The implementation of the ASCO/CAP guidelines in our laboratory was initiated in 2008. In 2002, only 49.4% (44 of 89) of the IHC 3+ cases were amplified compared to 95.0% (57 of 60) in 2011, a 92% improvement (Table 3). The change in true positive rate from 2002 to 2011 was highly significant (P < .001). The true negative rate was at least 95% throughout the study period.

The median rate of FISH positivity in the 3093 tumors tested was 24.7%. In the IHC 2+ and IHC- groups, the median rates of FISH positivity were 5.3% and 1.5%, respectively. In the IHC 2+ cohort, the FISH-positive rates increased from 2008 onward, and this was associated with an increase in the rate of IHC 2+ cases (Table 3).

We were able to retrieve the HER2/neu amplification ratios for 2987 of 3093 FISH tests (96.6%). In the FISH-amplified cohort, the level of HER2/neu amplification ratio showed significant association with IHC categories, with the highest ratio in the IHC 3+ and lowest in the IHC--group (P < .001) (Table 4). Interestingly, in the FISH--group, HER2/neu amplification ratio also showed significant association with IHC expression (P < .001).

For 698 of 2018 patients with primary breast cancer (34.5%), results of other biomarkers (ER, PR, and Ki-67), and pathologic features were available for comparison between the IHC 3+/FISH+ and the IHC 3+/FISH--group (Table 5). In the former, the tumors were more frequently high grade, with ductal morphology, than in the latter (P < .001, P < .001). The IHC 3+/FISH--group consisted more frequently of biopsies (145 of 211, 68.7%) than excisions (66 of 211, 31.3%) than did the IHC 3+/FISH+ group (P = .01). For the 132 cases with known duration of fixation, 124 (93.9%) were in the IHC 3+/FISH+ group versus 8 (6.1%) in the IHC 3+/FISH-- group (P < .001). There was no difference in the median duration of fixation in the 2 groups (P = .88). The ER and PR expression was more frequently positive in the IHC 3+/FISH-- than the IHC 3+/FISH+ group (P < .001), with higher ER and PR scores in the former than among the HER2-positive group. The Ki-67 index was higher in the IHC 3+/FISH+ than the IHC 3+/ FISH--cases.


In our patient population, the rate of HER2 positivity was 21.7%. This is within the range reported in the literature. (1-3) We found significant differences in rates of HER2 positivity among racial/ethnic groups with higher rates in Hispanic (28.3%) than white (19.4%) and African American (18.5%) individuals. In a recent study on breast tumor phenotypes among ethnic groups, Hispanic individuals had a strikingly higher proportion of HER2-positive tumors: 31.9% versus 14.3% among non-Hispanic white persons. (21) Our findings are supported by a recent population-based study that reported a higher prevalence of HER2 positivity among Hispanic women than other racial/ethnic groups. (22) There is a higher prevalence of aggressive breast cancer subtypes, such as triple-negative and HER2 subtypes, among minority women, and Hispanic individuals are more likely to have unfavorable tumor characteristics when compared to non-Hispanic white persons. (21,22)

To our knowledge, this is the largest study comparing quantitative HER2/neu IHC expression by IA with the FISH assay in a real-world setting. The overall agreement between IHC and FISH was 87.2%. When analyzed by year, the concordance rate was greater than 90% after 2007 (Figure). This finding is similar to that of a recent CAP survey on HER2 IHC-FISH concordance, which was 90% in most laboratories across the country. (20) The positive concordance rates improved significantly during the 10-year period, with rates of at least 95% in 2010-2011 (P < .001). On the other hand, the negative concordance rate was at least 95% throughout the test period. The median false-negative rate was 1.5%, which is much lower than the 3% to 5% rate reported previously. (23,24) Several studies (25,26) have shown that the concordance rate between IHC and FISH is highest for the IHC--cases and lowest for the IHC 2+ and 3+ cases. In several studies, (5,27) false-positive IHC findings were more common than false-negative results, whereas others (28) reported higher false-negative than false-positive rates.

The HER2/neu amplification rates in the 10-year period ranged from 15.4% to 28.3% with a median of 24.7%, which is within the range reported previously. (1-3) Although the IHC 3+ positive rates demonstrated a wider variation from 10% to 29.1% (data not shown) with higher rates before 2008, this finding was associated with higher IHC-FISH discordance, suggesting a lack of standardization of the IHC assay before 2008. We can infer from our findings that the FISH assay performed more consistently than IHC, because it is more objective and less affected by tissue fixation and processing. (8) On the other hand, IHC assessed by IA was a good predictor of HER2-negative status, since only 1.5% of the IHC-negative cases were positive by FISH. A recent study in 8 pathology centers across Canada showed a false-*negative rate of 0.98% even when different antibodies and IHC platforms were used. (29) Our findings support the view that false-positive rather than false-negative IHC results are a major issue with HER2/neu IHC testing. Several large trials using tumor samples with varying processing and fixation times have shown a higher success rate with the FISH assay than with IHC. (8)

In our laboratory, all IHC 3+ cases were confirmed with the FISH assay, and HER2/neu amplification is considered true positive. Also, most high-grade tumors that were triple negative were tested by the FISH assay. Several studies (5,7,8,26) suggest that FISH is superior to IHC, although it is not 100% sensitive or specific. Some laboratories have adopted the FISH-only approach since it is more objective than IHC. (5,7,8) We believe that IHC should be used as an initial screening test because of its high negative predictive value, and the IHC 2+ and 3+ results should be confirmed with the FISH assay. Although FISH is more expensive than IHC, the cost of treating patients who are not likely to benefit because of a false-positive IHC result far outweighs the cost of doing the FISH test. According to some authors, (26) the strategy with lowest cost effectiveness ratio for HER2 testing is to use IHC as a screening test and confirm 2+ and 3+ cases with the FISH assay.

In the FISH-positive cases, we have demonstrated a highly significant relationship between level of HER2/neu gene amplification ratio and scoring by IA. As expected, the highest mean amplification ratio was in the 3+ group followed by the 2+ and IHC--groups. Low level of amplification in the HER2--group has been shown by others. (29) This supports the argument that HER2 overexpression is the direct consequence of gene amplification. (8)

The improvement in positive concordance rate after 2008 may be due to several factors, including the experience gained over the years in performing HER2 testing, increasing the IA threshold for 3+ scoring, and more importantly, measures taken by the laboratory to be compliant with ASCO/CAP recommendations. (9) Rigorous standardization of fixation/processing of breast specimens and minimizing cold ischemic time was instituted. The minimum fixation time for biopsy samples was standardized at 6 hours and for large specimens, at 72 hours. Since 2009, we have made it mandatory for clinicians to document the time to fixation in the requisition forms. Before 2008, duration of fixation was not recorded and some biopsy specimens had less than 6 hours of fixation. Large specimens were sometimes placed in containers with insufficient volume of formalin, and without sectioning. We also educated residents/fellows, surgeons, and pathology staff about proper handling of breast specimens. Breast specimens were processed on the weekend in order to avoid prolonged fixation time. Calibration of the IA instrument and validation of IA results with the FISH assay were done on a regular basis. Before 2009 general pathologists performed HER2 interpretation. In 2009, breast pathology was subspecialized in our department and dedicated pathologists with experience in the field were responsible for all HER2 evaluation. Strict adherence to stain interpretation was followed, and cytotechnologists were retrained on image analysis technique. Vergara-Lluri et al (28) attributed the interpretation of HER2 results by breast subspecialty-trained pathologists as one of the factors for high concordance rate in their study. Similarly, others27 stressed the importance of experienced pathologists for accurate assessment of HER2 expression. Although the methodologic and interpretation requirements in HER2 testing have been well elucidated, the critical role of trained pathologist in HER2 interpretation has not been well emphasized. The ASCO/CAP Task Force does not specify how to monitor competency of the pathologists interpreting the HER2 test. (30) The interpretation of HER2 expression requires a high level of training and experience. Grimm et al (27) showed that 54% of IHC-FISH discordant cases were due to overinterpretation of weak positive staining as 3+. A review of some of our HER2 discordant cases (data not shown) showed overinterpretation of IHC 1+ and 2+ staining as 3+.

Studies analyzing the impact of the ASCO/CAP guidelines have shown improved concordance rate between IHC and FISH, while others reported no change. (28,31,32) Middleton et al (33) demonstrated that adherence to the ASCO/CAP guidelines resulted in greater IHC-FISH concordance, although the improvement was only 0.5%. Our data showed a significant improvement in concordance rates after implementation of the guidelines. We believe that poor concordance before 2008 was most likely due to the lack of test standardization and interpretation error.

It has been a little more than a decade since digital technology was developed for immunohistochemical quantitation of biomarkers. More recently, several imaging systems have become available for pathologists to assist in HER2 quantitation. Indeed, IA has been used for scoring of HER2/neu IHC since 2001 and the technique was shown to reduce the subjectivity associated with manual estimation, with better correlation with the FISH assay. (10-17) Wang et al (10) first reported HER2/neu quantification by IA, using the ACIS versus manual evaluation in 189 cases. The concordance rate with FISH was 91% versus 86% with the manual method. Bloom and Harrington (11) reported a concordance rate of 93% versus 71% by manual method with significant improvement in interobserver agreement. Tawfik et al (13) showed an overall concordance of 94% by IA with a positive concordance rate of 85% when using the ACIS. However, most of these studies included small number of cases and were not performed in a routine fashion.

Despite the reported objectivity of IA, the selection of areas to be imaged is subjective and operator dependent. Moreover, experience in imaging and HER2/neu IHC interpretation is critical for proper scoring. In an attempt to standardize the scoring by IA, Minot et al (15) developed a laboratory-validated scoring technique and correlated it with the FISH assay. The concordance with FISH ranged from 87% to 100% with fewer cases in the IHC 2+ category than for manual scoring.

The rate of IHC 2+ category varies widely in different studies and is reflective of the subjectivity in scoring this category. (26) A recent large study (34) reported the rate of IHC 2+ at 24% by manual scoring and 14% were amplified by FISH, using the ratio of greater than 2.0. The rate of IHC 2+ by IA in one study (17) was 11.2% and FISH was positive in 26% of the cases. In our study, the FISH+ rate ranged from 1% to 14% (median, 5.3%), although a higher FISH+ rate was seen from 2008 onward; however, this was associated with higher numbers of IHC 2+ cases. Studies comparing manual versus IA evaluation did not see a large reduction of IHC 2+ when using IA. (16,35) Others (10) reported fewer IHC 2+ cases by IA when compared to manual scoring. Improved concordance between FISH and IHC has resulted in a 40% increase in the rate of IHC 2+. (36)

Our findings demonstrated that the accuracy of IA is dependent on standardization of preanalytic and analytic variables in addition to operator experience. A switch to the VIAS digital imaging system in 2009 and the use of a different anti-HER2/neu antibody may have had little influence on our concordance rate, since issues related to tissue handling/processing and interpretation had a far greater impact on HER2 concordance than choice of antibody or imaging system. This is supported by the fact that duration of fixation was documented in only 3.8% of the IHC 3+/FISH- cases versus 25.5% in the IHC 3+/FISH+ group (P < .001), although there were no significant differences in the median duration of fixation between the 2 groups. We also observed that the IHC 3+/FISH- cases more frequently included biopsy (68.7%) than excision specimens (31.3%), P = .02. Crushed tumor cells and nonspecific staining at the edges are frequently seen in small biopsy specimens. (27,32) These artifacts may result in higher reading by the machine. Based on our experience, scoring of small biopsy specimens is more problematic than for excision specimens, because of limited tissue and overlapping fields. Accurate scoring by IA is critically dependent on good tissue and stain quality. (19) Others (13,17) have also reported a higher discordance rate in biopsy versus excision specimens. Furthermore, there is a misconception that smaller biopsy specimens require less time in formalin. Fixation is a chemical reaction and biopsy specimens require at least 6 hours of fixation.

The poor histologic grade, high Ki-67 index, and lower ER and PR expression in the IHC 3+/FISH+ group compared to the IHC 3+/FISH- group is consistent with the biology of HER2-positive disease. Our findings lend further support to the notion that IHC 3+/FISH+ tumors more accurately reflect positive HER2 status than IHC 3+/ FISH- tumors.

In conclusion, we have shown our experience with HER2 testing performed during a 10-year period and evaluated by IA. Before 2008 there was poor concordance with the FISH assay but with significant improvement in concordance rate afterward. This may be due to overall improvement in specimen handling, test standardization, and experience in HER2/neu scoring by IA.

The study was supported by a grant from Genentech, a member of the Roche Group, South San Francisco, California (project protocol No. ML25745).


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Venetia R. Sarode, MD; Qun Diane Xiang, MD; Alana Christie, MS; Rebecca Collins, MD; Roshni Rao, MD; A. Marilyn Leitch, MD; David Euhus, MD; Barbara Haley, MD

Accepted for publication September 26, 2014.

From the Departments of Pathology (Drs Sarode, Xiang, and Collins), Cancer Biology, Simmons Comprehensive Cancer Center (Ms Christie), Surgery (Drs Rao, Leitch, and Euhus), and Medical Oncology (Dr Haley), UT Southwestern Medical Center at Dallas, Dallas, Texas.

The authors have no relevant financial interest in the products or companies described in this article.

Presented in part at the San Antonio Breast Cancer Symposium; December 5, 2012; San Antonio, Texas.

Reprints: Venetia R. Sarode, MD, Department of Pathology, UT Southwestern Medical Center at Dallas, Harry Hines Blvd, Dallas, TX 75390 (e-mail:
Table 1. Distribution of HER2/neu Positivity by Fluorescence In Situ
Hybridization (FISH) in the Different Racial Groups With Diagnosis of
Primary Invasive Breast Cancer

                        FISH, No. Cases (Row %)          Total

                Amplified    Borderline   Nonamplified
White           167 (19.4)    32 (3.7)     662 (76.9)     861
Black           119 (18.5)    22 (3.4)     502 (78.1)     643
Hispanic         99 (28.3)    11 (3.1)     240 (68.6)     350
Other/unknown    48 (29.3)     3 (1.8)     113 (68.9)     164
Total              433           68           1517       2018

Abbreviation: HER2, human epidermal growth factor receptor 2.

Table 2. Comparison of Fluorescence In Situ Hybridization (FISH) and
Immunohistochemistry (IHC) Test Results in the 3093 Tumors Tested

                        FISH, No. Cases (Row %)           Total

IHC Positivity   Amplified    Borderline   Nonamplified

IHC 3+           594 (69.3)    29 (3.4)     234 (27.3)     857
IHC 2+            72 (7.1)     42 (4.2)     898 (88.7)    1012
IHC (0, 1+)       24 (2.0)     26 (2.1)    1174 (95.9)    1224
Total               690           97           2306       3093

Table 3. Frequency of HER2 Amplification and Positive
Immunohistochemistry by Year

                                 No. Amplified/Total Cases (%)

Year      Total Cases        IHC (3+)        IHC (2+)      IHC (0, 1+)
       With IHC and FISH

2002          197          44/89 (49.4)      4/48 (8.3)    1/60 (1.7)
2003          235          57/103 (55.3)     2/72 (2.8)    3/60 (5.0)
2004          223          59/90 (65.6)      1/70 (1.4)    3/63 (4.8)
2005          271          62/106 (58.5)     1/95 (1.1)    1/70 (1.4)
2006          260          64/90 (71.1)      3/95 (3.2)    1/75 (1.3)
2007          301          72/103 (69.9)     2/99 (2.0)    0/99 (0.0)
2008          437          68/92 (73.9)    14/131 (10.7)   3/214 (1.4)
2009          512          59/70 (84.3)    15/118 (12.7)   5/324 (1.5)
2010          336          52/54 (96.3)    11/149 (7.4)    2/133 (1.5)
2011          321          57/60 (95.0)    19/135 (14.1)   5/126 (4.0)

Year   Total Amplified,
           No. (%)

2002    49/197 (24.9)
2003    62/235 (26.4)
2004    63/223 (28.3)
2005    64/271 (23.6)
2006    68/260 (26.2)
2007    74/301 (24.6)
2008    85/437 (19.5)
2009    79/512 (15.4)
2010    65/336 (19.3)
2011    81/321 (25.2)

Abbreviations: FISH, fluorescence in situ hybridization; HER2, human
epidermal growth factor receptor 2; IHC, immunohistochemistry.

Table 4. Comparison of HER2/neu Amplification Ratios in the Different
IHC Categories

FISH              IHC (3+)                   IHC (2+)

               N     Mean [+ or -] SD     N     Mean [+ or -] SD

Amplified      567   5.74 [+ or -] 2.35   68    4.21 [+ or -] 1.91
Borderline     27    1.94 [+ or -] 0.12   39    1.97 [+ or -] 0.13
Nonamplified   225   1.23 [+ or -] 0.22   876   1.19 [+ or -] 0.19

FISH               IHC (0, 1+)             P Value

               N      Mean [+ or -] SD

Amplified      23     3.33 [+ or -] 1.93   <.001
Borderline     25     1.96 [+ or -] 0.12   .58
Nonamplified   1137   1.14 [+ or -] 0.18   <.001

Abbreviations: FISH, fluorescence in situ hybridization; HER2, human
epidermal growth factor receptor 2;IHC, immunohistochemistry; SD,
standard deviation.

Table 5. Comparison of Pathologic Features and ER, PR, and Ki-67
Expression in the IHC 3+/FISH+ Versus IHC 3+/FISH- Patients With
Primary Invasive Breast Cancer (a)

                         IHC 3+/FISH+       IHC 3+/FISH-     P Value
                          (n = 487)          (n = 211)

Histology                 (n = 487)          (n = 211)
  Ductal                 444 (91.2%)        177 (83.9%)       <.001
  Lobular                  9 (1.8%)          17 (8.1%)
  Other                   34 (7.0%)          17 (8.1%)
Grade                     (n = 428)          (n = 181)
  1                        8 (1.9%)          26 (14.4%)       <.001
  2                      199 (46.5%)         97 (53.6%)
  3                      221 (51.6%)         58 (32.0%)
Biopsy/excision           (n = 486)          (n = 211)
  Biopsy                 286 (58.8%)        145 (68.7%)        .01
  Excision               200 (41.2%)         66 (31.3%)
Duration of fixation      (n = 487)          (n = 211)
  Hours in formalin    9.5 (1.7, 102.0)   12.0 (4.0, 24.0)     .88
  Without duration       363 (74.5%)        203 (96.2%)       <.001
  With duration          124 (25.5%)          8 (3.8%)
Biomarker expression      (n = 480)          (n = 210)
levels (%)
  ER+                  78.0 (1.0-100.0)   98.0 (2.0-100.0)    <.001
  PR+                  27.5 (1.0-100.0)   65.0 (1.0-100.0)    <.001
  Ki-67                43.0 (2.0-100.0)   25.5 (0.0-99.0)     <.001

Abbreviations: ER, estrogen receptor;FISH, fluorescence in situ
hybridization; IHC, immunohistochemistry;n, number of cases; PR,
progesterone receptor.

(a) Continuous measures are displayed as median (range) and
categorical measures are displayed as frequency (percentage).

Sensitivity, specificity, and concordance rate
for HER2/neu immunohistochemistry compared
to fluorescence in situ hybridization
by year. Abbreviation: HER2, human epidermal
growth factor receptor 2.

Year            2002     2003      2004      2005      2006

Sensitivity     44/45    57/60     62/63     72/72     68/71
Specificity     59/104   54/83     67/106    69/92     97/121
Concordance    103/149   111/153   113/145   129/169   169/193

Year            2007     2008      2009      2010      2011

Sensitivity     72/72    68/71     68/71     52/54     57/62
Specificity     97/121   208/229   208/229   318/328   117/119
Concordance    169/193   276/300   276/300   377/392   174/181
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Author:Sarode, Venetia R.; Xiang, Qun Diane; Christie, Alana; Collins, Rebecca; Rao, Roshni; Leitch, A. Mar
Publication:Archives of Pathology & Laboratory Medicine
Date:Jul 1, 2015
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