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RESEARCH ARTICLE
J Pathol Inform 2020,  11:2

A validation study of human epidermal growth factor receptor 2 immunohistochemistry digital imaging analysis and its correlation with human epidermal growth factor receptor 2 fluorescence In situ hybridization results in breast carcinoma


1 Department of Pathology, The Ohio State University Wexner Medical Center, Columbus, OH 43210, USA
2 Department of NA, Jerome High School, Dublin, OH 43017, USA

Date of Submission09-Sep-2019
Date of Acceptance16-Oct-2019
Date of Web Publication04-Feb-2020

Correspondence Address:
Dr. Anil V Parwani
Department of Pathology, The Ohio State University Wexner Medical Center; 410 W, 10th Ave, Columbus, OH 43210
USA
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jpi.jpi_52_19

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   Abstract 


Background: The Visiopharm human epidermal growth factor receptor 2 (HER2) digital imaging analysis (DIA) algorithm assesses digitized HER2 immunohistochemistry (IHC) by measuring cell membrane connectivity. We aimed to validate this algorithm for clinical use by comparing with pathologists' scoring and correlating with HER2 fluorescence in situ hybridization (FISH) results. Materials and Methods: The study cohort consisted of 612 consecutive invasive breast carcinoma specimens including 395 biopsies and 217 resections. HER2 IHC slides were scanned using Philips IntelliSite Scanners, and the digital images were analyzed using Visiopharm HER2-CONNECT App to obtain the connectivity values (0–1) and scores (0, 1+, 2+, and 3+). HER2 DIA scores were compared with Pathologists' manual scores, and HER2 connectivity values were correlated with HER2 FISH results. Results: The concordance between HER2 DIA scores and pathologists' scores was 87.3% (534/612). All discordant cases (n = 78) were only one-step discordant (negative to equivocal, equivocal to positive, or vice versa). Five cases (0.8%) showed discordant HER2 IHC DIA and HER2 FISH results, but all these cases had relatively low HER2 copy numbers (between 4 and 6). HER2 IHC connectivity showed significantly better correlation with HER2 copy number than HER2/CEP17 ratio. Conclusions: HER2 IHC DIA demonstrates excellent concordance with pathologists' scores and accurately discriminates between HER2 FISH positive and negative cases. HER2 IHC connectivity has better correlation with HER2 copy number than HER2/CEP17 ratio, suggesting HER2 copy number may be more important in predicting HER2 protein expression, and response to anti-HER2-targeted therapy.

Keywords: Breast carcinoma, digital imaging analysis, fluorescence in situ hybridization, human epidermal growth factor receptor 2, immunohistochemistry, Visiopharm


How to cite this article:
Hartage R, Li AC, Hammond S, Parwani AV. A validation study of human epidermal growth factor receptor 2 immunohistochemistry digital imaging analysis and its correlation with human epidermal growth factor receptor 2 fluorescence In situ hybridization results in breast carcinoma. J Pathol Inform 2020;11:2

How to cite this URL:
Hartage R, Li AC, Hammond S, Parwani AV. A validation study of human epidermal growth factor receptor 2 immunohistochemistry digital imaging analysis and its correlation with human epidermal growth factor receptor 2 fluorescence In situ hybridization results in breast carcinoma. J Pathol Inform [serial online] 2020 [cited 2020 Jun 6];11:2. Available from: http://www.jpathinformatics.org/text.asp?2020/11/1/2/277789

#Ramon Hartage and Aidan C. Li contributed equally.





   Introduction Top


Human epidermal growth factor receptor 2 (HER2; ERBB2) gene amplification and/or protein overexpression occurs in approximately up to 20% of breast cancers.[1],[2],[3],[4] Anti-HER2 targeted drugs, such as trastuzumab and pertuzumab, are effective in treating HER2-positive breast cancers, but not HER2-negative breast cancers.[5],[6],[7],[8] Given anti-HER2 drugs' side effects and significant cost, accurate determination of HER2-positive status is mandatory before offering them to any breast cancer patient.[9]

HER2 status is usually assessed by immunohistochemistry (IHC) for HER2 protein expression and/or by fluorescence in situ hybridization (FISH) for HER2 gene amplification. IHC is used primarily and FISH is used as a reflex test on IHC equivocal cases by most laboratories in the United States.[9] HER2 IHCs are usually evaluated by pathologists in a nonquantitative manner and given a score from 0 to 3+ based on membranous staining of HER2 protein. Although the American Society of Clinical Oncology and the College of American Pathologists (ASCO/CAP) published guidelines on how to assess HER2 IHCs, interobserver variability does occur.[9],[10],[11]

Since the wide implementation of whole slide imaging (WSI), digital image analysis (DIA) has emerged as an objective and reproducible scoring method to assess HER2 IHC in a quantitative manner.[12],[13],[14],[15],[16] Studies have demonstrated DIA could reduce HER2 IHC equivocal cases.[12],[14],[17] The ASCO/CAP HER2 guideline has acknowledged DIA as a diagnostic modality for HER2 status assessment,[9] and CAP has created guidelines to facilitate adoption of HER2 DIA into routine pathology workflows.[18]

The Visiopharm HER2 IHC DIA algorithm evaluates cell membrane connectivity and the preliminary data have demonstrated accurate assessment of HER2 IHCs in breast carcinoma and gastric/esophageal adenocarcinoma.[12],[19],[20] We aimed to validate this DIA algorithm for clinical use by comparing with pathologists' scores and correlating with HER2 FISH results in breast carcinomas.


   Materials and Methods Top


Case selection

This study included 612 consecutive primary invasive breast carcinomas from the Ohio State University Wexner Medical Center between January 01, 2016, and January 31, 2017. The use of human materials was approved by the institutional review board at the Ohio State University.

Immunohistochemistry

HER2 IHC was performed using PATHWAY anti-HER2 (4B5) on Benchmark XT automated slide stainer according to the manufacturer's protocol (Roche Ventana Medical Systems, Tucson, AZ). An automated deparaffinization step was followed by cell conditioning and then rinsed and incubated with the prediluted anti-HER2 rabbit monoclonal primary antibody (clone 4B5) at 37°C. After rinsing, staining was visualized using the ultraView Universal DAB Detection Kit (Roche Ventana Medical Systems, Tucson, AZA). The slides were counterstained, then rinsed, and coverslipped.

Pathologists' scoring

HER2 IHC was manually scored by subspecialized breast pathologists according to ASCO/CAP guidelines: 0 (negative): no staining or faint/barely perceptible, incomplete membrane staining in ≤10% of tumor cells; 1+ (negative): faint/barely perceptible, incomplete membrane staining in >10% of tumor cells; 2+ (equivocal): weak/moderate complete membrane staining in >10% of tumor cells; and 3+ (positive): circumferential complete intense membrane staining in >10% of tumor cells.

Image acquisition and digital imaging analysis

Glass slides were scanned using Philips UltraFast Scanner (Philips, the Netherlands) at ×40 magnification with a single-focus layer. The tissue on slides was detected automatically with focus points to obtain the optimal image. Whole slide images were stored in a centralized server located at The Ohio State University's campus. HER2 IHCs were evaluated using the HER2-CONNECT algorithm in the Visiopharm Integrator System (Visiopharm, Hørsholm, Denmark) and recorded as a value from 0 to 1[12] [Figure 1].
Figure 1: Human epidermal growth factor receptor 2 immunohistochemistry and the connectivity analyzed by Visiopharm human epidermal growth factor receptor 2 immunohistochemistry algorithm. (a and b) One case with human epidermal growth factor receptor 2 immunohistochemistry 1+; (c and d) one case with human epidermal growth factor receptor 2 immunohistochemistry 2+. (e and f) one case with human epidermal growth factor receptor 2 immunohistochemistry 3+. (a, c, and e) Human epidermal growth factor receptor 2 immunohistochemistry; (b, d, and f) human epidermal growth factor receptor 2 connectivity (green color line) detected by Visiopharm

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HER2 DIA scores were categorized into four categories with the following cutoff values: (1) 0: connectivity = 0; (2) 1+: 0 <connectivity ≤0.12; (3) 2+: 0.12 <connectivity ≤0.49; and (4) 3+: connectivity >0.49. Similar to the HER2 scores used by pathologists, a HER2 DIA score 0 and 1 + were defined as negative, 2 + as equivocal, and 3 + as positive based on preliminary analysis and previously reported data.

Fluorescencein situ hybridization

The FISH analysis with the CEN17 probe was performed at our institution using the dual-color Vysis FDA-approved PathVysion HER2 DNA Probe Kit (Abbott Molecular, Des Plaines, IL). The signals for the HER2 gene and CEN17 were visualized under a fluorescence microscope using appropriate filters. The average numbers of HER2 and CEN17 signals per cell were recorded for at least 50 cells, and the HER2/CEN17 ratio was calculated for each case. The results were interpreted by specialized molecular pathologists and signed out by case pathologists with a specialization in breast pathology.

Statistical analysis

All clinicopathologic data were summarized using percentages and descriptive statistics. The HER2 DIA values were compared with the paired HER2 copy numbers/ratios to assess correlation (Pearson correlation). Kappa coefficient was calculated to measure the agreement between DIA scores and pathologists' scores using GraphPad Prism (San Diego, CA). This calculation used linear weights. The kappa result was interpreted as follows: ≤0 as no agreement; 0.01–0.20 as none to slight; 0.21–0.40 as fair; 0.41–0.60 as moderate; 0.61–0.80 as substantial; and 0.81–1.00 as almost perfect agreement. The correlations of HER2 IHC connectivity with HER2 copy number or HER2/CEP17 ratio were compared using cocor analysis at cocor website (http://comparingcorrelations.org/).[21] Other statistics were performed using SAS version 9.3 (SAS Institute, Cary, NC, USA). For all results, P < 0.05 was considered as statistically significant.


   Results Top


Demographic characteristics of the study cohort

The study cohort was composed of 612 invasive breast carcinomas, including 496 invasive ductal carcinomas, 65 invasive lobular carcinomas, 25 mixed ductal/lobular carcinomas, 7 metaplastic carcinomas, and 19 metastatic carcinomas in axillary lymph nodes [Table 1]. Four hundred and thirty-two cases had HER2 IHC scores of 0 or 1+ (negative), 101 cases had scores of 2+ (equivocal) and 79 cases had scores of 3+ (positive). Among the 101 HER2 2+ cases, 49 were not amplified, 41 were equivocal, and 11 were positive on FISH based on the 2013 HER2 guidelines. Based on the 2018 guidelines,[9] 90 cases were negative for FISH and 11 were positive for FISH.
Table 1: Demographic features of study cohort

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The correlation between human epidermal growth factor receptor 2 immunohistochemistry digital imaging analysis scores and pathologists' scores

Each HER2 IHC WSI slide was analyzed using a HER2 membrane algorithm in Visiopharm and the HER2 connectivity value was recorded. Four hundred forty-eight cases were categorized as negative (0/1+), 85 as equivocal (2+), and 79 as positive (3+). The HER2 DIA scores were correlated with pathologists' scores and 78 cases showed discordant results, including 25 pathologist-negative/Visiopharm-equivocal cases, 41 pathologist-equivocal/Visiopharm-negative cases, 6 pathologist-equivocal/Visiopharm-positive cases, and 6 pathologist-positive/Visiopharm-equivocal cases. All discordant cases (n = 78) were only one-step discordant (negative to equivocal/equivocal to positive, or vice versa) [Table 2]. The agreement between HER2 DIA and pathologists' read was “substantial” (kappa = 0.713, weighted kappa = 0.797, 87.3%). HER2 DIA decreased the HER2 IHC equivocal cases from 101 to 85 (16%).
Table 2: The correlation between human epidermal growth factor receptor 2 digital image analysis scores and pathologists' scores

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The correlation between human epidermal growth factor receptor 2 immunohistochemistry digital imaging analysis values and human epidermal growth factor receptor 2 fluorescencein situ hybridization copy numbers/ratios

Next, we analyzed the correlation between HER2 IHC DIA connectivity values with HER2 FISH copy numbers and ratios using the Pearson correlation in the 442 cases in which FISH results were available. The Pearson correlation coefficient (r) for HER2 IHC DIA connectivity and HER2 FISH copy number was 0.860594 (n = 442; P < 0.0001) with a mean coefficient of determination (R2) of 0.7245 [Figure 2]. The Pearson correlation coefficient (r) for HER2 IHC DIA connectivity and HER2 FISH copy number was 0.824927 (n = 442; P < 0.0001) with a mean coefficient of determination (R2) of 0.6701 [Figure 3]. The difference between these two correlation coefficients (DIA/copy number vs. DIA/ratio) was 0.0357 and was statistically significant based on cocor analysis using a backtransformed average Fisher's z procedure (z = 3.8429, P = 0.0001).
Figure 2: Correlation between human epidermal growth factor receptor 2 immunohistochemistry digital imaging analysis connectivity and human epidermal growth factor receptor 2 fluorescence in situ hybridization copy number. The Pearson correlation coefficient (r) for human epidermal growth factor receptor 2 immunohistochemistry digital imaging analysis connectivity and human epidermal growth factor receptor 2 fluorescence in situ hybridization copy number was 0.860594 (n = 442; P< 0.0001). (y = 20.498x + 1.7076. R2 =0.7245)

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Figure 3: Correlation between human epidermal growth factor receptor 2 immunohistochemistry digital imaging analysis connectivity and human epidermal growth factor receptor 2 fluorescence in situ hybridization ratio. The Pearson correlation coefficient (r) for human epidermal growth factor receptor 2 immunohistochemistry digital imaging analysis connectivity and human epidermal growth factor receptor 2 fluorescence in situ hybridization ratio was 0.824927 (n = 442; P <0.0001). (y = 7.1342x + 0.7798. R2 =0.6701)

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FISH results from all 442 cases were re-interpreted based on the 2018 ASCO/CAP HER2 guidelines to separate into five groups with groups 1 and 3 being FISH-positive, and groups 2, 4 and 5 being FISH-negative. Overall, only five cases (0.8%) showed discordant HER2 IHC DIA and HER2 FISH results, including 3 cases with negative IHC DIA but positive FISH, and 2 cases with positive IHC DIA but negative FISH [Table 3]. All five cases had HER2 copy numbers between 4 and 6, but the ratio was >2 in three FISH-positive cases (ISH group 1) and <2 in 2 FISH-negative cases (ISH group 4) [Table 4]. Four of these cases showed 2+ and 1 case showed 1+ on pathologists' scores. Additional FISH assays with or without alternative probe (D17S122) were performed, and the results were included in [Table 4].
Table 3: The correlation between human epidermal growth factor receptor 2 digital image analysis scores and fluorescence in situ hybridization results in 442 cases with fluorescence in situ hybridization

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Table 4: Five cases with discordant Human epidermal growth factor receptor 2 immunohistochemistry digital image analysis/fluorescence in situ hybridization results

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Pitfalls in human epidermal growth factor receptor 2 immunohistochemistry digital imaging analysis

In the process of analyzing HER2 IHC using DIA, we had identified several pitfalls and challenges. The causes for false positive DIA included air bubbles, pigments, ductal carcinoma in situ (DCIS) components, and inks [Figure 4]a, [Figure 4]b, [Figure 4]c, [Figure 4]d. On the other hand, out of focus or rare tumor cells caused false-negative DIA results [Figure 4]e and [Figure 4]f.
Figure 4: Pitfalls of human epidermal growth factor receptor 2 immunohistochemistry digital imaging analysis. (a). Air bubbles (red arrow); (b) pigments (red arrow); (c) ductal carcinoma in situ components (red arrow); invasive carcinoma was pointed with black arrow; (d) ink (red arrow); (e) out of focus; (f) rare tumor cells (red arrow). The human epidermal growth factor receptor 2 connectivity was labeled with green lines

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   Discussion Top


To the best of our knowledge, this study, including 612 invasive breast carcinomas, is one of the largest to validate HER2 IHC DIA using whole slide images. Our data have demonstrated that HER2 IHC DIA is a reliable measurement for HER2 protein expression based on HER2 membrane connectivity. HER2 DIA not only shows an excellent concordance with pathologists' manual scoring (87.3%), but also reduces HER2 IHC equivocal case numbers (16% reduction). All discordant cases (n = 78, 12.7%) show only one-step discordance (negative to equivocal/equivocal to positive, or vice versa). Our results are consistent with previous studies which have shown high agreement between HER2 DIA and manual scoring in breast cancer specimens, with 87.5%–94.2% agreement rates.[12],[13],[14],[15],[17],[22],[23]

Our data also reveal that HER2 IHC DIA can accurately discriminate between HER2 FISH positive and negative cases interpreted based on 2018 ASCO/CAP HER2 guidelines.[9] Overall, only five cases (0.8%) showed discordant HER2 IHC DIA and HER2 FISH results, including 3 cases with negative IHC DIA but positive FISH (false-negative), and 2 cases with positive IHC DIA but negative FISH (false-positive). Previous studies also revealed false-negative and/or false-positive cases with variable frequencies.[12],[13],[14],[17] All five cases had HER2 copy numbers between 4 and 6, and pathologist's score of 2+ (except one case with 1+), representing the difficult cases with borderline HER2 gene amplification/protein overexpression and lack of information regarding clinical outcomes.

Since the HER2 DIA used in this study also rendered an absolute value of HER2 connectivity from 0 to 1 for each case, we investigated its correlation with HER2 FISH copy number and ratio. The analysis reveals HER2 IHC connectivity has better correlation with HER2 copy number than HER2/CEP17 ratio (r: 0.860594 vs. 0.824927 ), suggesting HER2 copy number may be more accurate to predict HER2 protein expression, and even response to anti-HER2 targeted therapy than HER2/CEP17 ratio. The findings are consistent with previous literatures demonstrating breast cancers with ratio ≥2 and copy number <4 are predominantly HER2 IHC-negative and less responsive to HER2-targeted therapy than breast cancers with copy number u4.[24],[25],[26]

Lastly, we have identified several pitfalls in the HER2 DIA process, including air bubbles, pigments, DCIS components, and inks, that cause false-positive results and out of focus and rare tumor cells to cause false-negative results. These pitfalls are not infrequent (4%, 25/612), but most of them can be avoided by reprocessing glass slides (air bubble), rescanning (out of focus), and carefully annotating region of interest (excluding pigments, DCIS components, and inks).


   Conclusion Top


In summary, we have demonstrated that HER2 IHC DIA is a feasible and valid tool to determine HER2 status in breast carcinoma with high concordance when compared to pathologists' manual scoring and HER FISH. Our data have also revealed HER2 IHC connectivity has better correlation with HER2 copy number than HER2/CEP17 ratio, suggesting HER2 copy number may be more important to predict HER2 protein expression, and even response to anti-HER2-targeted therapy. Furthermore, HER2 IHC DIA measures HER2 protein in continuous quantitative values, providing useful information for correlating with clinical outcomes.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
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  [Table 1], [Table 2], [Table 3], [Table 4]



 

 
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