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HER-2 assessment in formalin-fixed paraffin-embedded breast cancer tissue by well-based reverse phase protein array.

Perry C, Conway CM, Ha JW, Braunschweig T, Morris J, Ylaya K, Cho H, Chung JY, Hewitt SM - Clin Proteomics (2014)

Bottom Line: HER-2 value of well-based RPPA significantly correlated with dot blotting results (R(2) = 0.939).When the cutoff level of HER-2 value was set to 0.689 (HER-2/GAPDH) on the basis of receiver-operating characteristic curve, the area under the curve was 0.975 (95% CI, 0.941-1.000).Sensitivity and specificity of well-based RPPA was 92.1% and 94.7%, respectively.

View Article: PubMed Central - PubMed

Affiliation: Tissue Array Research Program, Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 USA ; Antibody Characterization Laboratory, Advanced Technology Program, Leidos Biomedical Research, Inc, Frederick, MD USA.

ABSTRACT

Background: The human epidermal growth factor receptor-2 (HER-2) expression level is a critical element for determining the prognosis and management of breast cancer. HER-2 targeted therapy in breast cancer depends on the reliable assessment of HER-2 expression status but current standard methods are lacking a rigorous quantitative assay. To address this challenge, we developed an assessment of HER-2 expression method by well-based reverse phase protein array (RPPA).

Results: Well-based RPPA is based on a robust protein isolation methodology paired with a novel electrochemiluminescence detection system. HER-2 value of well-based RPPA significantly correlated with dot blotting results (R(2) = 0.939). By well-based RPPA, we successfully detected HER-2 expression in 76 human breast formalin-fixed paraffin-embedded tissue samples. We observed 93.4% (71/76) concordance between well-based RPPA and current HER-2 immunohistochemical assessment guideline. When the cutoff level of HER-2 value was set to 0.689 (HER-2/GAPDH) on the basis of receiver-operating characteristic curve, the area under the curve was 0.975 (95% CI, 0.941-1.000). Sensitivity and specificity of well-based RPPA was 92.1% and 94.7%, respectively.

Conclusions: HER-2 value by well-based RPPA was correlated with the current HER-2 status guideline, suggesting that this normalized HER-2 assessment may offer advantages over unnormalized current immunohistochemical assessment methods.

No MeSH data available.


Related in: MedlinePlus

HER-2 ROC curve and levels in breast cancer specimens. (A) ROC curve for well-based RPPA assay results in distinguishing between HER-2 postive and negative cases. (B) Individual HER-2 expression levels showed in 0/1+ (n = 32), 2+ (n = 15) and 3+ (n = 29) subgroups. Positive (●) and negative (○) were categorized according to the current ASCO/CAP guideline. A value of cutoff was 0.0689.
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Fig4: HER-2 ROC curve and levels in breast cancer specimens. (A) ROC curve for well-based RPPA assay results in distinguishing between HER-2 postive and negative cases. (B) Individual HER-2 expression levels showed in 0/1+ (n = 32), 2+ (n = 15) and 3+ (n = 29) subgroups. Positive (●) and negative (○) were categorized according to the current ASCO/CAP guideline. A value of cutoff was 0.0689.

Mentions: Next we evaluated the diagnostic performance of well-based RPPA to determine HER-2 expression status. Figure 4A shows the ROC curve for the discrimination HER-2 expression status with positive vs. negative. The area under the curve (AUC) of well-based RPPA was found to be 0.975 (95% CI, 0.941 -1.000). A cutoff value of 0.689 (ratio of HER-2/GAPDH) had the highest accuracy (minimal false negative and false positive results) for HER-2 detection. Figure 4B shows the individual relative HER-2 value in the different IHC groups. Concordance was excellent in 0/1+ subgroup (93.8%) and 3+ group (93.1%). In addition, the well-based RPPA technology was showed great concordance (93.3%) with FISH in IHC 2+ subgroup whereas IHC showed lower agreement (60%, 9/15). Overall, the well-based RPPA showed great sensitivity and specificity, especially this methodology could be used substantial HER-2 expression status confirmation assay with advantages of excellent positive predictive value (94.6%) (Table 1).Figure 4


HER-2 assessment in formalin-fixed paraffin-embedded breast cancer tissue by well-based reverse phase protein array.

Perry C, Conway CM, Ha JW, Braunschweig T, Morris J, Ylaya K, Cho H, Chung JY, Hewitt SM - Clin Proteomics (2014)

HER-2 ROC curve and levels in breast cancer specimens. (A) ROC curve for well-based RPPA assay results in distinguishing between HER-2 postive and negative cases. (B) Individual HER-2 expression levels showed in 0/1+ (n = 32), 2+ (n = 15) and 3+ (n = 29) subgroups. Positive (●) and negative (○) were categorized according to the current ASCO/CAP guideline. A value of cutoff was 0.0689.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4362651&req=5

Fig4: HER-2 ROC curve and levels in breast cancer specimens. (A) ROC curve for well-based RPPA assay results in distinguishing between HER-2 postive and negative cases. (B) Individual HER-2 expression levels showed in 0/1+ (n = 32), 2+ (n = 15) and 3+ (n = 29) subgroups. Positive (●) and negative (○) were categorized according to the current ASCO/CAP guideline. A value of cutoff was 0.0689.
Mentions: Next we evaluated the diagnostic performance of well-based RPPA to determine HER-2 expression status. Figure 4A shows the ROC curve for the discrimination HER-2 expression status with positive vs. negative. The area under the curve (AUC) of well-based RPPA was found to be 0.975 (95% CI, 0.941 -1.000). A cutoff value of 0.689 (ratio of HER-2/GAPDH) had the highest accuracy (minimal false negative and false positive results) for HER-2 detection. Figure 4B shows the individual relative HER-2 value in the different IHC groups. Concordance was excellent in 0/1+ subgroup (93.8%) and 3+ group (93.1%). In addition, the well-based RPPA technology was showed great concordance (93.3%) with FISH in IHC 2+ subgroup whereas IHC showed lower agreement (60%, 9/15). Overall, the well-based RPPA showed great sensitivity and specificity, especially this methodology could be used substantial HER-2 expression status confirmation assay with advantages of excellent positive predictive value (94.6%) (Table 1).Figure 4

Bottom Line: HER-2 value of well-based RPPA significantly correlated with dot blotting results (R(2) = 0.939).When the cutoff level of HER-2 value was set to 0.689 (HER-2/GAPDH) on the basis of receiver-operating characteristic curve, the area under the curve was 0.975 (95% CI, 0.941-1.000).Sensitivity and specificity of well-based RPPA was 92.1% and 94.7%, respectively.

View Article: PubMed Central - PubMed

Affiliation: Tissue Array Research Program, Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 USA ; Antibody Characterization Laboratory, Advanced Technology Program, Leidos Biomedical Research, Inc, Frederick, MD USA.

ABSTRACT

Background: The human epidermal growth factor receptor-2 (HER-2) expression level is a critical element for determining the prognosis and management of breast cancer. HER-2 targeted therapy in breast cancer depends on the reliable assessment of HER-2 expression status but current standard methods are lacking a rigorous quantitative assay. To address this challenge, we developed an assessment of HER-2 expression method by well-based reverse phase protein array (RPPA).

Results: Well-based RPPA is based on a robust protein isolation methodology paired with a novel electrochemiluminescence detection system. HER-2 value of well-based RPPA significantly correlated with dot blotting results (R(2) = 0.939). By well-based RPPA, we successfully detected HER-2 expression in 76 human breast formalin-fixed paraffin-embedded tissue samples. We observed 93.4% (71/76) concordance between well-based RPPA and current HER-2 immunohistochemical assessment guideline. When the cutoff level of HER-2 value was set to 0.689 (HER-2/GAPDH) on the basis of receiver-operating characteristic curve, the area under the curve was 0.975 (95% CI, 0.941-1.000). Sensitivity and specificity of well-based RPPA was 92.1% and 94.7%, respectively.

Conclusions: HER-2 value by well-based RPPA was correlated with the current HER-2 status guideline, suggesting that this normalized HER-2 assessment may offer advantages over unnormalized current immunohistochemical assessment methods.

No MeSH data available.


Related in: MedlinePlus