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Analytical Validation of a Highly Quantitative, Sensitive, Accurate, and Reproducible Assay (HERmark) for the Measurement of HER2 Total Protein and HER2 Homodimers in FFPE Breast Cancer Tumor Specimens.

Larson JS, Goodman LJ, Tan Y, Defazio-Eli L, Paquet AC, Cook JW, Rivera A, Frankson K, Bose J, Chen L, Cheung J, Shi Y, Irwin S, Kiss LD, Huang W, Utter S, Sherwood T, Bates M, Weidler J, Parry G, Winslow J, Petropoulos CJ, Whitcomb JM - Patholog Res Int (2010)

Bottom Line: We report here the results of the analytical validation of assays that measure HER2 total protein (H2T) and HER2 homodimer (H2D) expression in Formalin Fixed Paraffin Embedded (FFPE) breast cancer tumors as well as cell line controls.The accuracy of H2T measurements spans a broad dynamic range (2-3 logs) as evaluated by comparison with cross-validating technologies.The measurement of H2T expression demonstrates a sensitivity that is approximately 7-10 times greater than conventional immunohistochemistry (IHC) (HercepTest).

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Laboratory Operations, Monogram Biosciences, Inc., South San Francisco, CA 94080, USA.

ABSTRACT
We report here the results of the analytical validation of assays that measure HER2 total protein (H2T) and HER2 homodimer (H2D) expression in Formalin Fixed Paraffin Embedded (FFPE) breast cancer tumors as well as cell line controls. The assays are based on the VeraTag technology platform and are commercially available through a central CAP-accredited clinical reference laboratory. The accuracy of H2T measurements spans a broad dynamic range (2-3 logs) as evaluated by comparison with cross-validating technologies. The measurement of H2T expression demonstrates a sensitivity that is approximately 7-10 times greater than conventional immunohistochemistry (IHC) (HercepTest). The HERmark assay is a quantitative assay that sensitively and reproducibly measures continuous H2T and H2D protein expression levels and therefore may have the potential to stratify patients more accurately with respect to response to HER2-targeted therapies than current methods which rely on semiquantitative protein measurements (IHC) or on indirect assessments of gene amplification (FISH).

No MeSH data available.


Related in: MedlinePlus

Tissue Specificity testing of the H2T and H2D assays.  Tissue specificity was determined by measuring the H2T signal (a) or H2D signal (b) generated by the comparison of tumor tissue alone and with tumor tissue in addition to normal fat and stroma.  1006 = breast tumor; 878-1006-496 = breast tumor embedded with stroma and fat (a, b). 31274B2 = tumor; 272-31274B2-884 = breast tumor embedded with stroma and fat (a).
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fig9: Tissue Specificity testing of the H2T and H2D assays. Tissue specificity was determined by measuring the H2T signal (a) or H2D signal (b) generated by the comparison of tumor tissue alone and with tumor tissue in addition to normal fat and stroma. 1006 = breast tumor; 878-1006-496 = breast tumor embedded with stroma and fat (a, b). 31274B2 = tumor; 272-31274B2-884 = breast tumor embedded with stroma and fat (a).

Mentions: To analyze the effect of interfering substances on the H2T signal, two tumor samples positive for HER2 878/1006/496: 1006—high HER2; 272/31274B2/884: 31274B2—low HER2) were re-embedded with potential interfering substances from normal stroma tissue (496 and 884) and normal fat tissue (878 and 272). The blocks were then cut into slides and a subset of slides was sub-sectioned to remove the normal stroma and normal fat (tumor only: 1006 or 31274B2). These slides were then run with the corresponding whole (i.e., not sub-sectioned) re-embedded slides in the H2T assay. A pairwise comparison of final results was then obtained. For 878/1006/496, four samples of tumor only and four samples of tumor/stroma/fat were run in one batch, resulting in 16 pairwise comparisons. For 272/31274B2/884, six samples of tumor only and 9 samples of tumor/stroma/fat were run in one batch, resulting in 54 pairwise comparisons. A total of 70 pairwise comparisons were obtained and 100% (70/70) of the pairwise comparisons were within the stated acceptance criteria (2.5-fold). Further, 97% of the pairwise comparisons were within 2-fold, and 95% of the pairwise comparisons were within 1.9-fold. Results are detailed in Figure 9(a).


Analytical Validation of a Highly Quantitative, Sensitive, Accurate, and Reproducible Assay (HERmark) for the Measurement of HER2 Total Protein and HER2 Homodimers in FFPE Breast Cancer Tumor Specimens.

Larson JS, Goodman LJ, Tan Y, Defazio-Eli L, Paquet AC, Cook JW, Rivera A, Frankson K, Bose J, Chen L, Cheung J, Shi Y, Irwin S, Kiss LD, Huang W, Utter S, Sherwood T, Bates M, Weidler J, Parry G, Winslow J, Petropoulos CJ, Whitcomb JM - Patholog Res Int (2010)

Tissue Specificity testing of the H2T and H2D assays.  Tissue specificity was determined by measuring the H2T signal (a) or H2D signal (b) generated by the comparison of tumor tissue alone and with tumor tissue in addition to normal fat and stroma.  1006 = breast tumor; 878-1006-496 = breast tumor embedded with stroma and fat (a, b). 31274B2 = tumor; 272-31274B2-884 = breast tumor embedded with stroma and fat (a).
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2990097&req=5

fig9: Tissue Specificity testing of the H2T and H2D assays. Tissue specificity was determined by measuring the H2T signal (a) or H2D signal (b) generated by the comparison of tumor tissue alone and with tumor tissue in addition to normal fat and stroma. 1006 = breast tumor; 878-1006-496 = breast tumor embedded with stroma and fat (a, b). 31274B2 = tumor; 272-31274B2-884 = breast tumor embedded with stroma and fat (a).
Mentions: To analyze the effect of interfering substances on the H2T signal, two tumor samples positive for HER2 878/1006/496: 1006—high HER2; 272/31274B2/884: 31274B2—low HER2) were re-embedded with potential interfering substances from normal stroma tissue (496 and 884) and normal fat tissue (878 and 272). The blocks were then cut into slides and a subset of slides was sub-sectioned to remove the normal stroma and normal fat (tumor only: 1006 or 31274B2). These slides were then run with the corresponding whole (i.e., not sub-sectioned) re-embedded slides in the H2T assay. A pairwise comparison of final results was then obtained. For 878/1006/496, four samples of tumor only and four samples of tumor/stroma/fat were run in one batch, resulting in 16 pairwise comparisons. For 272/31274B2/884, six samples of tumor only and 9 samples of tumor/stroma/fat were run in one batch, resulting in 54 pairwise comparisons. A total of 70 pairwise comparisons were obtained and 100% (70/70) of the pairwise comparisons were within the stated acceptance criteria (2.5-fold). Further, 97% of the pairwise comparisons were within 2-fold, and 95% of the pairwise comparisons were within 1.9-fold. Results are detailed in Figure 9(a).

Bottom Line: We report here the results of the analytical validation of assays that measure HER2 total protein (H2T) and HER2 homodimer (H2D) expression in Formalin Fixed Paraffin Embedded (FFPE) breast cancer tumors as well as cell line controls.The accuracy of H2T measurements spans a broad dynamic range (2-3 logs) as evaluated by comparison with cross-validating technologies.The measurement of H2T expression demonstrates a sensitivity that is approximately 7-10 times greater than conventional immunohistochemistry (IHC) (HercepTest).

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Laboratory Operations, Monogram Biosciences, Inc., South San Francisco, CA 94080, USA.

ABSTRACT
We report here the results of the analytical validation of assays that measure HER2 total protein (H2T) and HER2 homodimer (H2D) expression in Formalin Fixed Paraffin Embedded (FFPE) breast cancer tumors as well as cell line controls. The assays are based on the VeraTag technology platform and are commercially available through a central CAP-accredited clinical reference laboratory. The accuracy of H2T measurements spans a broad dynamic range (2-3 logs) as evaluated by comparison with cross-validating technologies. The measurement of H2T expression demonstrates a sensitivity that is approximately 7-10 times greater than conventional immunohistochemistry (IHC) (HercepTest). The HERmark assay is a quantitative assay that sensitively and reproducibly measures continuous H2T and H2D protein expression levels and therefore may have the potential to stratify patients more accurately with respect to response to HER2-targeted therapies than current methods which rely on semiquantitative protein measurements (IHC) or on indirect assessments of gene amplification (FISH).

No MeSH data available.


Related in: MedlinePlus