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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

Precision of the HERmark assay. Fifteen replicates of each of 2 different cell lines spanning the dynamic range of the H2T and H2D assays were run in one batch for each assay to determine intra-assay variability.  95% of the values in the H2T and H2D assay are within 1.45-fold and 1.65-fold, respectively.
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fig4: Precision of the HERmark assay. Fifteen replicates of each of 2 different cell lines spanning the dynamic range of the H2T and H2D assays were run in one batch for each assay to determine intra-assay variability. 95% of the values in the H2T and H2D assay are within 1.45-fold and 1.65-fold, respectively.

Mentions: Assay precision was evaluated by determining the variability within a single assay or batch. Measurements were done by testing two independent cell lines spanning the high and low end of the assay dynamic range with 15 replicates per cell line per batch (MDA-MB-453 and MCF-7 for H2T; SKBR3 and MDA-MB-453 for H2D) and analyzing the results using pairwise comparisons. For the H2T assay, 100% (210/210) pairwise comparisons were within 1.7-fold and >95% of the pairwise comparisons were within 1.45-fold (Figure 4). For the H2D assay 100% (210/210) of the pairwise comparisons were within 2.3-fold and >95% of the pairwise comparisons were within 1.65-fold (Figure 4). The %CV for precision of all data in the H2T assay was 14.9% (MDA-MB-453) and 12.5% (MCF-7). The %CV for precision of 100% data in the H2D assay was 14% (SKBR3) and 21% (MDA-MB-453).


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)

Precision of the HERmark assay. Fifteen replicates of each of 2 different cell lines spanning the dynamic range of the H2T and H2D assays were run in one batch for each assay to determine intra-assay variability.  95% of the values in the H2T and H2D assay are within 1.45-fold and 1.65-fold, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4: Precision of the HERmark assay. Fifteen replicates of each of 2 different cell lines spanning the dynamic range of the H2T and H2D assays were run in one batch for each assay to determine intra-assay variability. 95% of the values in the H2T and H2D assay are within 1.45-fold and 1.65-fold, respectively.
Mentions: Assay precision was evaluated by determining the variability within a single assay or batch. Measurements were done by testing two independent cell lines spanning the high and low end of the assay dynamic range with 15 replicates per cell line per batch (MDA-MB-453 and MCF-7 for H2T; SKBR3 and MDA-MB-453 for H2D) and analyzing the results using pairwise comparisons. For the H2T assay, 100% (210/210) pairwise comparisons were within 1.7-fold and >95% of the pairwise comparisons were within 1.45-fold (Figure 4). For the H2D assay 100% (210/210) of the pairwise comparisons were within 2.3-fold and >95% of the pairwise comparisons were within 1.65-fold (Figure 4). The %CV for precision of all data in the H2T assay was 14.9% (MDA-MB-453) and 12.5% (MCF-7). The %CV for precision of 100% data in the H2D assay was 14% (SKBR3) and 21% (MDA-MB-453).

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