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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

Isotype control specificity of H2T and H2D assays. All 44 tumors and 11 cell lines were tested in the regular H2T assay.  Slide samples from adjacent sections were also run in an H2T assay where the anti-HER2-pro11 antibody conjugate was replaced with an IgG1-pro11 conjugate (“IgG1-pro11”) or where the anti-HER2-biotin antibody conjugate was replaced with an IgG1-biotin conjugate (“IgG1-biotin”). (a) is a graphical representation of the non-specific contribution of signal in the H2T assay whereas (b) represents the non-specific contribution of the signal in the H2D assay.
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fig8: Isotype control specificity of H2T and H2D assays. All 44 tumors and 11 cell lines were tested in the regular H2T assay. Slide samples from adjacent sections were also run in an H2T assay where the anti-HER2-pro11 antibody conjugate was replaced with an IgG1-pro11 conjugate (“IgG1-pro11”) or where the anti-HER2-biotin antibody conjugate was replaced with an IgG1-biotin conjugate (“IgG1-biotin”). (a) is a graphical representation of the non-specific contribution of signal in the H2T assay whereas (b) represents the non-specific contribution of the signal in the H2D assay.

Mentions: Assay specificity was demonstrated in a series of experiments designed to assess the frequency of false positive results from experiments using isotype control antibodies as well as false negative results due to the presence of commonly encountered interfering substances. 45 patient-derived tumor samples (data not shown) and 12 cell line samples (Table 2) were run using the H2T and isotype control antibodies. Isotype control antibodies were matched to the constant regions of the respective HER2 antibodies. For the HER2 Ab8-Pro11, the matched isotype control was IgG1-Pro11. For the HER2 Ab15-biotin, the matched isotype control was also IgG1-biotin. Signal generated from these reactions is not antigen specific, and represents nonspecific background. Samples were run in each of the following three formats for H2T and each format was run in a separate batch: Format 1: HER2 Ab8-Pro11/HER2 Ab15-biotin (assay specific format), Format 2: HER2 Ab8-Pro11/IgG1-biotin, and Format 3: IgG1-Pro11/HER2 Ab15-biotin. For H2D, the formats were: Format 1: HER2 Ab8-pro11/HER2 Ab8-biotin (assay specific format), Format 2: HER2 Ab8-pro11/IgG1-biotin, and Format 3: IgG1-pro11/HER2 Ab8-biotin. Sample results from each IgG1 format (Format 2 and Format 3) were compared to the negative control, MDA-MB-468, present in each batch. Samples results were also compared to the respective actual H2T or H2D signal. The results of the H2T assay demonstrated that 100% (112/112) of the results passed this criterion (either within 2-fold of the MDA-MB-468 background signal or less than or equal to 10% of the corresponding H2T signal). Results are shown in Figure 8(a). In the H2D assay, 99% (108/109) of the comparisons were within the stated acceptance criteria (either within 3-fold of the MDA-MB-468 background signal or less than or equal to 20% of the corresponding H2D signal). Results are shown in Figure 8(b).


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)

Isotype control specificity of H2T and H2D assays. All 44 tumors and 11 cell lines were tested in the regular H2T assay.  Slide samples from adjacent sections were also run in an H2T assay where the anti-HER2-pro11 antibody conjugate was replaced with an IgG1-pro11 conjugate (“IgG1-pro11”) or where the anti-HER2-biotin antibody conjugate was replaced with an IgG1-biotin conjugate (“IgG1-biotin”). (a) is a graphical representation of the non-specific contribution of signal in the H2T assay whereas (b) represents the non-specific contribution of the signal in the H2D assay.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig8: Isotype control specificity of H2T and H2D assays. All 44 tumors and 11 cell lines were tested in the regular H2T assay. Slide samples from adjacent sections were also run in an H2T assay where the anti-HER2-pro11 antibody conjugate was replaced with an IgG1-pro11 conjugate (“IgG1-pro11”) or where the anti-HER2-biotin antibody conjugate was replaced with an IgG1-biotin conjugate (“IgG1-biotin”). (a) is a graphical representation of the non-specific contribution of signal in the H2T assay whereas (b) represents the non-specific contribution of the signal in the H2D assay.
Mentions: Assay specificity was demonstrated in a series of experiments designed to assess the frequency of false positive results from experiments using isotype control antibodies as well as false negative results due to the presence of commonly encountered interfering substances. 45 patient-derived tumor samples (data not shown) and 12 cell line samples (Table 2) were run using the H2T and isotype control antibodies. Isotype control antibodies were matched to the constant regions of the respective HER2 antibodies. For the HER2 Ab8-Pro11, the matched isotype control was IgG1-Pro11. For the HER2 Ab15-biotin, the matched isotype control was also IgG1-biotin. Signal generated from these reactions is not antigen specific, and represents nonspecific background. Samples were run in each of the following three formats for H2T and each format was run in a separate batch: Format 1: HER2 Ab8-Pro11/HER2 Ab15-biotin (assay specific format), Format 2: HER2 Ab8-Pro11/IgG1-biotin, and Format 3: IgG1-Pro11/HER2 Ab15-biotin. For H2D, the formats were: Format 1: HER2 Ab8-pro11/HER2 Ab8-biotin (assay specific format), Format 2: HER2 Ab8-pro11/IgG1-biotin, and Format 3: IgG1-pro11/HER2 Ab8-biotin. Sample results from each IgG1 format (Format 2 and Format 3) were compared to the negative control, MDA-MB-468, present in each batch. Samples results were also compared to the respective actual H2T or H2D signal. The results of the H2T assay demonstrated that 100% (112/112) of the results passed this criterion (either within 2-fold of the MDA-MB-468 background signal or less than or equal to 10% of the corresponding H2T signal). Results are shown in Figure 8(a). In the H2D assay, 99% (108/109) of the comparisons were within the stated acceptance criteria (either within 3-fold of the MDA-MB-468 background signal or less than or equal to 20% of the corresponding H2D signal). Results are shown in Figure 8(b).

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