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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

HERmark Assay Workflow. Deparaffin/rehydration, antigen retrieval, and overnight antibody incubation steps are performed on Day 1.  On day 2, the tumor tissue is incubated with the photosensitizer molecule and illuminated to release VeraTags.  VeraTags are collected and separated on capillary electrophoresis and the tumors are H & E stained, tumor area identified and circled and the final sample and batch normalized data is typically available within a 7 day turnaround time.
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fig2: HERmark Assay Workflow. Deparaffin/rehydration, antigen retrieval, and overnight antibody incubation steps are performed on Day 1. On day 2, the tumor tissue is incubated with the photosensitizer molecule and illuminated to release VeraTags. VeraTags are collected and separated on capillary electrophoresis and the tumors are H & E stained, tumor area identified and circled and the final sample and batch normalized data is typically available within a 7 day turnaround time.

Mentions: The HERmark assay workflow has been described in detail in the Material and Methods and is depicted schematically in Figure 2. In brief, the FFPE tumor tissues are subjected to deparaffinization/rehydration, antigen retrieval, and the conjugated antibodies are then added and incubated overnight. The following day the tissues are processed as described in Materials and Methods and then illuminated on a chiller block for two hours. Following illumination, the VeraTag reporters are collected from the tumor specimens and run on capillary electrophoresis and quantified using proprietary informer software. The sections are then H & E stained and the invasive tumor area identified by a board certified pathologist and quantified using an image analysis system as described in Materials and Methods. Tumors that are morphologically more than 50% DCIS are excluded from the analysis. The tumor area is calculated by the identification and circling of the invasive tumor by a board certified pathologist and subsequently quantifying this area using an image analysis system as described in Materials and Methods. The final VeraTag data reported is normalized to the actual tumor area and the entire batch is normalized using cell line controls that have been previously characterized with expected values (data not shown).


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)

HERmark Assay Workflow. Deparaffin/rehydration, antigen retrieval, and overnight antibody incubation steps are performed on Day 1.  On day 2, the tumor tissue is incubated with the photosensitizer molecule and illuminated to release VeraTags.  VeraTags are collected and separated on capillary electrophoresis and the tumors are H & E stained, tumor area identified and circled and the final sample and batch normalized data is typically available within a 7 day turnaround time.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2990097&req=5

fig2: HERmark Assay Workflow. Deparaffin/rehydration, antigen retrieval, and overnight antibody incubation steps are performed on Day 1. On day 2, the tumor tissue is incubated with the photosensitizer molecule and illuminated to release VeraTags. VeraTags are collected and separated on capillary electrophoresis and the tumors are H & E stained, tumor area identified and circled and the final sample and batch normalized data is typically available within a 7 day turnaround time.
Mentions: The HERmark assay workflow has been described in detail in the Material and Methods and is depicted schematically in Figure 2. In brief, the FFPE tumor tissues are subjected to deparaffinization/rehydration, antigen retrieval, and the conjugated antibodies are then added and incubated overnight. The following day the tissues are processed as described in Materials and Methods and then illuminated on a chiller block for two hours. Following illumination, the VeraTag reporters are collected from the tumor specimens and run on capillary electrophoresis and quantified using proprietary informer software. The sections are then H & E stained and the invasive tumor area identified by a board certified pathologist and quantified using an image analysis system as described in Materials and Methods. Tumors that are morphologically more than 50% DCIS are excluded from the analysis. The tumor area is calculated by the identification and circling of the invasive tumor by a board certified pathologist and subsequently quantifying this area using an image analysis system as described in Materials and Methods. The final VeraTag data reported is normalized to the actual tumor area and the entire batch is normalized using cell line controls that have been previously characterized with expected values (data not shown).

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