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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

Sensitivity of the H2T and H2D assays.  (a) To assess the sensitivity of the H2T assay 2 different cell lines were utilized, a negative HER2 total protein cell line, MDA-MB-468, and a low HER2 receptor/cell MDA-MB-435 (IHC score  of 0, see Table 2). (b) Sensitivity of the H2D assay was assessed using the negative cell line MDA-MB-468 and the low receptor cell lineT47D (IHC score of 0/1+, see Table 2).
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fig6: Sensitivity of the H2T and H2D assays. (a) To assess the sensitivity of the H2T assay 2 different cell lines were utilized, a negative HER2 total protein cell line, MDA-MB-468, and a low HER2 receptor/cell MDA-MB-435 (IHC score of 0, see Table 2). (b) Sensitivity of the H2D assay was assessed using the negative cell line MDA-MB-468 and the low receptor cell lineT47D (IHC score of 0/1+, see Table 2).

Mentions: For the H2T assay, two cell lines (i.e., MDA-MB-435 and MDA-MB-468) were selected for sensitivity and the samples run in one batch. The batch consisted of six samples of MDA-MB-468 and nine samples of MDA-MB-435, yielding 54 potential pairwise comparisons. MDA-MB-468 has little or no HER2, while MDA-MB-435 has low signal levels, less than the lowest assay control, MCF7 [27, 28] as well as in house data (not shown). 100% (54/54) of the pairwise comparisons were within the stated acceptance criteria that all MDA-MB-435 values are greater than MDA-MB-468 values. Results are detailed in Figure 6(a), sorted from lowest signal to highest signal. Because the largest MDA-MB-468 signal (1.46) was less than the smallest MDA-MB-435 signal (3.59), detailed pairwise comparison was unnecessary. For the H2D assay, two cell lines (T47D and MDA-MB-468) were selected for sensitivity and the samples were run in one batch. The batch consisted of six samples of MDA-MB-468 and nine samples of T47D, yielding 54 pairwise comparisons. MDA-MB-468 has little or no HER2, while T47D has signal levels comparable to the low control, MCF7 [27, 28], and in, house data, (not shown). 100% (54/54) of the pairwise comparisons were within the stated acceptance criteria (T47D > MDA-MB-468). Results are detailed in Figure 6(b), sorted from lowest signal to highest signal. Since the largest MDA-MB-468 signal (0.82) was less than the smallest T47D signal (0.97), detailed pairwise comparison was unnecessary.


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)

Sensitivity of the H2T and H2D assays.  (a) To assess the sensitivity of the H2T assay 2 different cell lines were utilized, a negative HER2 total protein cell line, MDA-MB-468, and a low HER2 receptor/cell MDA-MB-435 (IHC score  of 0, see Table 2). (b) Sensitivity of the H2D assay was assessed using the negative cell line MDA-MB-468 and the low receptor cell lineT47D (IHC score of 0/1+, see Table 2).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2990097&req=5

fig6: Sensitivity of the H2T and H2D assays. (a) To assess the sensitivity of the H2T assay 2 different cell lines were utilized, a negative HER2 total protein cell line, MDA-MB-468, and a low HER2 receptor/cell MDA-MB-435 (IHC score of 0, see Table 2). (b) Sensitivity of the H2D assay was assessed using the negative cell line MDA-MB-468 and the low receptor cell lineT47D (IHC score of 0/1+, see Table 2).
Mentions: For the H2T assay, two cell lines (i.e., MDA-MB-435 and MDA-MB-468) were selected for sensitivity and the samples run in one batch. The batch consisted of six samples of MDA-MB-468 and nine samples of MDA-MB-435, yielding 54 potential pairwise comparisons. MDA-MB-468 has little or no HER2, while MDA-MB-435 has low signal levels, less than the lowest assay control, MCF7 [27, 28] as well as in house data (not shown). 100% (54/54) of the pairwise comparisons were within the stated acceptance criteria that all MDA-MB-435 values are greater than MDA-MB-468 values. Results are detailed in Figure 6(a), sorted from lowest signal to highest signal. Because the largest MDA-MB-468 signal (1.46) was less than the smallest MDA-MB-435 signal (3.59), detailed pairwise comparison was unnecessary. For the H2D assay, two cell lines (T47D and MDA-MB-468) were selected for sensitivity and the samples were run in one batch. The batch consisted of six samples of MDA-MB-468 and nine samples of T47D, yielding 54 pairwise comparisons. MDA-MB-468 has little or no HER2, while T47D has signal levels comparable to the low control, MCF7 [27, 28], and in, house data, (not shown). 100% (54/54) of the pairwise comparisons were within the stated acceptance criteria (T47D > MDA-MB-468). Results are detailed in Figure 6(b), sorted from lowest signal to highest signal. Since the largest MDA-MB-468 signal (0.82) was less than the smallest T47D signal (0.97), detailed pairwise comparison was unnecessary.

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