Developing a routine lab test for absolute quantification of HER2 in FFPE breast cancer tissues using Quantitative Dot Blot (QDB) method

Abstract

Developing immunoassay for absolute quantitation of protein biomarkers in Formalin Fixed Paraffin Embedded (FFPE) samples promises improved objectivity, consistency and accuracy in daily clinical practice. The feasibility of Quantitative Dot Blot (QDB) method for this purpose was explored in this study. We were able to measure HER2 protein levels using 0.5 µg/sample total protein lysate extracted from 2 × 5 µm FFPE slices absolutely and quantitatively using QDB method in 332 breast cancer FFPE samples. HER2 levels measured using two clinically validated antibodies for immunohistochemistry respectively were highly correlated (r = 0.963). We also achieved area under the curve (AUC) at 0.9998 ± 0.0002 (p < 0.0001, n = 224) with IHC analysis, and 0.9942 ± 0.0031 (p < 0.0001, n = 319) with combined results from IHC and Fluorescence in situ hybridization (FISH) analyses when analyzed with Receiver Operative Characteristics analysis (ROC) respectively. When the results were converted dichotomously with optimized cutoffs from ROC analyses, we achieved 99.5% concordance with IHC; and 96.9% with combined results from both IHC and FISH analyses. Therefore, we were able to demonstrate QDB method as the first immunoassay platform for absolute quantitation of protein biomarkers in FFPE samples to meet the need of daily clinical practice, especially for local laboratories or laboratories in developing countries.

Objective quantitation of EGFR protein levels using Quantitative Dot Blot (QDB) method for prognosis of gastric cancer patients

“…when measured with an anti-Her2 (EP3) antibody (21). In contrast, the distribution of EGFR was from 0 to 0.773 nmole/g (n=246) in gastric cancer specimens, and from 0 to…”

“…The QDB process was described elsewhere with minor modifications (21). In brief, the final concentration of the FFPE tissue lysates was adjusted to 0.25 µg/µl, and 2 µl/unit was used for QDB analysis in triplicate.…”

“…Recently, Quantitative Dot Blot method (QDB) has been used to measure Her2 and Ki67 protein levels absolutely and quantitatively in breast cancer specimens. This is an ELISA-like method ready to be adopted in routine clinical practice (19)(20)(21)(22). Compared with IHC method, QDB method is more simple, objective and consistent.…”

Improving Prognosis of Surrogate Assay for Breast Cancer Patients by Absolute Quantitation of Ki67 Protein Levels using Quantitative Dot Blot (QDB) Method

“…The QDB process was described in detail elsewhere with slightly modi cations [10,13]. Additional details are provided in Additional le 1.…”

“…We hypothesized the inherent inaccuracy associated with Ki67 IHC scores may negatively affect the performance of surrogate assay for Luminal-like patients. Therefore, we attempted to measure Ki67 levels absolutely and objectively using a recently developed Quantitative Dot Blot (QDB) method in Formalin Fixed Para n Embedded (FFPE) specimens [10][11][12][13]. The specimens were separated into luminal A (LumA q ) and luminal B subtypes (LumB q ) based on the absolute quantitated Ki67 levels in these specimens in a retrospective study (Table 1).…”