Abstract 3155: Multiple biomarker quantification as absolute and continuous variables in 1059 FFPE breast cancer specimens using QDB method
Authors: Zhang Jiandi , Tang Fangrong , Yu Guohua , Zhang Yunyun , Lv Jiahong , Zhang Wenfeng 
Published: August 2019

Abstract
Purpose: Although immunohistochemistry (IHC) based tissue biomarker assessment has become an integral part of solid tumor diagnosis and prognosis, the inherent problems with IHC, including its subjectivity and inconsistency, significantly limit its usage to its full potentials in clinical practice. The results from IHC analysis are also presented as discrete variables, unable to reflect the broad range distribution of biomarkers at protein level. Absolute quantification of tissue biomarkers for daily practice is needed to provide more accurate and reliable assessment of biomarkers at protein level.
Methods: Quantitative Dot Blot (QDB) method was used to measure several biomarkers including ER, PR, Ki67, Her2, p53, PCNA in 1059 FFPE breast cancer tissues. Total tissue lysates were extracted from 2X15μm FFPE slices for QDB measurement using antibodies well validated for IHC analysis (IHC antibodies), including 4B5 and EP3 for Her2, MIB and UMAB107 for Ki67, SP1 for ER, 1E2 for PR, DO-7 for p53, and PC10 for PCNA. Purified recombinant proteins were used as protein standard to achieve absolute quantification of these biomarkers. The results were validated indirectly with provided IHC/FISH results, and the correlation analysis were performed with other clinicopathological parameters using Spearman rank (rho) and/or Pearson (r) correlation analysis.
Results: QDB method was able to measure these biomarkers objectively and consistently with intra and inter-CV below 15% from three independent experiments, each in triplicate. Both Her2 and Ki67 levels were measured with two independent IHC antibodies with highly consistent results (r&gt;0.96). When converted Her2 levels into dichotomous variables using ROC analysis and provided IHC score, QDB achieved concordance with FISH at 94.2%. When analyzed as absolute and continuous variables, the p53, Ki67 and PCNA show strong correlation among themselves, with Ki67 and PCNA at rho=0.67, p=0.0000. Age was found to be positively associated with ER (p&lt;0.0001), negatively associated with PR (p&lt;0.01). Histological grade was best correlated with Ki67 (p&lt;0.0001), followed by PCNA (p&lt;0.0001), p53 (p&lt;0.0001) and Her2 (p&lt; 0.005) and negatively associated with ER levels (p&lt;0.0001). Both Ki67 and PCNA was found to be positively associated with PR (p&lt;0.001), but not ER.
Conclusions: This is the first large scale study with so many biomarkers measured as absolute and continuous variables simultaneously in FFPE specimens. QDB method was proved to be objective, consistent and in high throughput format to meet the daily need of clinical diagnosis and prognosis. It can be easily standardized to circumvent the limitations associated with current methods, and open path for database-based diagnosis and prognosis in the near future. The adoption of this method may have direct impact on precision medicine, especially on the field of targeted therapies.
Citation Format: Jiandi Zhang, Fangrong Tang, Guohua Yu, Yunyun Zhang, Jiahong Lv, Wenfeng Zhang. Multiple biomarker quantification as absolute and continuous variables in 1059 FFPE breast cancer specimens using QDB method [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3155.