Biomarker method validation in anticancer drug development

Abstract

Over recent years the role of biomarkers in anticancer drug development has expanded across a spectrum of applications ranging from research tool during early discovery to surrogate endpoint in the clinic. However, in Europe when biomarker measurements are performed on samples collected from subjects entered into clinical trials of new investigational agents, laboratories conducting these analyses become subject to the Clinical Trials Regulations. While these regulations are not specific in their requirements of research laboratories, quality assurance and in particular assay validation are essential. This review, therefore, focuses on a discussion of current thinking in biomarker assay validation. Five categories define the majority of biomarker assays from 'absolute quantitation' to 'categorical'. Validation must therefore take account of both the position of the biomarker in the spectrum towards clinical end point and the level of quantitation inherent in the methodology. Biomarker assay validation should be performed ideally in stages on 'a fit for purpose' basis avoiding unnecessarily dogmatic adherence to rigid guidelines but with careful monitoring of progress at the end of each stage. These principles are illustrated with two specific examples: (a) absolute quantitation of protein biomarkers by mass spectrometry and (b) the M30 and M65 ELISA assays as surrogate end points of cell death.

Figure 1

The Biomarkers spectrum from the perspective of different stakeholders. (a) The US Food and Drug Administration envisages three evidentiary stages towards biomarker approval (Goodsaid and Frueh, 2007). (b) Within the pharmaceutical industry biomarkers are utilized throughout the whole drug discovery process from compound selection to clinical trials (Lee et al., 2005). (c) In cancer medicine, biomarkers may be seen as being either diagnostic of tumour biology, or prognostic of disease or therapeutic outcome (Ludwig and Weinstein, 2005).

Figure 2

The stages of biomarker assay validation. Three stages are proposed for bioanalytical techniques referred to as Good Laboratory Practice (GLP) assays, each with a predefined purpose and goal. Biomarker assays require an additional phase where feasibility studies are conducted to determine whether or not to proceed with the particular technique.

Figure 3

The five categories of Biomarker assays. For a more comprehensive definition of each category, see main body of text.

Figure 4

Schematic representation of the cytokeratin 18 (CK18) epitope map targeted by the antibodies used in the M30 and M65 sandwich ELISA assays. In the case of M65 ELISA the M6 antibody acts as the catcher and M5 as detection antibody. For the M30 ELISA assay M5 is the catcher and HRP conjugated M30 the detection antibody. Proposed caspase cleavage sites of CK18 are also indicated.