Interpretation and molecular validation of biomarker studies

The development and implementation of biomarkers for the diagnosis and classification of breast cancers and stratification of breast cancer patients into clinically meaningful groups are essential for the realisation of individualised medicine. The accurate, robust and reproducible assignment of patients into subgroups of therapeutic relevance is of utmost importance. Breast cancer patient treatment decision-making currently relies on the analyses of a few immunohistochemical markers (for example, oestrogen receptor, progesterone receptor and HER2), fluorescence and/or chromogenic in situ hybridisation, protein analysis of lysates, and quantitative real-time PCR. It has become clear, however, that these markers are not sufficient for the potential of individualised therapy to be fully realised. The advent of high-throughput technologies and their use in fundamental and translational research endeavours have led to the development of diagnostic markers, potential prognostic and predictive factors, and therapeutic targets, which ultimately will need to be incorporated in clinical practice. Some of the major challenges in this process are to determine the accuracy of the research hypothesis, the exclusion of potential biases, and to define whether the reagents and methodologies are fit for purpose. This requires not only a thorough assessment of the accuracy, robustness and reproducibility of the markers and the methods for their analysis, but also an adequate contextualisation of the validity of a given biomarker. For instance, immunohistochemistry has become one of the major tools for the identification of expression of potential markers in cancer tissues; albeit at first glance trivial to perform, immunohistochemical analysis can be affected by numerous parameters that can affect its accuracy. Likewise, several gene expression profiling approaches for the identification of molecular subtypes of breast cancer have been shown not to assign the same patients into the same molecular subgroups consistently. Interpretation of in situ hybridisation is also fraught with difficulties. Discrepancies in the assessment of biomarkers have often been attributed to intra-tumour heterogeneity, without exclusion of sources of technical variation. The challenges for the interpretation of biomarker studies and validation of biomarkers in human tissues will be discussed.