6.2. What are prognostic factors and predictive markers?

From a biological standpoint, prognostic factor and predictive markers fall into one of three categories; patient-related characteristics, tumour intrinsic characteristics and time dependent factors ( table 6.1). prognostic and predictive markers have different roles in therapy decision making. Prognostic factors aim to predict independently and objectively the clinical outcome of a cancer patient. Prognostic markers are better defined in populations that did not receive systemic therapy. Predictive marker aim to forecast independently the effect of a particular therapeutic intervention in a patient. It should be noted that a predictive marker can have a prognostic impact and may even be the targert of the therapy it self; however, not all predictive markers have an impact on the outcome of breast cancer patients and not all predictive markers are the actual targets of a given targeted therapy. In this era of high throughput method(e.g. Proteomics, transciptomics and genomics), a plethora of potential novel prognostic and predictive markers have been reported (for review, the readers are reffered to weigelt et al,2009;Abdullah-sayani et al.2006; sotiriou and pusztai 2009) out of these, onlu a few have been thoroughly validated and even fewer have been translated into routine use in clinical practice. In fact, despite the many millions spent on translational research endeavours, only three markers have made prime-time to therapy decision making; immunohistochemistry for oestrogen receptor (ER) and progesterone receptor (PR), which are predictive markers for endocrine therapy, and immunohistochemistry and in situ hybridization for HER2, the therapeutic target and predictive marker for trastuzumab and lapatinib. Numerous factors, including sample size, rudimentary bioinformatics approaches and lack of a complete understanding of the sources of bias ( ransohoff2004,2005), may have contributed to this apparent failure to translate the biomarkers identified in high throughput studies into tools to guide breast cancer patient decision making. To classify the research studies on novel biomarkers, hayes et al. proposed a tumour marker utility grading system (TMUGS) with 6 different scales ()to +++) of grading (hayes et al 1996): 0 shows no evidence of utility in clinical pratice, biomarkers with ++or +++ can be considered for standar practice in special clinical situations or standar practice, respectively. For each biomarker a grade of utility is assigned, accompanied by a level of evidence (LOE) that scores the quality of the research. The LOE categories range from I to V (table 6.2). level V evidence is obtained from case reports and clinical experience and is considered weak, while level I evidence is derived either from at least one prospective randomized controlled trial specificallu designed to test the marker or from a meta-analysis and/or overview of level II or III studies and is considered definitive (Hayes et al 1996). This grading system has been endorsed by the American Society of clinical Oncology that regularly reports their recommendations for the clinical use of biomarkers (harris et al 2007). Using this system, only two markers have established powerful predictive value and are rountinely assessed in every breast cancer case, namely ER and HER2, In order to improve further research on biomarkers (McShane et al 2005) recently proposed a set of criteria that should be used when reporting the results of biomarker studies. Known as REMARK (Reporting Recommendations for tumour marker Prognostic Studies), these guidelines provide a description as to the minimum amount of information that should be reported for biomarker studies in cancer research. Specifically, they list 20 items that investigators should attempt to report in any tumour biomarkers study and that are grouped under heading: introduction, materials and methods, result,and discussion, reflecting the relevant sections of a scientific

article. Included are items such as a description of the patient population, a report of why some data are missing from the study, and a statistical report of how cut-points are determined for categorization of biomarkers. Notwithstanding the efforts in standardization of reporting and interpretation of biomarker-related studies, it is patently clear that more powerful prognostic and predictive markers are required for breast cancer individualized therapy to become a reality (weigelt et al. 2009; Geyer et al. 2009; Geyer and Reis-Filho 2009) 6.3 Technique and Methods Multiple techniques and tools for the molecular characterization of tumour samples are currently available. There are, however, several factors that limit the application of each easy assay in clinical practice. New microarray technologies for identification of tissue biomarkers require optimally preserved nucleic acids (e.g. fresh frozen tissue) and cannot be readily applied to formalin-fixed-paraffin-embedded(FFPE) specimens, the routine method of tissue fixation for diagnostic purposes. FFPE samples are highly stable at room temperature and expensive to store longterm. Furthermore, FFPE make up for a vast archive of clinical samples that can be promptly linked to clinical details and outcome, material from patients enrolled in previous randomized clinical trials, and is amenable for conducting both retrospective and prospective biomarker investigations. It should be noted, however, that formalin fixation and paraffin embedding are some of the main sources of spurious results in biomarkers discovery and validation, as different protocols for tissue handling, composition of fixatives, fixation conditions and fixation times introduce confounding factors that have often hindered the standarisation of methods for biomarker assessment.