Tejpar, Bertagnolli, Bosman et al. rently, in the E5202 clinical trial (discussed below) [52], 18q AI status is being used to differentiate between low- and high-risk stage II tumors in an extrapolation of the stage III data, which in reality may not be biologically correct. In addition, when the PETACC group evaluated the effect of 18q loss of heterozygosity (LOH) in univariate, compared with multivariate, models (containing MSI and tumor node status), it was found that 18q LOH status lost significance if MSI was included in the model [36], suggesting that these markers do not act independently and correct prognostica- tion will have to take into account several markers. A further problem in assessing tumor 18q AI status is determining what is actually being measured by 18q AI, which is currently generally unclear. Unless carefully ana- lyzed, AI can be scored as the consequence of a number of different genetic events arising from different molecular causes, with possibly different functional and biological consequences. Thus, AI may be generated by loss or gain of chromosomal material. Where loss is the proven mecha- nism of the AI, the assumption commonly made is that the clinical significance is a result of the loss of function of spe- cific genes within the chromosomal region (SMAD7, SMAD4, DCC, and SMAD2). If this is indeed the case, then 18q AI association studies should incorporate data derived from quantitative assays measuring target gene or protein expression, as has been reported in metastatic CRC (mCRC) for SMAD4 [53]. In stage III colon cancer, lower expression of SMAD4 was reported to be associated with poor prognosis in patients treated with 5-FU– based chemo- therapy [54 –56]. However, it is also possible that chromosome 18q AI may simply be a surrogate marker for the complex CIN phe- notype found in the majority of colon tumors [57, 58]. Thus, AI assays restricted to chromosome 18q are not able to dis- criminate between 18q-related gene-inactivation events and more general aneuploidy (a characteristic of CIN, which may also nevertheless lead to the inactivation or dim- inution of 18q gene function). This has implications in re- lation to our understanding of the contribution of chromosome 18q imbalance to colon tumor biology and re- sponse to therapy, and its role as a biomarker. Thus, tumor phenotypes might be masked or conflated using one tech- nology to assess imbalance at 18q, which may in turn ex- plain the contradictory findings on 18q AI and prognosis in colon cancer in the literature [34, 45, 46, 49]. Examining CIN as a prognostic marker in colon cancer has proven difficult, first because the phenotype is poorly defined and second because a number of different technol- ogies, including AI, flow cytometry, and array-based com- parative genomic hybridization (a-CGH), have been used to measure CIN. A recent meta analysis of 63 studies (10,126 www.TheOncologist.com 395 CRC patients of all stages) found CIN to be associated with a worse prognosis in CRC, including patients with locally advanced disease [59]. In that analysis, CIN was assessed in studies using techniques to measure chromosome ploidy (flow cytometry and image analysis), and hence the data in- clude chromosome 18 numerical alterations. Further, the predictive value for patients receiving 5-FU– based chemo- therapy could not be determined. The authors called for CIN to be evaluated as a prognostic marker together with MSI status in clinical trials of colon cancer patients involv- ing adjuvant therapy. Candidate Genes as Biomarkers A number of important colon cancer genes have been iden- tified and extensively studied as candidate biomarkers in colon cancer in the adjuvant setting and are reviewed below. TP53 The TP53 gene encodes a transcription factor, and in re- sponse to a variety of cellular stresses, including DNA dam- age, activated TP53 protein binds to the regulatory sequences of a number of target genes to initiate a program of cell cycle arrest, DNA repair, apoptosis, and angiogene- sis [60]. Loss of function of TP53 is critical in tumorigen- esis, and alterations to the TP53 gene (mutations, often resulting in protein overexpression) are frequent events in colon cancer, often associated with the CIN phenotype and inversely correlated with the MSI tumor phenotype [61, 62]. Associations of TP53 tumor alterations with patient prognosis and response to adjuvant chemotherapy have been widely studied, and findings are contradictory (Table 2) [63, 64]. For example, TP53 protein expression and gene mutation have been associated with poor prognosis in colon cancer patients, although other studies report no prognostic value [63, 68]. In clinical studies in which adjuvant chemo- therapy–treated and nontreated groups could be analyzed, stage III CRC patients whose tumors demonstrated no TP53 alterations experienced significantly longer survival fol- lowing 5-FU– based chemotherapy than patients whose tu- mors overexpressed p53 [61, 67, 69]. However, other studies in colon cancer patients failed to demonstrate cor- relations between TP53 alterations and benefit from adju- vant therapy [66, 70]. The contradictory nature of these studies may reflect differences in the methodologies used to assess TP53 status, including different antibodies used to detect the protein (with varying sensitivities for wild-type or mutant protein), different immunostaining techniques, and different scoring systems used for assessing expres- sion. Indeed, the reported value for TP53 overexpression in the literature covers a wide range (27%–76%), which may