Tejpar, Bertagnolli, Bosman et al. TYMS The thymidylate synthase gene TYMS encodes a key en- zyme for pyrimidine biosynthesis and is an essential com- ponent of the DNA synthesis pathway. TYMS protein activity is inhibited by 5-FU (a pyrimidine analog), leading to cell cycle arrest and apoptosis [98]. In vitro data indicate that TYMS expression is a determinant of 5-FU sensitivity, suggesting that the expression of the gene may also deter- mine tumor sensitivity in vivo [99, 100]. However, conflict- ing data make the role of this gene as a prognostic or predictive marker in the adjuvant setting controversial (Ta- ble 2). High levels of tumor TYMS protein are reported to be associated with poor prognosis in CRC patients, partic- ularly in those receiving surgery alone, although the rea- sons for this remain unclear [65, 66, 101–104]. Study of the expression of other enzymes in the pyrimidine biosynthesis pathway— dihydropyrimidine dehydrogenase (DPD) and thymidine phosphorylase— has shown low tumor expres- sion of TYMS and DPD to be associated with worse prog- nosis in stage II and stage III CRC patients treated with surgery alone [105]. Patients receiving adjuvant 5-FU– based chemotherapy with high levels of tumor TYMS ex- pression were reported to experience significantly longer survival times [101, 102, 106], with TYMS expression re- ported to be predictive of response to adjuvant chemother- apy [101, 102]. However, other studies found no prognostic [68, 107, 108] or predictive [66, 108] value of response to adjuvant chemotherapy for TYMS expression in colon cancer. Some studies have investigated TYMS mRNA levels in tumors, and high levels of tumor TYMS mRNA and failure to respond following 5-FU– based chemotherapy have been reported [109, 110]. Germline variants in the TYMS gene have been shown to alter TYMS protein and gene expres- sion [111, 112], and have been associated with response, time to tumor progression, OS, and time to tumor recur- rence after 5-FU– based chemotherapy, although the data are conflicting [113–115]. The clinical significance and re- lationships between mRNA and protein levels in tumors and between germline variation and TYMS gene function remain to be elucidated in colon cancer. The Use of Randomized Clinical Trials for Biomarker Validation in Adjuvant Colon Cancer: The PETACC-3 Study The PETACC-3 trial encompassed a translational study to validate current candidate biomarkers in a large colon can- cer cohort of 3,278 patients. The main aims of the transla- tional study were: (a) to assess the feasibility of biomarker analysis on archival formalin-fixed, paraffin-embedded (FFPE) material collected prospectively from 368 collabo- www.TheOncologist.com 397 rating centers in 31 European countries, (b) to evaluate or confirm the prognostic relevance of selected biological markers using 3-year DFS and OS endpoints, and (c) to as- sess the predictive utility of specific markers in patients re- ceiving irinotecan in combination with 5-FU and LV, compared with those receiving 5-FU and LV alone [10, 36, 42]. FFPE tissue blocks were available from 1,564 patients and were processed in a central laboratory, where 20 –25 sections were cut per patient tissue block for subsequent analysis. Biomarkers were assessed using validated and ro- bust methodologies [10]. All data were collected and ana- lyzed at the Swiss Group for Clinical Cancer Research. Biomarker data were available from 1,452 cases, with 1,401 evaluable for matched normal and tumor tissue. The success rate for the number of samples evaluable for spe- cific markers was high: 80% for IHC analysis and 95% for DNA mutation analysis using techniques optimized for use on degraded DNA extracted from FFPE tissues. The frequency of specific biomarker alterations in the PETACC-3 study was consistent with that found in the lit- erature [10], with sufficient statistical power to detect an HR of 0.7 for DFS if the proportion of single-marker detec- tion is 80% [10, 116]. Thus, a reassessment of the signifi- cance of TP53 mutation and IHC, KRAS mutation, TYMS genotype and IHC, and MSI in this cohort is ongoing [36, 42, 77], on which many of the same biomarkers are being tested [8, 41, 51, 78]. Clearly, these two studies provide useful independent test and validation cohorts of patients in which to investigate candidate biomarker utility. The Current Status of Biomarkers in Adjuvant Colon Cancer: A Summary Extensive colon cancer research over the last decade has provided some promising biomarkers. In some cases, we are close to using these in meaningful prospective clinical studies. For example, the E5202 study is currently deter- mining the role of MSI and 18q AI as predictive factors to guide decision making for stage II colon cancer patients. In that trial, the risk for relapse after adjuvant treatment for stage II CRC is being assessed based on initial stratification by MSI status and 18q AI; low-risk patients are subject to observation whereas high-risk cases receive FOLFOX and bevacizumab [52]. Other candidate biomarkers are a long way from having their utility in the clinical setting con- firmed, with many of the studies providing evidence of their suitability being limited by the following common features: 1. Many studies are retrospective analyses of single-arm investigations performed in small and often heteroge- neous cohorts of patients in which rectal tumors have