Molecular Genetics of Colon Cancer
The knowledge of the molecular etiology of colon cancer has facilitated the identification of a number of promising prognostic and/or predictive biomarkers. A simplified model of tumor progression from adenoma to carcinoma has been proposed, which includes the stepwise accumulation of genetic events to several key genes and genetic loci: disruption to WNT signaling, activation of the KRAS protooncogene, allelic imbalance (AI) on chromosome 18q, reduced expression of SMAD4, and mutation of the TP53tumor suppressor gene. A more detailed molecular analysis of colon cancers revealed colon tumors to be heterogeneous with regard to molecular alterations and potentially categorizable into specific tumor phenotypes based on their molecular profiles.
Two of these represent genetic instability classes. The majority of sporadic cases (up to 85%) display chromosomal instability (CIN), which manifests as aneuploid and polyploid karyotypes and multiple structural chromosomal changes. This phenotype is thought to arise through defects in a number of processes, including aberrant expression or mutation of mitotic checkpoint genes, microtubule spindle defects, and telomere dysfunction . In contrast, the remaining 15% of sporadic colon cancers demonstrate a microsatellite instability (MSI) phenotype, in which tumors display insertion– deletion mutations, most commonly in short tandemly repeated nucleotides (microsatellites) . Chromosome losses are rarer in these tumors, which tend to have a diploid karyotype . The underlying genetic mechanism responsible for this phenotype is loss of function, predominantly through gene silencing of DNA mismatch repair (MMR) genes, in particular, MLH1 in sporadic CRC. Consequently, this phenotype is also often referred to as the MMR deficient (dMMR) phenotype, and in 2%–3% of CRC is caused by germline mutations to one of a number of MMR gene(MLH1, MSH2, MSH6, and PMS2) that form part of the presentation of including TGFBR2, IGF2R, and PTEN, and is associated with gain-of-function mutations in oncogenes such as BRAF; this phenomenon, in turn, is often referred to as a “mutator” phenotype. Finally, the analysis of CpG island methylation in the silencing of genes in colon tumors has led to the identification of the CpG island methylator phenotype, which appears to partially overlap the MSI phenotype .
Genomic Instability Phenotypes as Biomarkers
p<>. MSI can be detected in tumors by a number of complementary approaches. Using the polymerase chain reaction (PCR) to amplify specific microsatellite repeats, the presence of instability can be monitored through a comparison of the length of repeats obtained from normal DNA (typically extracted from adjacent normal mucosa cells) with those from the DNA extracted from the tumor cells. A reference panel of 5–10 microsatellite loci is used to diagnose MSI cases , for which three categories have been established: MSI-High (MSI-H), unstable for 30% of markers used; MSI-Low (MSI-L), unstable for 10%–30% of markers used; and microsatellite stable (MSS), for cases that display no MSI. Lack of expression of MMR proteins as assessed by immunohistochemistry (IHC) (primarily using antibodies to the MLH1 protein) is diagnostic for dMMR and is often used in MSI tumor analysis as an alternative to PCR, and additionally in the clinical setting to complement genetic testing for Lynch syndrome patients .
In clinical studies, MSI rates have been shown to vary with tumor stage—22% reported in stage II, 12% reported in stage III, and 2% reported in stage IV disease. In the adjuvant setting, MSI tumor status has been shown to be a significant prognostic marker. The majority of retrospective studies (Table 2) demonstrate that patients with MSI-H (or dMMR) colon cancers have higher survival rates than those with MSS tumors . These findings were confirmed in a meta-analysis of 32 trials, which confirmed the prognostic advantage in patients with MSI-H tumors and those treated with 5-FU–based adjuvant therapy . In the PETACC-3 study, the prognostic value of MSI status was found to be more significant in patients with stage II disease than in stage III cases. In addition, in a multivariate analysis of stage II colon cancer patients from the Quick and Simple And Reliable (QUASAR) study, Kerr and colleagues demonstrated that MMR deficiency (hazard ratio [HR], 0.31; 95% confidence interval [CI], 0.15– 0.63; p .001) and T4 stage (HR, 1.94; 95% CI, 1.35–2.79; p.005) (together accounting for 25% of patients) were independent prognostic factors for tumor recurrence . Similar findings were reported in a multivariate analysis of the PETACC-3 data .
The value of MSI tumor status as a predictive marker of adjuvant therapy is less clear. An early study suggested that
MSI-H was predictive of response to 5-FU–based adjuvant therapy in patients with stage III colon cancer . However,
an accumulating body of evidence suggests that patients with MSI-H tumors do not benefit from 5-FU–based adjuvant therapy, compared with patients with MSS tumors. This is particularly relevant for patients with stage II disease, for whom adjuvant chemotherapy (5-FU alone) is reported to increase survival by approximately 3%, and has led some investigators to recommend that stage II colon tumors should be analyzed for dMMR status to guide decisions on the use of adjuvant therapy. Recently the CALGB 89803 study reported a higher 5-year DFS rate in stage III colon cancer patients with MMR-deficient/MSI-H tumors treated with irinotecan plus 5-FU than in patients treated with the same regimen with intact MMR proteins: this was not observed in patients treated with 5-FU and leucovorin (LV) alone, suggesting that tumor MSI status might be predictive of response to irinotecan in stage III colon cancer . In contrast, the PETACC-3 study, in 1,327 patients, failed to demonstrate a predictive effect of tumor MSI status for patients treated with irinotecan, 5-FU, and LV, compared with those receiving 5-FU alone .
To date, MSI is considered to be a strong and wellvalidated prognostic marker in adjuvant CRC, and it is currently the only such biomarker in this setting. In the appropriate clinical setting, we would advocate that MSI data may be used in clinical decision making, particularly in stage II colon cancers, for which a favorable outcome of patients with MSI-H tumors suggests that these patients should not receive adjuvant chemotherapy . The assessment of MSI tumor status as a predictive marker for adjuvant therapy requires more data. It should also be considered, however, that the value of MSI tumor status as a prognostic or predictive marker in the adjuvant setting may be effected by mutations to other genes involved in colon cancer etiology, such the BRAF gene.
- Chromosome 18q AI/CIN
p<>. Chromosome 18q AI has been associated with poor prognosis in stage II and stage III CRC patients in some studies, but not others . Watanabe and colleagues reported that patients with stage III MSS colon tumors with no 18q AI had a higher survival rate following 5-FU–based treatment (70% versus 50%) than those whose tumors displayed 18q AI. In the CALGB 89803 study, stage III colon cancer patients with 18q AI had lower 5-yearDFS (0.78 versus 0.93) and OS (0.85 versus 0.98) rates than patients whose tumors displayed no 18q AI . However, drawing conclusions from comparing chromosome 18q AI studies in colon cancer is difficult, and differences in the methodologies used, including the scoring of AI, possibly explains the contradictory findings reported. Thus, the inconsistency of the genetic markers used among studies leads to analysis of AI in different regions on chromosome 18q.
An additional complication comes from the stage-specific effects of biomarkers. The PETACC group presented, at the 2009 American Society of Clinical Oncology Annual Meeting , that tumor 18q AI status was not found to be prognostic in stage II tumors, whereas an effect was found in stage III tumors on univariate analysis. This is important because the patient population most in need of prognostic markers is stage II patients, for whom treatment versus no treatment is based on the inherent prognostic features. Currently, in the E5202 clinical trial , 18q AI status is being used to differentiate between lowand 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 , suggesting that these markers do not act independently and correct prognostication 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 analyzed, 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 mechanism of the AI, the assumption commonly made is that the clinical significance is a result of the loss of function of specific 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 . In stage III colon cancer, lower expression of SMAD4 was reported to be associated with poor prognosis in patients treated with 5-FU–based chemotherapy.
However, it is also possible that chromosome 18q AI may simply be a surrogate marker for the complex CIN phenotype found in the majority of colon tumors . Thus, AI assays restricted to chromosome 18q are not able to discriminate between 18q-related gene-inactivation events and more general aneuploidy (a characteristic of CIN, which may also nevertheless lead to the inactivation or diminution of 18q gene function). This has implications in relation to our understanding of the contribution of chromosome 18q imbalance to colon tumor biology and response to therapy, and its role as a biomarker. Thus, tumor phenotypes might be masked or conflated using one technology to assess imbalance at 18q, which may in turn explain the contradictory findings on 18q AI and prognosis in colon cancer in the literature .
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 technologies,including AI, flow cytometry, and array-based comparative genomic hybridization (a-CGH), have been used to measure CIN. A recent meta analysis of 63 studies found CIN to be associated with a worse prognosis in CRC, including patients with locally advanced disease . In that analysis, CIN was assessed in studies using techniques to measure chromosome ploidy (flow cytometry and image analysis), and hence the data include chromosome 18 numerical alterations. Further, the predictive value for patients receiving 5-FU–based chemotherapy 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 involving adjuvant therapy.
Candidate Genes as Biomarkers
A number of important colon cancer genes have been identified and extensively studied as candidate biomarkers in colon cancer in the adjuvant setting.
p<>. The TP53 gene encodes a transcription factor, and in response to a variety of cellular stresses, includingDNAdamage,
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 angiogenesis . Loss of function of TP53 is critical in tumorigenesis, and alterations to the TP53 gene (mutations, often in protein overexpression) are frequent events in colon cancer, often associated with the CIN phenotype and inversely correlated with the MSI tumor phenotype . Associations of TP53 tumor alterations with patient prognosis and response to adjuvant chemotherapy have been widely studied, and findings are contradictory. 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 . In clinical studies in which adjuvant chemotherapy– treated and nontreated groups could be analyzed, stage III CRC patients whose tumors demonstrated no TP53 alterations experienced significantly longer survival following 5-FU–based chemotherapy than patients whose tumors overexpressed p53 . However, other studies in colon cancer patients failed to demonstrate correlations between TP53 alterations and benefit from adjuvant therapy . 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 expression.
Indeed, the reported value for TP53 overexpression in the literature covers a wide range (27%–76%), which may reflect these issues. It is generally accepted that the detection of p53 protein by IHC is a poor indicator of TP53 gene mutation status, because alternative molecular mechanisms can lead to protein stabilization in tumors, and some mutations lead to loss of protein stability . Studies in which TP53 mutations were detected by gene sequencing report associations with poor prognosis in colon cancer patients . It has been suggested that, to analyze the gene properly in clinical studies, TP53 mutation status should be assessed by DNA sequencing and data must be combined with TP53 protein expression information as determined by IHC.
p<>. The KRAS proto-oncogene encodes a 21-kDa guanosine triphosphate/guanosine diphosphate binding protein involved in facilitating cellular response to extracellular stimuli. Mutations within the KRAS gene (primarily at codons 12 and 13) abrogating GTPase activity and leading to downstream activation of RAS/RAF signaling are common (35%–42%) and early events in colon tumorigenesis . However, the role of KRAS mutation status as a prognostic and predictive biomarker in the adjuvant setting is controversial (Table 1). In a large meta-analysis, codon 12 glycine-to-valine mutations were found to be prognostic in patients with stage III disease . Smaller studies have shown KRAS mutation status to be associated with poor prognosis in patients with stage II and stage III disease. However, recent analyses from the CALGB 89803 (stage III colon cancer) and PETACC-3 (stage II and III) trials demonstrated KRAS mutation to not be a prognostic marker for patients treated with adjuvant 5-FU–based chemotherapy . In addition, the National Cancer Institute of Canada CO.17 trial recently demonstrated that tumor KRAS mutation status had no prognostic effect for OS in pretreated stage IV patients receiving best supportive care. As a predictive marker in the adjuvant setting, most studies report no association between KRAS mutations and response to standard chemotherapy . In a Southwest Oncology Group trial, patients with stage III tumors with KRAS mutations gained no additional benefit from receiving 5-FU/LV compared with observation or LV alone. In contrast, patients with KRAS wild-type tumors significantly benefited from 5-FU/LV therapy . Data from the CALGB 89803 study suggest that KRAS tumor mutation status is not prognostic or predictive for treatment with irinotecan plus 5-FU and LV in stage III colon tumors . In contrast, because of the central role of KRAS downstream in the EGFR signaling pathway, there is currently interest in KRAS mutation status as a predictive biomarker in patients with advanced CRC treated with therapies targeted to EGFR. KRAS gene mutations activate the EGFR signaling pathway independently of ligand stimulation of the receptor, and thus bypass the efficacy of EGFRtargeting drugs. Single-arm studies and large randomized studies in first-line, and in previously treated mCRC patients have demonstrated KRAS tumor mutations to be predictive of a lack of response to the EGFR-targeted antibodies cetuximab and panitumumab. It is now common practice to reserve treatment with EGFR-targeting agents to wild-type KRAS CRC patients.
p<>. The BRAF gene encodes a serine–threonine protein kinase that acts as a downstream effector of KRAS signaling and belongs to the RAS–RAF–mitogen-activated protein kinase/ extracellular signal–related kinase kinase (MEK)– extracellular signal–related kinase (ERK) kinase pathway . BRAF gene mutations are important in colorectal tumorigenesis . The most frequently reported BRAF tumor mutation is a valine-to-glutamic acid amino acid (V600E) substitution that leads to the aberrant activation of the MEK–ERK pathway . BRAF and KRAS mutations tend to be mutually exclusive events in tumors , with BRAF mutations occurring more frequently in MSI than in MSS tumors .
In patients with stage IV CRC, BRAF mutations have been reported to be associated with poor prognosis , and in chemotherapy-refractory mCRC patients BRAF mutations have been reported to be predictive of a lack of response to EGFR-targeted agents . In stage II and stage III colon cancer patients in the PETACC-3 study, BRAF mutations occurred in 7.9% of tumors and were found to not be prognostic of relapse-free survival, but they were prognostic for OS, particularly in patients with MSI-L and MSS tumors (HR, 2.2; p_.0003) . Other retrospective studies have also demonstrated an association between BRAF mutation and poor prognosis in stage II–III and stage I–IV CRC patients. Interestingly, in those studies the good prognosis associated with patients with MSI-H tumors was abrogated in the presence of coincident BRAF mutations .
In the adjuvant setting, BRAF mutation status appears to be a valid prognostic marker; however, associations of BRAF tumor mutations with different molecular subgroups may have to be considered in order to assess the impact of BRAF mutation status as a predictive marker for treatment in future studies in this setting.
p<>. The thymidylate synthase gene TYMS encodes a key enzyme for pyrimidine biosynthesis and is an essential component of the DNA synthesis pathway. TYMS protein activity is inhibited by 5-FU (a pyrimidine analog), leading to cell cycle arrest and apoptosis . In vitro data indicate that TYMS expression is a determinant of 5-FU sensitivity, suggesting that the expression of the gene may also determine tumor sensitivity in vivo . However, conflicting data make the role of this gene as a prognostic or predictive marker in the adjuvant setting controversial . High levels of tumor TYMS protein are reported to be associated with poor prognosis in CRC patients, particularly in those receiving surgery alone, although the reasons for this remain unclear . Study of the expression of other enzymes in the pyrimidin biosynthesis pathway—dihydropyrimidine dehydrogenase (DPD) and thymidine phosphorylase—has shown low tumor expression of TYMS and DPD to be associated with worse prognosis in stage II and stage III CRC patients treated with surgery alone . Patients receiving adjuvant 5-FU– based chemotherapy with high levels of tumor TYMS expression were reported to experience significantly longer survival times , with TYMS expression reported to be predictive of response to adjuvant chemotherapy . However, other studies found no prognostic or predictive 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 . Germline variants in the TYMS gene have been shown to alter TYMS protein and gene expression , and have been associated with response, time to tumor progression, OS, and time to tumor recurrence after 5-FU–based chemotherapy, although the data are conflicting . The clinical significance and relationships 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 cancer cohort of 3,278 patients. The main aims of the translational study were: (a) to assess the feasibility of biomarker analysis on archival formalin-fixed, paraffin-embedded (FFPE) material collected prospectively from 368 collaborating 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 © to assess the predictive utility of specific markers in patients receiving irinotecan in combination with 5-FU and LV, compared with those receiving 5-FU and LV alone .
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 robust methodologies . All data were collected and analyzed 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 specific 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 literature, with sufficient statistical power to detect an HR of 0.7 for DFS if the proportion of single-marker detection is 80% . Thus, a reassessment of the significance of TP53 mutation and IHC, KRAS mutation, TYMS genotype and IHC, and MSI in this cohort is ongoing , on which many of the same biomarkers are being tested . Clearly, these two studies provide useful independent test and validation cohorts of patients in which to investigate candidate biomarker utility.
FUTURE CONSIDERATIONS FOR BIOMARKER INVESTIGATIONS IN THE ADJUVANT SETTING IN THE POSTGENOMIC ERA
In this postgenomic era, technological developments have occurred in which the whole genome can be rapidly and cost-effectively investigated with high-throughput approaches. We now review how a combination of functional genomics and molecular profiling in conjunction with carefully designed clinical trials can be applied to identifying biomarkers and provide a vision for the future for adjuvant colon cancer.
A number of technology platforms have been developed to detect genomewide alterations in tumors. These tools also have the potential to provide predictive profiles for patient prognosis and response to chemotherapy and are being applied to CRC in general. Gene expression microarrays allow the analysis of global gene-expression patterns in mRNA extracted from tissue samples. Changes in tumor DNA copy number have been traditionally characterized using a-CGH, based on bacterial artificial chromosome construct probes . Although copy number–dependent AI and copy number–neutral AI have been assessed by SNP microarrays , often the two technologies are combined to allow a more precise definition of the molecular basis of AI events occurring in tumors . It has also been reported that SNP-bead arrays can discriminate between both copy number–dependent and copy number–neutral AI events . Furthermore, with the development of high-throughput gene sequencing and mutation detection capabilities, a detailed picture of the mutation spectrum of many genes in individual tumors can be realistically achieved . Recently, many of these technology platforms and methods were adapted for use withDNAor RNA extracted from FFPE tissues. Following the identification of candidate genes of interest through expression profiling, the feasibility of developing quantitative reverse transcription PCR assays for use on FFPE material from colon cancer patients in a clinical setting was demonstrated . These developments are important for biomarker validation in large retrospective analyses, in which FFPE material is often the only tissue available for study.