Use of Molecular Biomarkers to Inform Adjuvant Therapy for Colon Cancer
Use of Molecular Biomarkers to Inform Adjuvant Therapy for Colon Cancer
ABSTRACT: The decision about who may derive benefit from adjuvant chemotherapy in colon cancer is often a difficult one for clinicians. While multiple trials have demonstrated that adjuvant chemotherapy reduces the risk of recurrence and improves overall survival in patients with stage III disease, the data supporting the use of adjuvant chemotherapy in patients with stage II disease are not as compelling. Because adjuvant therapy can have significant toxicity, tools to help clinicians determine who may derive a benefit from therapy are of the utmost importance. Recent advances in high throughput technologies have led to the identification of molecular biomarkers—including microsatellite instability (MSI), loss of heterozygosity (LOH), p53, Kirsten rat sarcoma viral oncogene homolog (KRAS), v-raf murine sarcoma viral oncogene homolog B1 (BRAF), thymidylate synthase (TS), and excision repair cross-complementation group 1 (ERCC1)—as well as various multigene assays that are being studied for their ability to offer both prognostic and predictive information to clinicians. Here we review the current knowledge about molecular biomarkers that may aid the clinician in offering personalized cancer therapy based on the genetic landscape of an individual patient’s tumor.
The detailed elucidation of molecular mechanisms of pathogenesis in cancer has fueled an interest in developing therapies that are tailored to the unique molecular characteristics of a patient’s tumor, in order to improve outcomes and minimize toxicity. This approach to cancer therapy, known as personalized medicine, strives to target genetic alterations that are responsible for driving tumor growth, survival, and metastasis by pairing the right drug with the right patient. With the advances in high throughput technologies that characterize genetic alterations and gene expression, as well as an expanding repertoire of molecularly targeted therapies, the goal of personalized medicine is becoming increasingly attainable.
The personalized medicine approach is of particular importance in colorectal cancer, which is the third most common cancer in the world. In 2013, there are estimated to be 142,820 new cases and 52,390 deaths from colorectal cancer in the United States alone. While non-targeted or cytotoxic chemotherapy used after complete removal of the tumor has been shown to prevent disease recurrence, it is not without significant potential toxicity, cost, and inconvenience to patients. The prognostic information offered by the TNM staging system is useful in that it has been shown that patients with stage I disease, who have a risk of recurrence of 5%, do not benefit from adjuvant chemotherapy, whereas patients with stage III disease who receive it can have up to a 50% reduction in the risk of recurrence. However, there is significant controversy about which patients with stage II disease, who have a widely variable risk of recurrence ranging between 20% and 40%, stand to derive benefit from adjuvant therapy. For this group of patients, there is a need for molecular biomarkers to help guide decision making regarding whether to offer adjuvant therapy.
Development and integration of molecular biomarkers to guide treatment and improve clinical outcomes for patients with colorectal cancer have become major focuses of research over the past few years. These biomarkers can be categorized as prognostic markers, which indicate the likelihood of patient outcome regardless of the specific treatment the patient receives, or predictive markers, which indicate the likelihood of benefit from a specific therapy.[3,4] Both predictive and prognostic biomarkers are now becoming integral parts of the treatment of cancer patients. Particularly for patients with colorectal cancer for whom the decision about whether to offer adjuvant therapy is not straightforward, these molecular markers can be used to guide clinical decision making. In this review, we will discuss adjuvant chemotherapy in non-metastatic colon cancer, the existing prognostic and predictive molecular biomarkers in the field, and how to integrate these molecular biomarkers into the decision about whether to administer adjuvant therapy.
Molecular Biomarkers in Colorectal Cancer
A number of studies have demonstrated improved outcomes with 5-fluorouracil (5-FU) and oxaliplatin administered in the adjuvant setting, but no benefit for irinotecan, bevacizumab, or cetuximab (Table 1).[5-21] While the benefit of chemotherapy in an adjuvant setting for patients with stage III disease is clear, the magnitude of benefit to patients with stage II disease is small at best, and although there may be a benefit in terms of disease-free survival (DFS), none of these trials is powered to show a benefit in overall survival (OS). Therefore, the decision about which stage II patients stand to derive a greater degree of benefit from adjuvant chemotherapy must depend on other mechanisms of stratifying a patient’s risk of recurrence.
It has been shown that clinicopathologic variables, including T4 disease, poorly differentiated histology (including signet ring and mucinous tumors), lymphovascular invasion (LVI), perineural invasion, bowel obstruction or perforation, positive margins, inadequately sampled lymph nodes, high levels of carcinoembryonic antigen (CEA), and occult nodal micrometastases all portend a worse prognosis, although the risk conferred by each of these factors is not easy to assess.[22,23] The College of American Pathologists, in their consensus guidelines from 2000, only recognized T4 disease, nodal status, LVI, high preoperative CEA level, and positive margins as factors definitively proven to be of prognostic significance based on evidence from published trials. However, although these factors are of prognostic significance, there is no evidence to indicate that they hold any predictive significance. An active area of study is the identification of molecular biomarkers that are both prognostic and predictive. Currently, molecular biomarkers that are potentially clinically useful are microsatellite instability (MSI), loss of heterozygosity at chromosome 18q (LOH 18q), p53, Kirsten rat sarcoma viral oncogene homolog (KRAS), v-raf murine sarcoma viral oncogene homolog B1 (BRAF), thymidylate synthase (TS), excision repair cross-complementation group 1 (ERCC1), and multigene assays (Table 2). Here, we will review these molecular biomarkers and the data supporting their utility in clinical practice.
One of the first biomarkers to be identified as having prognostic value was microsatellite instability (MSI) status. Patients with stage II and III colon cancer with MSI, a measure of deficient DNA mismatch repair (dMMR), may derive benefit from adjuvant therapy. In one of the first studies to evaluate MSI status in colorectal cancer, MSI assay or immunohistochemistry for MMR proteins was performed in 457 patients who were previously randomly assigned to 5-FU–based chemotherapy (either 5-FU/levamisole or 5-FU/leucovorin [LV]) vs observation. Overall, 70 patients (15%) exhibited MSI. Adjuvant chemotherapy significantly improved DFS in patients with microsatellite stable (MSS) or proficient MMR tumors, whereas patients with MSI had no improvement in DFS with 5-FU (hazard ratio [HR] = 1.10; 95% confidence interval [CI], 0.42–2.91; P = .85) compared with those randomly assigned to surgery alone. In patients with stage II disease and with MSI tumors, treatment was associated with reduced OS (HR = 2.95; 95% CI, 1.02–8.54; P = .04). Thus, stratifying patients by MSI status may provide a more tailored approach to colon cancer adjuvant therapy.
In another study that investigated the usefulness of MSI status as a predictor of the benefit of adjuvant chemotherapy with 5-FU in stage II and stage III colon cancer, tumor specimens were collected from patients with colon cancer who were enrolled in randomized trials of 5-FU–based adjuvant chemotherapy. Of the 570 tissue specimens, 95 (16.7%) exhibited high-frequency MSI. Among 287 patients who did not receive adjuvant therapy, those with tumors displaying high MSI had a better 5-year OS than patients with MSS or low MSI (HR = 0.31; 95% CI, 0.14–0.72; P = .004). Among patients who received adjuvant chemotherapy, high MSI was not correlated with increased OS (HR = 1.07; 95% CI, 0.62–1.86; P = .8). The benefit of treatment differed significantly according to MSI status (P = .01). Adjuvant chemotherapy improved OS among patients with MSS tumors or tumors exhibiting low MSI, according to multivariate analysis adjusted for stage and grade (HR = 0.72; 95% CI, 0.53–0.99; P = .04). However, there was no benefit of adjuvant chemotherapy in the group with high MSI. Therefore, 5-FU adjuvant chemotherapy benefited patients with stage II or stage III colon cancer with MSS tumors or tumors exhibiting low MSI, but it did not benefit those with tumors exhibiting high MSI. Furthermore, the benefit with 5-FU/LV/oxaliplatin (FOLFOX) vs 5-FU alone, and the benefit in patients with stage II vs stage III disease, are not yet known.
Loss of a region on the long arm of chromosome 18 is seen frequently in colon cancer. This region contains three genes that may influence intestinal tumorigenesis: DCC, SMAD4, and SMAD2. Several studies have looked at how LOH 18q affects response to adjuvant chemotherapy. Tumor tissue from 460 patients with stage III and high-risk stage II colon cancer who had been treated with various combinations of adjuvant 5-FU, LV, and levamisole was analyzed to determine the ability of various markers to predict survival. The study evaluated loss of heterozygosity from chromosomes 18q, 17p, and 8p; cellular levels of p53 proteins and p21 WAF1/CIP1 proteins; and MSI. The investigators found that LOH 18q was present in 155 of 319 cancers (49%), and high levels of MSI were found in 62 of 298 tumors (21%). Among patients with MSS stage III cancer, 5-year OS after 5-FU-based chemotherapy was 74% in those whose cancer retained 18q alleles and 50% in those with loss of 18q alleles. The relative risk of death with LOH 18q was 2.75 (95% CI, 1.34–5.65; P = .006). Therefore, this study demonstrated that retention of 18q alleles in MSS cancers points to a favorable outcome after adjuvant chemotherapy with 5-FU-based regimens for stage III colon cancers. However, this study did not address the significance of LOH 18q in patients with stage II disease.
When patients from the Cancer and Leukemia Group B (CALGB) 9581 and 89803 trials were analyzed for MMR status and LOH 18q, more stage II tumors than stage III tumors had MSI (21.3% vs 14.4%, P < .001) and were intact at 18q (24.2% vs 15.1%, P = .001). For the combined cohort, patients with MSI tumors had better 5-year DFS (0.76 vs 0.67, P < .001) and OS (0.81 vs 0.78, P = .029) than those with MSS. However, LOH18q did not affect outcome among patients with MSS.
The Eastern Cooperative Oncology Group (ECOG) 5202 study is a randomized phase III trial comparing 5-FU + LV + oxaliplatin + bevacizumab to 5-FU + LV + oxaliplatin or to observation only following surgery in patients with stage II colon cancer at high risk of recurrence, to determine prospectively the prognostic value of molecular markers (Figure 1). The primary outcome measure of this study is DFS based upon a stratified log-rank test, with stratification by stage (IIA vs IIB) and MSI status (MSS vs MSI-low). Patients with disease that is high risk for MSI and LOH 18q were randomized to one of the two treatment arms (plus or minus bevacizumab), while patients with disease that is low risk for MSI and LOH 18q were assigned to an observation-only arm. Secondary outcome measures are OS; rates of toxicity; and the impact of tumor biologic characteristics, including their relationship to OS in high- and low-risk populations. This trial is ongoing, although accrual has been closed, and results are expected in the near future.
The tumor suppressor p53 responds to cellular stresses, including DNA damage, to initiate a program of cell cycle arrest, DNA repair, apoptosis, and angiogenesis. Loss of p53 is critical for tumorigenesis in a variety of cancers, and is found frequently in colon cancer, where it often is associated with carcinoma in situ and inversely correlated with MSI status.[30,31] Many studies have investigated the role of p53 mutations on prognosis and response to adjuvant chemotherapy, but the results are conflicting. A retrospective multivariate analysis in 233 unselected patients with stage III colon cancer treated with 5-FU/LV or with FOLFOX demonstrated that the addition of oxaliplatin to the treatment regimen for patients with tumors that overexpressed p53 was an independent factor predictive of benefit from FOLFOX (P = .03). The TP53 Colorectal Cancer International Collaborative Study investigated 3,583 patients from 25 different research groups in 17 countries and found that Dukes’ C tumors with wild-type p53 and those with mutated p53 showed significantly better prognosis when treated with adjuvant chemotherapy. However, in another analysis of 135 patients treated with adjuvant FOLFOX after curative resection, p53 positivity was not significantly associated with DFS or OS. Therefore, the role of p53 as a prognostic or predictive marker remains unclear.
KRAS, a member of the Ras family of genes, is an oncogene that encodes a GTPase involved in many signal transduction pathways, such as the epidermal growth factor receptor (EGFR) signaling pathway. Located on chromosome 12, KRAS has been found to be mutated in 40% to 50% of colorectal cancers, with 90% of the mutations occurring in codons 12 or 13.[35-38] Currently, two drugs are available that target the EGFR pathway in colon cancer: cetuximab is a chimeric mouse-human monoclonal antibody that inhibits EGFR, and panitumumab is a fully human monoclonal antibody specific to EGFR. The use of KRAS as a prognostic marker was first evaluated in the RASCAL study (utilizing a database of The Kirsten Ras-in-Colorectal-Cancer Collaborative Group), in which 2,721 patients were evaluated for a KRAS mutation and its impact on clinical outcome. There was not any association between the presence of a KRAS mutation and tumor location, tumor stage, and pattern of recurrence, but in the multivariate analysis, DFS was decreased in patients with a KRAS mutation. RASCAL II, the 2001 follow-up study, confirmed the findings that a KRAS mutation had a significant negative impact on DFS.
In the CALGB 89803 trial, 1,264 patients with stage III colon cancer were enrolled in a randomized adjuvant trial of 5-FU/LV with or without irinotecan. Mutations of KRAS were detected in 178 tumors. Compared with patients with KRAS–wild-type tumors, patients with KRAS-mutated tumors did not experience any difference in DFS, OS, or recurrence-free survival. The effect of a KRAS mutation on patient survival did not differ significantly according to clinical features, chemotherapy arm, or MSI status, and the effect of adjuvant chemotherapy assignment on outcome did not differ according to KRAS status.
The prognostic role of a KRAS mutation in stage II and III resected colon cancer was evaluated in a translational study of the Pan European Trials Adjuvant Colon Cancer (PETACC)-3, the European Organisation for the Research and Treatment of Cancer (EORTC) 40993, and Swiss Group for Clinical Cancer Research (SAKK) 60-00 trials. In this study, 1,564 formalin-fixed paraffin-embedded tissue blocks were prospectively collected and DNA extracted from tissue sections from 1,404 cases. The KRAS mutation rate was 37% and did not differ significantly according to tumor stage. In a multivariate analysis controlling for stage, site of tumor, nodal status, sex, age, grade, and MSI status, KRAS mutation was associated with grade (P = .0016) but did not have a major prognostic value regarding relapse-free survival or OS. Recent investigations have shown the utility of KRAS as a predictive biomarker in metastatic colorectal cancer, and assessment of KRAS status is routinely being used in the metastatic setting to identify patients who may benefit from both cetuximab and panitumumab.[41,42] A discussion of markers in the metastatic setting is beyond the scope of this review.
BRAF is a member of the RAF kinase family of serine/threonine kinases, and it is involved in regulating the mitogen-activated protein (MAP) kinase/extracellular signal–regulated protein kinase (ERK) signaling pathway. The activating mutation of BRAF (V600E) is seen in approximately 10% of colorectal cancers and is mutually exclusive of the KRAS mutation. In the translational study of the PETACC-3, EORTC 40993, and SAKK 60-00 trials, the BRAF tumor mutation rate was 7.9%; was significantly associated with female sex (P = .017); and was highly significantly associated with right-sided tumors, older age, high grade, and MSI-high tumors (all P < .0001). BRAF status was not prognostic for recurrence-free survival, but it was prognostic for OS, particularly in patients with MSI-low and MSS tumors (HR = 2.2; 95% CI, 1.4–3.4; P = .0003). Although studies have demonstrated that the BRAF mutation portends a worse prognosis than wild-type BRAF tumors, these have been limited to patients with metastatic disease, and thus are outside the scope of this review.
Thymidylate synthase is an enzyme that is the dominant target for the active metabolite of 5-FU, or fluorodeoxyuridine monophosphate (5-FdUMP). Because initial studies investigating the relationship between TS expression and survival in colorectal cancer showed poorer OS and progression-free survival with higher TS expression, a meta-analysis was undertaken that investigated 13 studies with 887 patients with advanced colorectal cancer and 7 studies with 2,610 patients with localized colorectal cancer. The combined HR estimate for OS was 1.74 (95% CI, 1.34–2.26) and 1.35 (95% CI, 1.07–1.8) in the advanced and adjuvant settings, respectively, despite the evidence of heterogeneity and possible publication bias. Therefore, this meta-analysis concluded that patients with tumors expressing high TS levels had a poorer OS compared with those whose tumors expressed lower TS levels.
ERCC1 is a key molecule in the nucleotide excision repair (NER) pathway, which is responsible for repairing DNA adducts induced by platinum drugs. Silencing ERCC1 gene expression increases sensitivity to platinum agents, and low ERCC1 gene expression is predictive of oxaliplatin cytotoxicity in colon cancer cell lines.[45,46] Because of these findings and other preclinical data, many studies have sought to evaluate whether high ERCC1 levels predict a poor outcome in patients whose colorectal cancer is treated with oxaliplatin-based chemotherapy. However, all of these data were generated in the metastatic setting. One group reported a significantly shorter survival time in patients with progressive stage IV disease and high tumor ERCC1 mRNA levels vs those with low ERCC1 mRNA levels (relative risk [RR] = 4.24; 95% CI = 1.35–13.29; P = .008). A phase I trial in 91 patients, evaluating escalating doses of capecitabine with a fixed dose of oxaliplatin, confirmed that higher ERCC1 mRNA levels were associated with a shorter time to treatment failure compared with lower ERCC1 mRNA levels (85 days vs 162 days; P = .046). Furthermore, ERCC1 gene expression was associated with response and OS in 191 of the more than 2,000 patients enrolled in the Colorectal Oral Novel Therapy for the Inhibition of Angiogenesis and Retarding of Metastases (CONFIRM)-1 and CONFIRM-2 trials. In these trials, low ERCC1 gene expression was significantly associated with better response to first- and second-line chemotherapy (P = .047) and high ERCC1 gene expression was associated with shorter OS in patients receiving first-line chemotherapy (P = .014).[49,50] Both studies strongly supported that ERCC1 gene expression evaluated by reverse transcriptase polymerase chain reaction (RT-PCR) correlates with resistance to oxaliplatin. However, ERCC1 protein expression as assessed by immunohistochemistry was not linked with outcome in two studies, including a large prospective analysis of FOCUS, a randomized trial looking at 1,197 patients that compared treatment with 5-FU, 5-FU/irinotecan, and 5-FU/oxaliplatin.[51,52] Although ERCC1 has a role as a prognostic marker in non–small-cell lung cancer, only a few studies have evaluated its role as a prognostic marker in colorectal cancer.