CDX2 as a Prognostic Biomarker in Stage II and Stage III Colon Cancer

Abstract

Background The identification of high-risk stage II colon cancers is key to the selection of patients who require adjuvant treatment after surgery. Microarray-based multigene-expression signatures derived from stem cells and progenitor cells hold promise, but they are difficult to use in clinical practice. Methods We used a new bioinformatics approach to search for biomarkers of colon epithelial differentiation across gene-expression arrays and then ranked candidate genes according to the availability of clinical-grade diagnostic assays. With the use of subgroup analysis involving independent and retrospective cohorts of patients with stage II or stage III colon cancer, the top candidate gene was tested for its association with disease-free survival and a benefit from adjuvant chemotherapy. Results The transcription factor CDX2 ranked first in our screening test. A group of 87 of 2115 tumor samples (4.1%) lacked CDX2 expression. In the discovery data set, which included 466 patients, the rate of 5-year disease-free survival was lower among the 32 patients (6.9%) with CDX2-negative colon cancers than among the 434 (93.1%) with CDX2-positive colon cancers (hazard ratio for disease recurrence, 3.44; 95% confidence interval [CI], 1.60 to 7.38; P=0.002). In the validation data set, which included 314 patients, the rate of 5-year disease-free survival was lower among the 38 patients (12.1%) with CDX2 protein-negative colon cancers than among the 276 (87.9%) with CDX2 protein-positive colon cancers (hazard ratio, 2.42; 95% CI, 1.36 to 4.29; P=0.003). In both these groups, these findings were independent of the patient's age, sex, and tumor stage and grade. Among patients with stage II cancer, the difference in 5-year disease-free survival was significant both in the discovery data set (49% among 15 patients with CDX2-negative tumors vs. 87% among 191 patients with CDX2-positive tumors, P=0.003) and in the validation data set (51% among 15 patients with CDX2-negative tumors vs. 80% among 106 patients with CDX2-positive tumors, P=0.004). In a pooled database of all patient cohorts, the rate of 5-year disease-free survival was higher among 23 patients with stage II CDX2-negative tumors who were treated with adjuvant chemotherapy than among 25 who were not treated with adjuvant chemotherapy (91% vs. 56%, P=0.006). Conclusions Lack of CDX2 expression identified a subgroup of patients with high-risk stage II colon cancer who appeared to benefit from adjuvant chemotherapy. (Funded by the National Comprehensive Cancer Network, the National Institutes of Health, and others.).

Figure 1. Study Design

A database containing 2329 human gene–expression arrays from both 214 normal colon tissue samples and 2115 colorectal-cancer tissue samples was mined to identify genes that fulfilled the “X-negative implies activated leukocyte-cell adhesion molecule (ALCAM)–positive” Boolean implication. The search yielded 16 candidate genes, of which only 1 (CDX2) encoded for a clinically actionable biomarker. The association between CDX2 expression and disease-free survival was tested in two independent patient cohorts: a discovery data set (National Center for Biotechnology Information Gene Expression Omnibus [NCBI-GEO]) and a validation data set (Cancer Diagnosis Program of the National Cancer Institute [NCI-CDP]). The association between CDX2 expression and benefit from adjuvant chemotherapy was tested in a pooled database of 669 patients with stage II disease and 1228 patients with stage III disease from four independent data sets (NCBI-GEO, NCI-CDP, National Surgical Adjuvant Breast and Bowel Project [NSABP] C-07 trial [NSABP C-07], and the Stanford Tissue Microarray Database [TMAD]).

Figure 2. Relationship between CDX2 Expression and Disease-free Survival in the NCBI-GEO Discovery Data Set

Analysis of CDX2 messenger RNA (mRNA) expression in the NCBI-GEO discovery data set revealed the presence of a minority subgroup of CDX2-negative colon cancers that were characterized by high ALCAM mRNA expression levels (Panel A) and that were associated with a lower rate of 5-year disease-free survival than CDX2-positive colon cancers (Panel B). In Panel A, each circle in the scatter plot represents one patient sample. The association between CDX2-negative cancers and a lower rate of disease-free survival remained significant in a multivariate analysis that excluded tumor stage, tumor grade, age, and sex as confounding variables (Panel C).

Figure 3. Relationship between CDX2 Protein Expression and Disease-free Survival in the NCI-CDP Validation Data Set

Analysis of CDX2 protein expression in the NCI-CDP validation data set confirmed the existence of a minority subgroup of CDX2-negative cancers (Panel A) that lacked the distinctive CDX2 nuclear expression that is characteristic of epithelial cancer cells in the majority of colon cancers (Panel B). CDX2-negative cancers were associated with a lower rate of 5-year disease-free survival than CDX2-positive cancers (Panel C). The association between the absence of CDX2 expression and a lower rate of 5-year disease-free survival was confirmed by means of a multivariate analysis (based on the Cox proportional-hazards method) that excluded tumor stage, tumor grade, age, and sex as confounding variables (Panel D). CDX2-negative tumors were associated with a lower rate of survival independent of their sub-classification with regard to low or intermediate (G1 or G2) or high (G3 or G4) pathological grade (Panel E).

Figure 4. Relationship between CDX2 Expression and Disease-free Survival among Patients with Stage II Disease

In the NCBI-GEO discovery data set (Panel A), CDX2-negative cancers were associated with a rate of 5-year disease-free survival that was lower than the rate associated with CDX2-positive cancers. In the NCI-CDP validation data set (Panel B), CDX2-negative cancers were associated with a rate of 5-year disease-free survival that was lower than the rate associated with CDX2-positive cancers.

Figure 5. Relationship between CDX2 Expression and Benefit from Adjuvant Chemotherapy

The relationship between CDX2 expression and benefit from adjuvant chemotherapy was evaluated in a pooled database of 669 patients with stage II disease (Panel A) and 1228 patients with stage III disease (Panel B) from four independent data sets (NCBI-GEO, NCI-CDP, NSABP C-07, and Stanford TMAD). Among all patients with stage II disease in the entire database, treatment with adjuvant chemotherapy was not associated with a higher rate of 5-year disease-free survival. However, treatment with adjuvant chemotherapy was strongly associated with a higher rate of 5-year disease-free survival in the CDX2-negative subgroup, but it was not associated with a higher rate of 5-year disease-free survival in the CDX2-positive subgroup. Among patients with stage III disease, treatment with adjuvant chemotherapy was associated with a higher rate of 5-year disease-free survival in the entire database and in both the CDX2-negative and CDX2-positive subgroups. A test for an interaction between the biomarker and the treatment indicated that in both stage II and stage III disease, the benefit associated with adjuvant chemotherapy was superior among CDX2-negative patients than among CDX2-positive patients.