CDX2 Polymorphisms, RNA Expression, and Risk of Colorectal Cancer

Colorectal carcinogenesis is defined by a complex multistep process that involves genetic, epigenetic, and environmental factors (1). Despite the identification of several important genes and the 2-fold increase in risk of colorectal cancer in first-degree relatives of colorectal cancer patients, the genetic basis of the majority of sporadic colorectal cancers is unknown. A candidate gene for colorectal cancer is the caudal-type homeobox gene CDX2 (13q12-13), which is exclusively expressed in adults in intestinal epithelium cells and plays an important role in epithelial cell proliferation, differentiation, and determination of cell fate in different organs along the lower gastrointestinal tract (2, 3). There are multiple lines of evidence that implicate CDX2 in colorectal carcinogenesis. Whereas mouse Cdx2 homozygote null mutants die between 3.5 and 5.5 days post coitum, mice heterozygous for Cdx2 null mutations have a variable phenotype including the development of multiple intestinal adenomatous polyps in the proximal colon, some of which contain areas of true neoplasia (4). CDX2 expression is reduced in colorectal cancers from rats and humans (5–7) and may define a distinct set of minimally differentiated colorectal cancers (7). A few studies have evaluated somatic and germ line variants in CDX2. No pathogenic mutations of CDX2 were found in 49 colorectal cancer cell lines (8) or in 51 sporadic colorectal cancer tumors (9). Single nucleotide polymorphism (SNP) discovery in the Sivagnanasundaram study (9) identified six polymorphisms in 51 sporadic British colorectal cancer cases (47 Caucasian, 4 Asian). The frequency of these polymorphisms did not differ between these cases and 60 unrelated Centre d'Etude du Polymorphisme Humain controls and 50 unrelated British controls. However, there has been no thorough evaluation of population risk associated with germ line CDX2 variants in a population-based case-control study. Here, we did SNP discovery in 35 Ashkenazi Jewish colorectal cancer subjects with a family history of colorectal cancer and typed the discovery variants in 455 matched Ashkenazi Jewish cases and controls. We considered both allelic and haplotype associations with colorectal cancer in these subjects, as haplotype analysis may be useful for detecting putative variants in this founder population. CDX2 expression was evaluated with respect to the SNPs and histopathologic characteristics of the tumors in a subset of the colorectal cancer subjects.

Subjects. The Molecular Epidemiology of Colorectal Cancer study is a population-based, matched case-control study that includes 1,947 incident colorectal cancer cases and 1,947 matched controls. A sequentially selected subset of the first 455 Ashkenazi Jewish cases and 455 Ashkenazi Jewish population-based controls were used for these analyses, as power calculations indicated that we had 86.3% power to detect an odds ratio of 1.5 using the same test set of cases and controls we used for a previous analysis (10). The Molecular Epidemiology of Colorectal Cancer study participants have previously been described (10, 11). Eligible cases include any person newly diagnosed with colorectal cancer between March 31, 1998 and April 1, 2004 in Northern Israel. Cases were identified through rapid case ascertainment in the hospitals and Kupat Holim Clalit National Center for Cancer Control tumor registry by International Classification of Diseases code for cancer of the colon or rectum and were invited to participate and interviewed. Potential controls were matched for exact year of birth, sex, and primary-care clinic code. Structured interviews assessed demographic information, personal and family history of cancer, reproductive history and medical history, medication use, health habits, and a food-frequency questionnaire. Individuals previously diagnosed with cancer of the colorectum were not eligible to participate. Overall, 3,181 potentially eligible cases of colorectal cancer were ascertained in the study period before this analysis. Of them, 618 (19.4%) could not be located or approached, including 275 (8.6%) who died before being approached. Thus 2,563 were approached and invited to participate. Forty-two were subsequently excluded as too sick to participate or unable to communicate in Hebrew, Russian, Arabic, or English. Of the 2,521 remaining eligible cases, 335 declined to participate (13.3%). Therefore, 2,186 cases were eligible and consented, and 2,146 completed the in-person interview, which corresponds to a response rate of 67% of all eligible cases. In addition, 2,162 consented matched controls were interviewed. Among the matched controls who were first approached, 52.1% participated in the study. The study was approved by the Institutional Review Boards at the University of Michigan and Carmel Medical Center in Haifa. Written informed consent was required for eligibility.

Study design. The SNP study was done in two stages: SNP discovery and SNP screening. SNP discovery was done on 35 Molecular Epidemiology of Colorectal Cancer colorectal cancer patients with at least one first-degree relative with a history of colorectal cancer through resequencing of all exons and overlapping intron boundaries. All variant alleles identified in the SNP discovery stage were screened in the matched sample of 455 Ashkenazi cases and 455 Ashkenazi controls. A partially overlapping set of 201 frozen tumors with expression profiling data from the same study permitted joint evaluation of CDX2 expression and CDX2 SNPs for 82 Molecular Epidemiology of Colorectal Cancer cases.

Single nucleotide polymorphism genotyping. SNP genotyping was done using Masscode Technology (Qiagen GmbH, Valencia, CA), which uses highly multiplexed detection of low molecular weight photocleavable Mass Tags in a single quadrupole mass spectrometer. Briefly, the assay used a two-step PCR process for signal amplification and SNP discrimination. Allele-specific incorporation of the Mass Tags occurs during the second amplification step as previously described (12). SNP assays were carried out using 4 ng of genomic DNA. Positive and negative (no DNA) controls were included on every 96-well plate analyzed.

Microsatellite instability analyses. Microsatellite instability (MSI) analyses were done as previously described (13). Normal and tumor DNA were extracted from microdissected DNA and analyzed for the consensus panel of five markers (14). Briefly, forward primers for BAT25, BAT26, D2S123, and D5S346 and reverse primers for D17S250 were labeled with [γ-³²P]ATP, and included in a 20 μL PCR reaction that included 1 μL of microdissected DNA. PCR products were run on 6% polyacrylamide gels for ∼3 hours at 65 W, and exposed to film at −80°C for 12 to 20 hours. Films were double scored and entered as stable, unstable, or loss of heterozygosity. Tumors were designated as MSI-high if there was instability at two or more markers. For uninformative markers, a sixth marker from the consensus panel was substituted.

CDX2 RNA expression. Primary tumors for expression analysis were obtained at the time of surgical resection and snap frozen, embedded in optimum cutting temperature freezing media (Miles Scientific, Naperville, IL), cryotome sectioned, stained with H&E, and evaluated by a single surgical pathologist (T.J.G.) as previously described (15). Areas with >70% tumor cellularity were identified for RNA isolation. Selected sections of tumor samples were homogenized in Trizol (Life Technologies, Gaithersburg, MD), and total cellular RNA was purified according the instructions of the manufacturer, with additional purification using RNeasy spin columns (Qiagen). RNA quality was assessed by 1% agarose gel electrophoresis, and samples were included only if the 18S and 28S bands were discrete and approximately equal.

Expression levels were measured using commercially available Affymetrix high-density microarrays (Affymetrix, Santa Clara, CA), with sample preparation of cRNA, hybridization, and scanning all done according to the protocols of the manufacturer. Tumors were evaluated on either HuGeneFL or U133 chips.

Statistical analyses. Matched univariate analyses were done on each individual SNP using conditional logistic regression as implemented in SAS version 8.2 (SAS Institute, Cary, NC, 1999). Each SNP was analyzed using both additive and dominant models. T tests, ANOVA, and generalized linear models as implemented in SAS were used to compare the distribution of CDX2 expression between subjects with and without a minor allele, and with and without histopathologic characteristics. PHASE version 2.0.1 (16) was used to infer haplotypes for individual subjects. Linkage disequilibrium between SNP pairs was calculated as D′ and Δ² using the GOLD program (17). Haplotypes were also inferred using the EM algorithm, and associations between haplotypes and colorectal cancer and CDX2 expression were determined using the haplo_score program in Splus (18). Family history of colorectal cancer was self-reported and coded as “colorectal cancer reported in any first-degree relative” or “colorectal cancer reported in any relative.”

Expression analysis was done as previously described (15). In brief, we subtracted the mismatch probe values from the perfect match values and averaged the middle 50% of these differences as the expression measure for the CDX2 probe set. The data were quantile normalized to adjust for intensity differences across chips. A monotone linear spline was applied to each chip that mapped quantiles 0.02 up to 0.98 (in increments of 0.02) exactly to the corresponding median quartiles for all samples. The data were then transformed for each chip, using log(100 + max(X + 100;0)).

Nine unique SNPs were identified in the 35 colorectal cancer cases with a first-degree family history of colorectal cancer. In comparison to the Sivagnanasundaram study which also looked at all exon/intron boundaries of CDX2, the majority of the SNPs identified were in the intronic regions: eight in noncoding regions and one in exon 3 (Table 1). The T877C change results in serine (uncharged polar) to proline (nonpolar) substitution in exon 3. Two of the SNPs, IVS1 −33T>A and IVS1 −34T>C, are very similar to polymorphisms described in the Sivagnanasundaram et al. study (IVS1 −31A>T, IVS1 −32C>T). Although it is not possible to recognize whether these polymorphisms represent the exact same polymorphisms previously reported by Sivagnanasundaram from the published report, this possibility seems highly likely. The data we report here find no evidence of a significant association with colorectal cancer for these two polymorphisms, and this interpretation is entirely consistent with the conclusions drawn by Sivagnanasundaram et al. None of the other SNPs were identified in previous studies, which is likely due to population differences between Ashkenazi Jews and the other groups studied. All identified SNPs were typed in the screening population of 455 matched cases and controls. The observed genotype data were consistent with Hardy-Weinberg equilibrium as measured in control subjects. The mean age was similar between cases and controls by design, where the mean age of cases was 73.7 (SD = 9.7) versus 74.4 (SD = 9.7) for controls. Slightly more than half (55.6% of cases and 55.1% of controls) of the subjects were male. The SNPs were highly frequent, with the least frequent minor allele found in 17% of the subjects (Table 1). All SNPs were seen equally frequently in cases and controls. Odds ratios for these SNP associations with colorectal cancer from matched analyses ranged from 0.94 to 1.06 (modeling presence or absence of the minor allele and risk of colorectal cancer), indicating that none of these SNPs were associated with risk of colorectal cancer in this population. This result did not change when modeling an additive genetic model and risk of colorectal cancer (data not shown).

Although there were no putative SNP-colorectal cancer associations, we also tested whether haplotypes were associated with colorectal cancer. Specifically, we evaluated haplotypes of SNPs by including only a single SNP from any set of associated SNPs, defined as Δ² > 0.90. The SNPs in this gene were in strong linkage disequilibrium, implying that there was very little historical recombination in this gene in the Ashkenazi Jewish population (D′ = 1 for 27 of the 36 pairs of SNPs). IVS1 −1575 and IVS1 −34 had pairwise measures of Δ² ranging from 0.059 to 0.423 with each of the other SNPs and each other, and T877C exon 3 had measures of Δ² of 0.250 and 0.082 with IVS1 −1575 and IVS1 −34, respectively, and a minimum Δ² of 0.955 with each of the remaining SNPs. The remaining six SNPs were in very strong linkage disequilibrium with each other (Δ² > 0.91). The PHASE program inferred three common haplotypes of the nine SNPs with relative frequencies of 55%, 25%, and 17% (Table 2). The three SNPs effectively distinguish or tag these three haplotypes, which account for 97% of the predicted haplotypes in our sample. Score statistics were used to compare haplotype frequencies between cases and controls for all combinations of these three SNPs to determine if there was any association between haplotypes and colorectal cancer. Although there were a limited number of significant differences in haplotype frequencies between cases and controls, there were no discernable patterns and these differences were likely due to chance (data not shown).

It is possible that variants in CDX2 that are not associated with an increased risk of colorectal cancer may have a functional effect on CDX2 expression. Global gene expression derived from microarrays of CDX2 correlates well with CDX2 protein expression (19). We analyzed these variants with respect to differences in CDX2 expression in an overlapping subset of 82 colorectal cancer case subjects with both expression and CDX2 variant data available. None of the SNPs or derived haplotypes of the three tag SNPs predicted significant differences in CDX2 expression in these subjects. Previously, Hinoi et al. (7) reported reduced CDX2 expression in large-cell minimally differentiated carcinomas, which are predominantly right sided. Data on specific tumor location was available for 65 of the 82 colorectal cancer cases with expression data, 28 of which were left-sided and 37 right-sided tumors. Right-sided tumors showed significantly lower mean CDX2 expression (27% decrease, P = 0.018) than left-sided tumors. In addition, the larger data set of 201 tumors with CDX2 expression data (only 82 of which were genotyped for CDX2 variants) were analyzed to evaluate the expression with respect to histopathologic characteristics. These analyses show that CDX2 is underexpressed in right-sided, poorly differentiated tumors, consistent with previous reports (Table 3). It is clear that, whereas overall levels seem to be highest in the sigmoid colon, rectosigmoid junction, and rectum, some tumors in these areas have low expression levels comparable to those found in the cecum and other right-sided areas (Fig. 1A and B). As an expected consequence of tumor heterogeneity, a small number of right-sided tumors have high expression levels, with low levels of expression found in some left-sided tumors.

Twelve (6.0%) of the 199 cases with both CDX2 expression data and family history data from Molecular Epidemiology of Colorectal Cancer interviews reported a first-degree relative with a history of colorectal cancer, and these subjects had significantly lower CDX2 expression (817.1 versus 1,283.6, P = 0.019; Table 2). The 18 (9.0%) subjects who reported any relative with a family history of colorectal cancer had lower CDX2 expression than those who did not report a relative with colorectal cancer (1,008.4 versus 1,280.0), but this result was not statistically significant (P = 0.101; Table 2).

Microsatellite unstable (MSI) tumors have significantly lower expression than microsatellite stable tumors (737.5 versus 1,343.6, P < 0.001; Table 2; Fig. 1C), which is consistent with the Hinoi et al. study that reported a high frequency of the MSI phenotype (60%) in a limited panel of tumors with reduced CDX2 expression (7). In a multivariate model that included both MSI status and tumor location, MSI status is more strongly associated with CDX2 expression (P < 0.001) than location (P = 0.192), suggesting loss of CDX2 expression through somatic alterations in MSI-high tumors.

The nature and implications of CDX2 alterations in colorectal cancer are not clearly defined. Here, we investigated germ line variants found in colorectal cancer cases with a family history of colorectal cancer to determine if these are associated with susceptibility to sporadic colorectal cancer in this population. However, no variants or combination of variants were associated with risk of colorectal cancer in this population. Furthermore, we did not identify any germ line variants that predicted CDX2 expression levels, but confirmed the previously described patterns of CDX2 expression observed in poorly differentiated, right-sided tumors. However, reduced CDX2 expression is not limited to right-sided tumors, and further molecular studies are needed to clarify the nature and extent of CDX2 alterations in colorectal tumorigenesis. We provide additional evidence here that MSI status may be more strongly associated with CDX2 expression than tumor location. Hinoi et al. (20) propose a dominant repressor pathway, where reduced or absent CDX2 expression in MSI tumors is due to repression of a transcription factor or protein required for CDX2 expression commonly altered in MSI colorectal cancers. This same study showed that demethylating agents were not sufficient to reactivate CDX2 expression, showing that CDX2 is unlikely, in these colorectal cancers, to be silenced by methylation. Here, we show that it is also unlikely that germ line variants play a role in reduced CDX2 expression in sporadic colorectal cancers and there is little genetic diversity in CDX2 in this population. To our knowledge, this is the only population-based study of CDX2 germ line variants published to date. Given the number of subjects in this study, germ line variants in CDX2 are unlikely to be associated with risk of colorectal cancer in this population.

Grant support: A grant (1R01CA81488) from the National Cancer Institute and a gift from the Weinstein Foundation, a non profit charitable organization.

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