Abstract
Multiple GWAS have identified several susceptibility variants for colon cancer at 8q24. However, the functional roles of these variants have yet to be elucidated. Here, we evaluated the potential role of these markers in tumor progression and examined association with commonly observed structural abnormalities in this region, c-MYC amplification and chromosome fragility at FRA8C and FRA8D. We first replicated the previously reported association by testing 1178 cases and 1009 clinic-based controls with eight markers localized to three specific regions at 8q24. We observed significant associations with colon cancer risk with markers rs13254738 (ordinal OR=0.82, 95% CI=0.072-0.94, Ptrend=0.0037) and rs6983267 (ordinal OR=1.17, 95% CI=1.03-1.32, Ptrend=0.013). Survival analysis was performed using a separate set of 460 cases to evaluate the clinical significance of these markers. Overall, univariate analysis did not detect survival differences for any of the markers. We also tested a subset of the 460 cases (N=380) for structural abnormalities at or near the c-MYC locus using FISH analysis. Furthermore, we evaluated a small number of cases homozygous for the rs6983267 alleles to test for differences in fragile site induction. None of the 8q markers correlated with amplification at the c-MYC locus as detected by FISH, and no clear pattern of breakage was observed at the FRA8C and FRA8D sites. In this study, we confirm the association for several SNPs at 8q24 in colon cancer but have not detected any structural role relating to c-MYC amplification or chromosomal fragility. Finally, these risk alleles do not appear to be associated with survival.
Keywords: 8q, SNP, association, survival, FISH, fragile site, c-MYC
Introduction
Multiple independent genome-wide association studies (GWAS) have localized several cancer susceptibility variants to five distinct but adjacent regions on chromosome 8q24 (128.14 -128.62 Mb) (1-10). The single nucleotide polymorphism (SNP) rs1447295 was the first marker at 8q24 to be identified as a risk factor for prostate cancer (1, 3, 4, 7, 11). Subsequent GWAS in colorectal cancer identified a second independent genetic susceptibility marker (rs6983267) on chromosome 8q (5, 6, 8, 12, 13). These SNPs are located in different linkage disequilibrium (LD) blocks (D̀=0.25) and are separated by a recombination hotspot (6, 7). Markers in this region were also associated with prostate and ovarian cancer. In addition, SNPs located on two other regions seemed to be specific to prostate cancer (3, 4). Finally, SNPs in an adjacent region have been reported to be associated with breast cancer only (2). All these multiple regions with different cancer specifities were summarized in a recent report (9).
Despite the strong association reported for the various SNPs in these regions, it is unknown if these are the true causal variants. It is likely that some or all of these are in LD with the etiologically relevant markers that have not yet been identified. Additionally, these data argue for several independent risk markers mapping to this particular chromosomal region. In an effort to obtain additional fine mapping data, Yeager et al. sequenced a 136 kb region, including the rs6983267 and rs1447295 variants, and generated a detailed map of genetic variation (14). Analyses suggested rs6983267 as the best candidate SNP for functional studies as it lies within a highly conserved region and has a regulatory potential.
The functional variants responsible for susceptibility within this 8q24 region, as well as their mechanism of action, have not yet been identified. As these markers lie in a chromosomal gene desert, the identification of specific target genes for the risk-markers has been difficult. One of the known genes in close proximity is c-MYC, located telomeric to the 8q SNP cluster (~220 Kb). However, no significant relationship has yet been observed between the 8q24 variants and c-MYC mRNA expression (6, 8, 12). Given that there are independent markers in at least five separate regions, these data suggest that altered chromatin structure may play an important role is disease susceptibility. c-MYC, as well as other closely localized genes, is frequently involved in structural chromosomal alterations in a variety of tumor types. This 8q24 region has been reported to be a frequent site of somatic amplification in both prostate (15) and colorectal cancer (16, 17). Somatic gains in copy number of 8q24 have been reported in 18% of colorectal adenomas and 34% of colorectal cancers (18).
Interestingly, in addition to amplification at c-MYC, the 8q24 chromosomal region contains another common structural alteration. Two fragile sites, FRA8C and FRA8D, lie in close proximity to the SNP cluster. This fragile site has also been reported to be one of the preferred integration sites for Human Papillomavirus (HPV) (19). The relationship, if any, of the various SNP genotypes to 8q amplification and/or to chromosome fragility is unknown.
In this study, we directed our efforts to understand the biological mechanism and possible functional importance of this region in colon cancer. Our main goal was to determine whether there were any structural changes at 8q24 that might correlate with the variants reported for this region. We also examined the implication of these susceptibility markers for colon cancer case-control status as well as survival.
Materials and Methods
Association Study
Cases
Colon cancer cases were selected from two prospective collections. The first, the Colorectal Neoplasia Repository, is an ongoing collection of biospecimens from Mayo Clinic Rochester patients with colorectal neoplasia. A subset of these patients underwent a surgical resection for colon cancer during a three-year period 1995-1998. The collection was re-initiated in 2000. Cases for the association study were selected from both of these two time periods. Of the consented patients, US resident Caucasians who had only colon cancer (not rectal) were included in this study. Patient chart reviews were performed to obtain clinical characteristics of the tumor, including tumor site, stage and age at diagnosis. For tumor site, tumors of the proximal colon were defined as those occurring in the cecum, the ascending colon, or the transverse colon (including both flexures). Distal tumors were defined as those occurring in the descending or sigmoid colon.
The second group was derived from a subset of the North Central Cancer Treatment Group (NCCTG) clinical trial NO147. Cases for the clinical trial were enrolled at about 65 NCCTG sites in the US and Canada, which included Mayo Clinic (Rochester, Arizona, and Florida). As part of the clinical trial protocol, biospecimens have been collected on an ongoing basis starting in March, 2004. Patients aged ≥18 years with histologically confirmed and surgically removed stage III colon cancer were eligible. Patients undergoing chemotherapy through the multicenter NCCTG study were included if they had only colon cancer and not rectal, had surgery at most eight weeks prior to treatment, had no distant metastasis, and no previous radiation or chemotherapy for colon cancer.
Controls
Clinic-based controls were described in detail elsewhere (20). Briefly, the control group was obtained from patients being seen for a routine annual general physical examination in the Department of Medicine, Mayo Clinic, Rochester, MN. This ongoing prospective collection of biospecimens was initiated in 2000. Control subjects had no prior history of cancer (except non-melonoma skin-cancer) at the time of enrollment.
Overall, there were 1483 cases ascertained from the two sources described in the previous section: Colorectal Neoplasia Repository (N=834) and NCCTG_N0147 (N=649). After removing 110 individuals with non-Caucasian ancestry, 60 patients who did not live in the continental US, and 6 others for miscellaneous reasons, there were 1307 remaining Caucasian cases. Of the 1307 available cases, 1244 were matched on age, gender and state of residence to the general medicine controls (all Caucasian). DNA was not available for 129 cases and 235 controls. Genotyping was performed on a total of 1178 cases and 1009 controls that comprised the final analytic cohort.
Survival Study
A second independent group of cases for the survival study (N=460) were participants of a Phase III adjuvant therapy trial (NCCTG, 914653) (21). DNA was available from only the proficient mismatch repair (pMMR) cases for this study. Patients had undergone complete surgical resection of high-risk stage II/III colon cancer during a five-year period, from April, 1993 to January, 1998. Patients with surgically removed primary, histologically confirmed colon cancer with no evidence of residual disease but high risk of recurrence were eligible. Patients could not have prior exposure to 5-FU, any prior radiation therapy or chemotherapy, or any concurrent or previous malignant tumor within the previous three years, except superficial squamous or basal cell carcinoma of the skin or in situ carcinoma of the cervix. Patients aged between 31 and 85 years were randomized to receive either high dose (N=224) or standard dose (N=236) of treatment regimens.
Genotyping
Genotyping on blood DNA was carried out using multiple platforms. The TaqMan assay (Applied Biosystems, Foster City, CA) was performed according to the manufacturer's guidelines, using 10-20ng DNA. Primers and probes were Assay-by-Design (Applied Biosystems); rs1447295 (C_2160574_30), rs10090154 (C_26755480_10), rs13254738 (C_31232916_10), rs6983561 (C_1645354_10), rs16901979 (C_33280526_10), rs6983267 (C_29086771_20), rs7000448 (C_2160622_10). Following PCR amplification, end reactions were read on the ABI Prism 7900HT using Sequence Detection Software (Applied Biosystems). The Illumina custom GoldenGate (Illumina Inc., San Diego, CA) genotyping protocol was performed according to the manufacturer's guidelines. Arrays were imaged using an Illumina BeadArray Reader and the data analyzed using BeadStudio software 2.0 (Illumina). The microsatellite polymorphism DG8S737 was detected using ABI 3100 fragment length analysis (Applied Biosystems, Foster City, CA).
Genotyping on paraffin DNA was carried out using allele-specific PCR for the markers rs1447295, rs16901979, rs13254738, rs6983267, and DG8S737. The forward primers were fluorescently labeled by HEX or FAM (primers used are available on request). The PCR product was mixed with formamide containing ROX dye size standard (GeneScan 400HD; Applied Biosystems) and analyzed on an ABI 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA). The PCR product fragment lengths were determined by GeneScan software.
To ensure quality control of genotyping results, one CEPH DNA and three negative controls, along with two duplicate samples were included on each 96-well plate. Genotypes were determined blinded to phenotype status. The overall average call rate for the markers was 97% (range, 86.4-99.8%). In addition, Chi-square goodness of fit test for Hardy-Weinberg equilibrium was applied to the genotype proportions in the controls. All markers were in Hardy-Weinberg equilibrium, except rs10090154 (P=0.02) that also had a low call rate of 86.4%.
Mismatch Repair Status
Defective mismatch repair (dMMR) was defined by presence of microsatellite instability (MSI-H) and/or the absence of protein expression for hMLH1, hMLH2, or hMSH6. Proficient mismatch repair (pMMR) was defined as tumors having normal protein expression for hMLH1, hMSH2, and hMSH6 and the presence of MSS (microsatellite stable) or MSI-L (low level instability).
Microsatellite instability (MSI) in colon cancer cases was performed with paired normal and tumor DNA isolated from formalin-fixed, paraffin-embedded (FFPE) material (22). Tumors were classified as MSI-H if ≥30% markers demonstrated instability and as MSS/MSI-L if <30% demonstrated MSI (23, 24). Immunohistochemical analysis of hMLH1, hMSH2, and hMSH6 expression was performed on FFPE samples, as previously described (25).
Fluorescence In Situ Hybridization (FISH) Analysis
Paraffin sections were baked and deparaffinized using a process of 2 five minute incubations in Citrosolv at room temperature and 2 five minute incubations in 100% ethanol at room temperature. The air dried slides were microwaved in 10mM citric acid until boiling then pepsinized for 15 min in 4mg Pepsin/L 0.9% NaCl at 37°C followed by a dehydration series in 75%, 80% and 100% ethanol for two minutes each. Vysis Provysion (LSI c-MYC 8q24.12-24.13 in Spectrum Green, CEP 8 in Spectrum Aqua and LSI LPL 8p22 in Spectrum Orange) probe was applied to each slide, denatured on a HyBrite and hybridized overnight. The slides were then washed in 2xSSC with 0.1% NP-40, counterstained with Dapi and coverslips were applied.
Fifty nuclei per sample were scored and the number of signals per nuclei recorded for each probe. A ratio of c-MYC copy number to CEP 8 copy number was established for each sample. The normal range was considered to be from 0.8 to 1.29. Duplication of the c-MYC region was determined to be any specimen with a ratio between 1.3 and 2.0. Amplification was defined as a ratio of greater than 2.0 with more than 10 copies of c-MYC. A sample was considered to have gain of c-MYC if at least 30% of the nuclei had 3 or more signals for c-MYC but had a ratio in the normal range.
Cell Culture and Fragility Induction
EBV transformed cells were grown in RPMI 1640 medium with Glutamax, 10% fetal bovine serum and 1% penicillin/streptomycin (100 units/mL, 100 µg/mL) (All reagents from Invitrogen). Aphidicolin (Sigma) diluted in DMSO was added 24 hours prior to harvest at a final concentration of 0.4 µM to induce fragile sites. Cell harvest was performed according to standard methods. Briefly, cells were treated with colcemid (0.075 µg/mL) for one hour followed by a potassium chloride/sodium citrate hypotonic solution (0.075M, 0.8%) treatment for 20 minutes. Two changes of fixative (3:1 methanol/glacial acetic acid) were performed prior to slide preparation.
Chromosome band 3p14.2 contains a common fragile site that is highly inducible through aphidicolin treatment. A commercially available DNA probe for centromere 3 labeled in SpectrumOrange (Abbott Molecular) was used. A DNA probe for 8q24.2 was created at the Mayo Clinic from the following BAC DNAs: RP11-382A18, RP11-327N12, RP11-367L7, RP1136L8, RP11-55J15 and RP263C20. Slides were pretreated with 2x standard saline citrate (SSC) at 37°C, dehydrated in an ethanol series and air dried. Probe solution was placed onto the slide. The probe and target DNA were co-denatured using a ThermoBrite system (Abbott Molecular) set at 73°C for 3 minutes followed by hybridization at 37°C overnight. Slides were washed with 0.4xSSC at 72°C to remove excess probe and counterstained with 10% 4’,6’-diamidino-2-phenylindole dihydrochloride (DAPI).
Metaphase cells were located using a fluorescence microscope equipped with a 100-W mercury lamp and a DAPI single-pass filter. The FISH signals were viewed using a 100x oil immersion objective along with a dual-pass filter for SpectrumOrange-SpectrumGreen, a single-pass filter for SpectrumOrange or a single pass filter for SpectrumGreen. FISH signal patterns were documented with a Cytovision workstation (Applied Imaging, Santa Clara, CA, USA). A maximum of 100 metaphases were analyzed per sample.
Statistical Analysis
All categorical variables were summarized using frequencies and percents. Continuous variables were summarized using means and standard deviations. The case and control genotype frequencies were calculated and then tested for Hardy-Weinberg equilibrium (HWE) within the controls group using Chi-square goodness of fit or Fisher's exact tests. Pairwise linkage disequilibrium measures were estimated using Haploview software (v4.1). Logistic regression analyses were performed to test interactions among SNPs across 3 regions. Estimates of relative risk (OR; odds ratio) and 95% confidence intervals were estimated for the association between genotypes and colon cancer using unconditional logistic regression, adjusted for age. In our analyses, an ordinal model (log-additive) was used in which homozygotes for the common allele was the reference category. Marker rs6983267 was an exception to this and the homozygotes for the rare allele was used as the reference category to be consistent with the literature. In addition to testing for the main effect of each marker, the analyses were stratified by the cases’ tumor characteristics at diagnosis; mismatch repair status, tumor site and stage.
Overall survival (OS) (censored at 8 years) was calculated as the number of years from random assignment to the date of death or last contact. Disease-free survival (DFS) (censored at five years) was calculated as the number of years from random assignment to the first of either disease recurrence or death. The distributions of OS and DFS among the 460 independent cases were estimated using Kaplan-Meier methodology. Univariate and multivariate Cox proportional hazards ratios from an additive model (26) were calculated to explore the association of clinical and genetic factors with OS and DFS. Due to rarity of homozygous DG8S737 -8/-8 and -10/-10 genotypes, they were combined with those heterozygous for DG8S737 -8 and -10, respectively, in the Cox model. The log-rank and likelihood ratio test p-values were used to test the significance of each covariate in the univariate and multivariate models, respectively. According to the graphical and statistical methods used, all the underlying model assumptions were satisfied (e.g., proportional hazards) (27).
All statistical tests were two sided, with P ≤ 0.05 considered significant. P-values were not adjusted for multiple comparisons. Statistical analyses were performed using SAS software (SAS Institute, Cary, NC).
Results
Association Study
Our goal was to first replicate the previously described associations at 8q24 for colon cancer. The demographic and clinical characteristics of the participants used for these studies are given in Table 1. There were 1178 cases and 1009 controls in the association analysis. Cases and controls were closely matched for age, gender, and state of residence at the time of diagnosis. Two-thirds of the cases had tumors of the proximal colon. The frequency of stage III cases was much higher compared to other stages because a subset of the cases were participants of the NCCTG clinical trial protocol NO147 which restricted enrollment to stage III colon cancer patients. Of the 1073 tumors analyzed, 878 (81.8%) were pMMR (MSS/MSI-L, and/or the presence of normal protein expression) and the remaining were dMMR (MSI-H and/or absence of protein expression).
Table 1.
Characteristics of colon cancer cases and clinic-based controls*
| Variable | Level | Association Study | Survival Study | |
|---|---|---|---|---|
| Controls N= 1009 | Cases N =1178 | Cases N= 460 | ||
| Age range | Range (min, max) | 24-95 | 29-98 | 31-85 |
| Age category | <40 | 19 (1.9%) | 21 (1.8%) | 18 (3.9%) |
| 40-49 | 86 (8.5%) | 92 (7.8%) | 38 (8.3%) | |
| 50-59 | 179 (17.7%) | 227 (19.3%) | 113 (24.6%) | |
| 60-69 | 303 (30.0%) | 364 (30.9%) | 188 (40.9%) | |
| 70+ | 422 (41.8%) | 474 (40.2%) | 103 (22.4%) | |
| Mean age | Mean (SD) | 65.3 ± 11.6 | 65.5 ± 11.5 | 61.6 ± 10.2 |
| Gender | Male | 495 (49.1%) | 608 (51.6%) | 249 (54.1%) |
| Female | 514 (50.9%) | 570 (48.4%) | 211 (45.9%) | |
| Tumor Site | Proximal | -- | 717 (62.1%) | 201 (43.9%) |
| Distal | -- | 432 (37.4%) | 257 (56.1%) | |
| Tumor Stage | I | -- | 163 (14.5%) | -- |
| II | -- | 245 (21.7%) | 122 (26.5%) | |
| III | -- | 624 (55.3%) | 338 (73.5%) | |
| IV | -- | 96 (8.5%) | -- | |
| MMR† | dMMR | -- | 195 (18.2%) | -- |
| pMMR | -- | 878 (81.8%) | 460 (100%) | |
| Treatment‡ | Standard | -- | -- | 236 (51.3%) |
| High dose | -- | -- | 224 (48.7%) | |
Tumor site, tumor stage and MMR data were missing from a subset of cases and therefore do not add to 100%.
MMR = Mismatch repair status; dMMR = defective mismatch repair; pMMR = proficient mismatch repair.
Treatment arms based on ref. (21).
Eight previously reported markers, 7 SNPs and 1 microsatellite, in three chromosomal regions at 8q24 were evaluated for susceptibility for colon cancer risk in a case-control study. Regions 1 and 2 contain three markers each, DG8S737, rs1447295, rs10090154 andrs13254738, rs6983561, rs16901979, respectively; and region 3 containstwo markers, rs6983267 and rs7000448 (Figure 1). The SNPs in regions 1 and 2 were highly correlated with each other (r2>78).Region 3 SNPs presented a more moderate correlation (r2=52).No significant interactions were observed among SNPs across these 3 regions (data not shown).
Figure 1.
The 8q24 locus and linkage disequilibrium (LD) structure showing tested markers (DG8S737, not shown in region 1) on three cancer susceptibility regions in our population. Diamond boxes represent the pairwise D’ values between SNPs, as derived by Haploview software (v.4.1). Darker the shading stronger the correlation between SNPs.
Results of the association study with risk of colon cancer are provided in Table 2. We observed two significant associations with case-control status. At region 2, SNP rs13254738 had an ordinal OR=0.82 (95% CI=0.072-0.94, Ptrend=0.0037); the frequency of the risk allele C was 0.34 in controls. The association of the SNP rs6983267 at region 3 showed significance with an increased risk of 1.17 (95% CI=1.03-1.32, Ptrend=0.013).
Table 2.
Association of SNP variants on chromosome 8q24 with colon cancer and tumor characteristics*.
| Marker Position(bp) Region |
MAF‡ | All cases | Stratified by Tumor Characteristics | |||||
|---|---|---|---|---|---|---|---|---|
| Mismatch Repair Status | Tumor Site | Tumor Stage | ||||||
| OR§ (95% CI) p-value |
dMMR cases |
pMMR cases |
Distal cases |
Proximal cases |
Stages I-II |
Stages III-IV |
||
| rs13254738 128173525 region 2 | C(0.34) | 0.82 (0.72,0.94) 3.7×10−3 | 0.62 (0.47,0.81) 3.8×10−4 | 0.86 (0.75,0.99) 0.036 | 0.85 (0.71,1.01) 0.07 | 0.82 (0.70, 0.95) 8.8×10−3 | 0.77 (0.64,0.93) 6.7×10−3 | 0.85 (0.73,0.98) 0.03 |
| rs6983561 128176062 region 2 | C(0.03) | 1.14 (0.82,1.60) 0.43 | 0.85 (0.44, 1.65) 0.64 | 1.25 (0.87, 1.79) 0.23 | 1.13 (0.71,1.79) 0.60 | 1.16 (0.80, 1.68) 0.44 | 0.90 (0.56,1.46) 0.67 | 1.28 (0.88,1.86) 0.21 |
| rs16901979 128194098 region 2 | A(0.03) | 1.02 (0.72,1.46) 0.91 | 0.54 (0.24, 1.22) 0.14 | 1.15 (0.79, 1.68) 0.45 | 1.09 (0.68,1.76) 0.71 | 0.99 (0.66, 1.48) 0.94 | 0.78 (0.46,1.31) 0.34 | 1.15 (0.77,1.71) 0.49 |
| rs6983267 128482487 region 3 | G(0.51)∥ | 1.17 (1.03,1.32) 0.013 | 0.92 (0.73, 1.16) 0.46 | 1.23 (1.08, 1.41) 1.9×10−3 | 1.30 (1.10,1.54) 2.1×10−3 | 1.08 (0.94, 1.24) 0.30 | 1.06 (0.89,1.26) 0.50 | 1.22 (1.06,1.41) 4.9×10−3 |
| rs7000448 128510352 region 3 | T(0.37) | 0.95 (0.84,1.07) 0.39 | 0.84 (0.66, 1.07) 0.15 | 0.96 (0.84, 1.09) 0.52 | 0.96 (0.82,1.14) 0.67 | 0.92 (0.80, 1.06) 0.25 | 0.92 (0.77,1.09) 0.33 | 0.98 (0.85,1.13) 0.75 |
| rs1447295 128554220 region 1 | A(0.10) | 0.99 (0.81,1.21) 0.95 | 0.81 (0.54, 1.21) 0.31 | 1.01 (0.81, 1.25) 0.96 | 1.16 (0.90,1.51) 0.25 | 0.89 (0.71, 1.13) 0.33 | 0.97 (0.74,1.29) 0.85 | 0.98 (0.78,1.23) 0.85 |
| rs10090154 128601319 region 1 | T(0.10) | 0.98 (0.79,1.23) 0.90 | 1.00 (0.67, 1.50) 0.99 | 0.93 (0.72, 1.20) 0.57 | 1.09 (0.81,1.48) 0.56 | 0.90 (0.69, 1.17) 0.44 | 1.14 (0.86,1.52) 0.37 | 0.87 (0.66,1.15) 0.32 |
The bold font refers to significant p-values (p<.05).
MAF; minor allele frequency in control subjects.
Odds ratios (ORs) based on the trend test are shown with their 95% confidence intervals (95% CI) and p-values.
rs6983267 G allele is the common allele. We used the minor T allele as the reference to be consistent with the literature.
In subset analysis, the associations between these markers were evaluated with a number of tumor characteristics. Results of the analysis for SNP rs6983267 demonstrated statistically significant associations based on the tumor MMR status, tumor site and tumor stage. Significant associations were observed in patients whose tumors were pMMR, occurred in the distal colon and were higher stage (Table 2). No statistically significant associations were observed for the remaining markers tested.
Survival Study
In addition to the role of these markers in tumor etiology, we examined their potential role in tumor progression. To perform this analysis, a separate set of 460 cases were analyzed (Table 1). Subjects were dominantly Caucasians with a mean age of 62 years. The gender distribution slightly favored males. Fifty-six percent of the cases had tumors of the distal colon (44% proximal). About three-fourths of the cases had TNM stage III, the remaining were stage II. These cases were a subset of the participants of a Phase III adjuvant therapy (NCCTG, 914653), which recruited stage II and III colon cancer patients. All 460 tumors analyzed were pMMR.
Survival analysis was performed for 5 of the 8 markers. All three chromosomal regions were covered by utilizing markers that were in LD with adjacent markers (Figure 1). Univariate analysis did not detect any significant differences in overall survival (OS) or disease-free survival (DFS) for any of the markers, although the rare alleles showed a trend for decreased survival rate. As expected, patients with stage III tumors had significantly decreased OS and DFS compared to stage II cases (OS OR=2.08, 95% CI=1.38-3.15, P=4x10-4) (Table 3).
Table 3.
Five-year survival rates and associations with tumor stage and variants on chromosome 8q24.
| Total N | 5-year DFS | DFS HR (95% CI)* | P-value† | 5-year OS | OS HR (95% CI)* | P-value† | |
|---|---|---|---|---|---|---|---|
| Clinical Variable | |||||||
| Tumor Stage_ II | 121 | 81.0% | 85.9% | ||||
| III | 336 | 61.3% | 2.32 (1.49, 3.61) | 1.0×10−4 | 66.5% | 2.08 (1.38, 3.15) | 4.0×10−4 |
| Variant | |||||||
| rs13254738_ A/A | 202 | 66.6% | 72.2% | ||||
| A/C | 197 | 67.4% | 73.1% | ||||
| C/C | 55 | 62.9% | 1.05 (0.83, 1.32) | 0.70 | 64.6% | 1.14 (0.91, 1.43) | 0.25 |
| rs16901979_ C/C | 393 | 67.5% | 73.2% | ||||
| A/C | 35 | 57.1% | 60.0% | ||||
| A/A | 3 | 66.7% | 1.32 (0.83, 2.11) | 0.23 | 66.7% | 1.34 (0.86, 2.09) | 0.20 |
| rs6983267_ ‡ T/T | 87 | 63.2% | 66.6% | ||||
| G/T | 184 | 70.1% | 74.9% | ||||
| G/G | 122 | 63.4% | 1.00 (0.79, 1.28) | 0.97 | 71.3% | 1.00 (0.79, 1.27) | 0.99 |
| rs1447295_ C/C | 363 | 66.1% | 72.6% | ||||
| C/A | 87 | 67.8% | 67.8% | ||||
| A/A | 6 | 66.7% | 0.97 (0.67, 1.39) | 0.85 | 66.7% | 1.11 (0.79, 1.55) | 0.55 |
| DG8S737_§ */* | 408 | 66.4% | 72.2% | ||||
| */−8 or −8/−8 | 42 | 71.4% | 0.80 (0.45, 1.41) | 0.44 | 71.4% | 1.10 (0.67, 1.79) | 0.71 |
| DG8S737_§ */* | 427 | 67.2% | 72.7% | ||||
| */−10 or −10/−10 | 23 | 60.9% | 1.31 (0.67, 2.58) | 0.43 | 60.9% | 1.57 (0.85, 2.90) | 0.15 |
Disease free survival (DFS) and overall survival (OS) hazard ratios (HR) from an additive model of the number of the rare allele copies.
Log-rank test p-value.
This variant was assessed to be consistent with the literature and so the rs6983267 G allele number of copies has been tested even though this is the common allele in our cases.
Rare allele groups combined since only 1 and 0 cases had −8/−8 and −10/−10 alleles, respectively.
Structural Study
The functional variant responsible for susceptibility to colorectal cancer within this 8q24 region remains unknown. As the region of interest is very gene poor, our goal was to determine whether there were any structural changes on this chromosomal segment that might be correlated with the variants reported.
First, we tested for abnormalities at or near the c-MYC locus using fluorescently labeled DNA probes for FISH analysis (Figure 2). A summary of results for the 380 cases that had both FISH and 8q24 marker genotype data are shown in Table 4. Seventy-one cases (19%) showed normal copy number at or near c-MYC, 129 cases (34%) had gain, 164 cases (43%) had c-MYC duplication and 16 cases (4%) had c-MYC amplification. Only histologic grade and tumor site were significantly associated with the four FISH categories (P=0.02 and P=0.03, respectively) (Table 4). No differences were found among the four c-MYC categories for either the allele or genotype frequencies for any of the 8q markers tested (Table 4).
Figure 2.
-A schematic view of the fragile sites, cancer susceptibility regions and BACs used for FISH DNA probes in 8q24 region. A. Positioning of presumed boundaries of FRA8C and FRA8D. B. Detailed view of a segment from 8q24 region showing the positions of markers tested within independent regions and c-MYC gene on chromosome 8q24. C. Representation of BAC clones that are used to characterize the fragile site regions
Table 4.
Distribution of FISH categories by clinical characteristics and 8q24 markers.
| Normal (N=71) | Gain (N=129) | c-MYC Duplication (N=164) | c-MYC Amplification (N=16) | Total (N=380)* | p-value† | |
|---|---|---|---|---|---|---|
| Clinical Variable | ||||||
| Histologic Grade | 0.02 | |||||
| Grade 1 or 2 | 55 (20.1%) | 101 (36.9%) | 110 (40.1%) | 8 (2.9%) | 274 (72.1%) | |
| Grade 3 or 4 | 16 (15.1%) | 28 (26.4%) | 54 (50.9%) | 8 (7.5%) | 106 (27.9%) | |
| Stage | 0.17 | |||||
| Stage II | 18 (16.7%) | 45 (41.7%) | 43 (39.8%) | 2 (1.9%) | 108 (28.4%) | |
| Stage III | 53 (19.5%) | 84 (30.9%) | 121 (44.5%) | 14 (5.1%) | 272 (71.6%) | |
| Tumor Site | 0.03 | |||||
| Distal | 35 (16.4%) | 79 (36.9%) | 96 (44.9%) | 4 (1.9%) | 214 (56.6%) | |
| Proximal | 35 (21.3%) | 49 (29.9%) | 68 (41.5%) | 12 (7.3%) | 164 (43.4%) | |
| Age | 0.30 | |||||
| Mean (SD) | 59.9 (11.72) | 62.0 (10.34) | 61.2 (9.82) | 64.8 (9.19) | 61.4 (10.36) | |
| Median | 61.0 | 63.0 | 63.0 | 68.0 | 63.0 | |
| Range | (31.0-79.0) | (34.0-85.0) | (31.0-77.0) | (47.0-78.0) | (31.0-85.0) | |
| Gender | 0.77 | |||||
| F | 35 (20.1%) | 61 (35.1%) | 72 (41.4%) | 6 (3.4%) | 174 (45.8%) | |
| M | 36 (17.5%) | 68 (33.0%) | 92 (44.7%) | 10 (4.9%) | 206 (54.2%) | |
| Variant | ||||||
| rs13254738 | 0.78 | |||||
| A/A | 35 (20.7%) | 58 (34.3%) | 69 (40.8%) | 7 (4.1%) | 169 (44.6%) | |
| A/C or C/C | 36 (17.1%) | 70 (33.3%) | 95 (45.2%) | 9 (4.3%) | 210 (55.4%) | |
| rs16901979 | ||||||
| C/C | 63 (19.0%) | 109 (32.9%) | 143 (43.2%) | 16 (4.8%) | 331 (91.2%) | 0.37 |
| A/A or A/C | 5 (15.6%) | 14 (43.8%) | 13 (40.6%) | 0 (0%) | 32 (8.8%) | |
| rs6983267 | ||||||
| G/G | 15 (14.6%) | 40 (38.8%) | 44 (42.7%) | 4 (3.9%) | 103 (31.3%) | 0.13 |
| G/T or T/T | 45 (19.9%) | 70 (31.0%) | 102 (45.1%) | 9 (4.0%) | 226 (68.7%) | |
| DG8S737 | ||||||
| −8 allele | ||||||
| ** | 66 (19.2%) | 116 (33.7%) | 147 (42.7%) | 15 (4.4%) | 344 (92%) | 0.58 |
| */−8 or −8/−8 | 4 (13.3%) | 11 (36.7%) | 14 (46.7%) | 1 (3.3%) | 30 (8%) | |
| −10 allele | ||||||
| ** | 65 (18.3%) | 119 (33.5%) | 155 (43.7%) | 16 (4.5%) | 355 (94.9%) | 0.82 |
| */−10 or −10/−10 | 5 (26.3%) | 8 (42.1%) | 6 (31.6%) | 0 (0%) | 19 (5.1%) | |
| rs1447295 | ||||||
| C/C | 60 (19.7%) | 99 (32.5%) | 134 (43.9%) | 12 (3.9%) | 305 (80.3%) | 0.19 |
| C/A or A/A | 11 (14.7%) | 30 (40.0%) | 30 (40.0%) | 4 (5.3%) | 75 (19.7%) |
380 come from merging the FISH data with the 8q markers data and only including cases that had marker data.
Chi-square or Fisher's exact p-value for categorical data, and the ANOVA F-test for the continuous age data.
Second, we evaluated whether cases with a certain genotype were more susceptible to fragile site induction at FRA8C and FRA8D (Figure 2). The fragility test was conducted on cases homozygous for the candidate SNP demonstrating the most significant association results, rs6983267. Overall, ten samples were tested, five cases that were homozygous for the common G allele and five cases that were homozygous for the rare T allele. Four of the five cases in each group were pMMR. A FISH probe for the centromere of chromosome 3 was included as a locator of an internal control for breakage assay (28). As expected, all ten cases exhibited 3p14 breakage (proportion range 6-32%). Of the ten cases tested, 3 TT and 2 GG homozygous samples exhibited low-level fragility in 1-4% of cells (Table 5). None of the samples in either group had breakage in both 3p and 8q in the same metaphase spread.
Table 5.
Fragility assay by FISH on homozygous cases for rs6983267.
| Genotype* | Total† | 3p | 3p & 8q | 8q | Normal |
|---|---|---|---|---|---|
| GG | 100 | 20 | 0 | 0 | 80 |
| GG | 68 | 4 (6%) | 0 | 0 | 64 |
| GG | 100 | 9 | 0 | 0 | 91 |
| GG | 87 | 5 (6%) | 0 | 2 (2%) | 80 |
| GG | 54 | 8 (15%) | 0 | 2 (4%) | 44 |
| TT | 100 | 21 | 0 | 0 | 79 |
| TT | 100 | 9 | 0 | 0 | 91 |
| TT | 100 | 32 | 0 | 1 | 67 |
| TT | 100 | 6 | 0 | 1 | 93 |
| TT | 100 | 16 | 0 | 1 | 83 |
T is the rare allele.
A maximum of 100 metaphases were analyzed per sample.
Discussion
Polymorphisms on chromosome 8q24 have been studied in relation to risk for a range of cancers, including prostate, colon, breast and ovarian (1, 3, 6-9, 29). These studies have identified a number of independent risk-markers that cluster within a fairly narrow region at 8q24.
In this study, we replicate the findings reported by others showing an association between two of the eight polymorphic markers, rs6983267 and rs13254738, on 8q24 region and colon cancer risk. The results for rs6983267 were similar to those first observed from the GWAS (6, 8) and were in agreement with the other replication studies (5, 9, 13, 30-32). Our data also agree with studies using a different SNP (rs10505477) that is in high LD with the rs6983267 (8, 12). The observed minor allele frequency (T allele) of 0.49 among controls was similar to the reported study populations. However, it should be stressed that the association observed in our study is for the G allele, which differs from other markers as we used the rare allele as the reference (T allele for this specific SNP). The analyses were performed in this manner in order to be consistent with other published studies.
The SNP, rs13254738, has not been extensively evaluated by other groups. Available data from the three recent publications did not show a significant association between this SNP and risk of colorectal cancer (5, 9, 13). Earlier studies have different case and control selection criteria. Cases were selected for our study only if they had colon but not rectal cancer. It might be important to test rs13254738 on rectal only cases as this may help us understand whether this SNP is specific to colon only cases.
We tested all eight markers stratified by tumor characteristics and detected a significant association of rs6983267 with MMR status, stage and site. No significant evidence of association was reported in the previous studies with respect to these tumor characteristics (5, 6, 12, 30) except that a trend was found towards the association of rs6983267 with MSS colorectal cancer cases in a Finnish population (OR=1.37; 95% CI=1.02-1.85; P=0.04) (33). Similarly, our findings in the pMMR cases showed an association for SNP rs6983267, with an OR of 1.17. In addition, pMMR cases with stage III-IV distal tumors were at increased colon cancer risk for SNP rs6983267 (Table 2). Although, the overall effects are modest, the data suggests that this SNP has an effect in the development of colon cancer among subjects with distinct tumor characteristics profiles. Additional studies will be required to confirm these findings and to fully understand the precise mechanism.
In addition to their potential etiologic role, the clinical significance of these 8q24 variants in colon cancer was examined. Although there was a trend toward an association between the rare alleles and decreased survival, none of the associations were statistically significant. No survival differences between carriers and the non-carriers of the rare allele were also reported by Gruber et al. (12) in a dominantly Jewish population.
Finally, we examined the possible functional importance of this region. Our main goal was to determine whether there were any structural changes on this chromosomal segment that might correlate with the variants reported for this region. The functional and structural role of the 8q24 markers is not well understood. Defining their role has been particularly challenging as these markers lie in a chromosomal region in which there are no known genes. The c-MYC oncogene is the only well known gene that is in close proximity. c-MYC is frequently deregulated by structural chromosomal alterations in various tumor types. In addition, chromosomal region 8q24 contains two fragile sites, FRA8C and FRA8D, which suggests that 8q24 region, may be prone to structural alterations due to its fragile nature. However, we were not able to detect any correlation between the 8q24 variants and any structural change in this region. Similarly, the susceptibility to breakage was not strongly correlated with the SNP variant.
There are several limitations to our study. First, our association study was restricted to Caucasians that had colon cancer only and thus our results may not be generalized to other ethnicities or rectal cancers. Second, the low event rate (30-35%) in our survival data limited our power for associations. Third, our fragility data was derived from a small number of samples and the prevalence of 8q24 fragility in at risk patients should be interpreted with caution.
Clearly, additional functional studies will be needed to understand the importance of this 8q24 region. In vitro studies may help to investigate the role of these SNPs in regulating gene expression. Although such a mechanism has not yet been implicated for genes located nearby, one cannot rule out an interaction for genes located at some distance away either in cis or trans. In addition, searching and identifying sequence conservation within and flanking regions might be important in determining the region's role in regulation and transcription.
In summary, we have further confirmed the association of two 8q24 markers with colon cancer risk. Our data, however, suggest that the 8q variants do not influence patient survival and are not implicated in the two chromosomal changes tested – c-MYC amplification in tumor tissue and induction of fragility at FRA8C and FRA8D.
Acknowledgements
Grant Sponsor: We wish to thank the generous support of the Richard M. Schulze Family Foundation.
References
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