High Incidence of Microsatellite Instability in Colorectal Cancer from African Americans¹ Free

CRC³ remains the second deadliest killer among cancers in the United States, with 56,600 deaths predicted for 2002 (1). In the United States, AAs have the highest incidence and mortality rates for colon cancer among United States racial and ethnic groups (1, 2). The reason for this disparity is not known.

Our current understanding of colorectal tumorigenesis is based on the development of genomic instability. One form of genomic instability, MSI, has been identified in colorectal tumors from HNPCC patients and from 15–20% of sporadic CRC patients (3). MSI is defined as a change in DNA microsatellite length because of insertion or deletion of nucleotides in tumor DNA when compared with normal tissue (4). This phenomenon is caused by a failure of the DNA MMR system to correct errors that occur during the replication of DNA. This leads to the accumulation of single nucleotide mutations and alterations in the length of microsatellite sequences that occur ubiquitously throughout the genome or in genes that contain microsatellite DNA in their coding regions (4, 5). MSI represents a hypermutable phenotype and has been suggested as a mechanism for rapid neoplastic progression for MSI tumors (6).

Unlike HNPCC patients who have germ-line mutations in one of the DNA MMR genes (i.e., hMSH2, hMLH1, or hMSH6) causing tumor MSI-H, sporadic CRCs with MSI-H have biallelic hypermethylation of the promoter of hMLH1 as the mechanism (7). MSI-H tumors tend to occur proximal to the splenic flexure, are often mucinous (3), and in vitro studies suggest that MSI-H tumors may be more resistant to chemotherapy (8). Some studies have shown that individuals with MSI-H tumors have improved survival (9), whereas others have not (10). Other factors downstream of DNA MMR inactivation could also influence survival.

Because evidence suggests that CRCs in AA patients are more frequently in the right colon (11) and that MSI-H may occur more commonly among American blacks (12), we evaluated the frequency of MSI in sporadic CRCs among consecutive AA CRC patients. We also correlated MSI findings with methylation of p16 to determine whether methylation was a common mechanism for inactivation of both the DNA MMR system and cell cycle regulation. We found that nearly half of the consecutive AA patients had tumors that demonstrated MSI-H, most were females, but p16 methylation did not correlate with DNA MMR inactivation.

Twenty-two AA patients with CRC who serially underwent surgical resection at Howard University Hospital between 1998 and 2000 are included in this study. After obtaining approval from the Howard University Institutional Review Board, the formalin-fixed, paraffin-embedded archival tissue was collected. Clinical data collected on each patient included race, age, site of primary tumor, mucin production, tumor differentiation, and family history of cancer. Family history was used to identify those families that met either the Amsterdam I or Amsterdam II criteria for HNPCC.

Independent pathologists who were not aware of the MSI status evaluated specific histopathologic characteristics. Tumors were classified as proximal (proximal to the splenic flexure) and distal tumors. The Tumor-Node-Metastasis system of the International Union against cancer was used for tumor staging. Mucin production was evaluated using the modified criteria of Wiggers et al. (13) as absent (no extracellular mucin production), focal (when extracellular mucin production was present in <50% of the cells), and predominant (when the area of extracellular mucin production was present in ≥50% of the cells).

Two methodologies were used for the MSI studies. Nontumor and tumor tissue domains were microdissected, then deparaffinized and purified. Radiolabeled primers designed to amplify specific DNA microsatellite sequences were used. A reference panel of five pairs of microsatellite primers recommended for CRC specimens to determine the presence of MSI was used (4).

Each primer pair was optimized for efficient amplification. One primer from each pair was end-labeled with γ-³³P in a reaction containing the primer, kinase buffer, T4 polynucleotide kinase, and [γ-³³P]ATP. PCR reactions were carried out on the cell line template DNA (200 ng) in a reaction containing 0.125 pmol each of the end-labeled and “cold” primers, 0.25 units of Taq DNA polymerase, 40 μm of dNTP stock solution, and final concentration of 1.5–2.0 μm magnesium. In general, each reaction was carried out for 35 cycles, and had annealing temperatures ranging from 50°C to 60°C. PCR products were denatured in 95% formamide and electrophoresed on a 6% polyacrylamide gel using fluoromer-labeled primers containing 7.5 m urea. The gels were dried and exposed to X-ray film (Fig. 1).

A second method using fluorescent-labeled primers was used. PCRs were carried out in 10-μl volumes containing 60 ng of genomic DNA, 1 pmol each of fluorescently labeled primer, 50 mm KCl, and 10 mm Tris; 0.4 mm each dNTP and 1.5 mm MgCl2; and 0.5 units of Taq DNA polymerase (Promega, Madison, WI). PCRs consisted of an initial denaturation step at 94°C for 4 min, followed by 30 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s, and a final extension step at 72°C for 6 min. The annealing temperature varied among primer sets but was between 50°C and 60°C. Simultaneous gel electrophoresis of PCR products was performed on 5.25% Long Ranger gels (Bio-Rad, Hercules, CA) containing 6 m urea. Samples were loaded onto 96-lane gels and electrophoresed on an ABI Prism 377 automated DNA sequencer (Perkin-Elmer, Norwalk, CT). Gels were scored in two steps using separate programs. The first program-Genesca (ABI), checked for consistency across all of the lanes. Consistency was also checked manually. The second program, Genotyper (ABI), assigned allele sizes and derived peak heights in arbitrary fluorescent units. Peak heights were then evaluated by hand (data not shown), with the color and size of each peak identifying the product being evaluated and the presence of MSI. Lesions were characterized as MSI-H if they manifested instability at two or more of the five loci, MSI-L if unstable at only one locus, or MSS if showing no instability at any loci.

p16 methylation was analyzed according to the manufacturer’s recommendations (Introgene, Gaithersburg, MD). Presence or absence of p16 methylation in cancers was determined by comparing the signals in the tumor versus nontumor lanes (Fig. 2). Primer sequences conditions for PCR and restriction enzymes were used according to manufacturer recommendation (Introgene). The protocol is based on MSP. MSP distinguishes unmethylated from methylated alleles based on sequence alterations produced by bisulfite treatment of DNA, which converts unmethylated, but not methylated, cytosines to uracil, and subsequent PCR using primers specific to methylated or unmethylated DNA. Briefly, 1 μg of genomic DNA was denatured by treatment with NaOH and was modified by sodium bisulfite. DNA samples were purified using Wizard DNA purification resin (Promega), again treated with NaOH, precipitated with ethanol, and resuspended in water. PCR was then performed using the primer pairs described below under the following conditions: the PCR mix contained 10× PCR buffer [16.6 mm ammonium sulfate, 67 mm Tris (pH 8.8), 6.7 mm MgCl2, and 10 mm 2-mercaptoethanol], dNTPs (each at 1.25 mm), primers (50 ng each per reaction), and bisulfite-modified DNA (50 ng) in a final volume of 50 μl. Reactions were hot-started at 95°C for 5 min before the addition of 1.25 units of Taq polymerase (Life Technologies, Inc.). Amplification was carried out in a MJ Research temperature cycler for 40 cycles (30 s at 95°C, 30 s at 59°C, then 30 s at 72°C), followed by a final 4-min extension at 72°C. Control PCRs lacking genomic DNA were performed for each set of reactions. Positive control (hypermethylated) and negative control DNA was used from the kit. Ten μl of each PCR reaction product were directly loaded onto 2% agarose gels stained with ethidium bromide and visualized under UV illumination.

Age and cancer stage were treated as continuous variables, whereas MSI, gender, location, mucin production, differentiation, and p16 were treated as binary, categorical variables. The associations among MSI classes and age, cancer stage, gender, location, mucin production, differentiation, and p16 were quantified using FREQ and LOGISTIC procedures of the SAS System (SAS/STAT 2001). Ps were reported based on asymptotic χ² or Fisher’s exact test as appropriate.

The clinical and pathologic characteristics of the patients are given in Table 1. Of 22 tissue samples analyzed, 13 were obtained from males and 9 from females. The mean age of patients in the study group was 62.6 years. All of the patients in the study self-identified themselves as AAs. The samples were subdivided into three groups: 11 MSS (52%), 1 MSI-L (5%), and 9 MSI-H (43%). Patients in the MSI-H group were older with a mean age of 67.9 years compared with the study population with a mean age of 62 years, and the MSS group with a mean age of 58.9 years. However, this did not reach statistical significance (P = 0.18).

There was a significant association between MSI-H and gender (Table 1). Seventy percent of the MSI-H cancers were seen in females. As indicated by odds ratio, the chance of female patients having MSI-H was 11.7 times greater than that of male patients (P < 0.03). Interestingly, the majority of the tumors in females were in the MSI-H group (77%; Table 2), compared with males (23%). Patient number 8 (Table 2) with MSI-H had a strong family history of colon cancer and met the Amsterdam criteria. Two patients (numbers 20 and 21; Table #2) based on their age met the Bethesda criteria MSI listing (14). However, tissue samples from these patients were MSS.

Seventeen of the tissue samples were from cancers arising in the proximal colon (77%). The incidence of proximal lesions in the MSI-H group (90%) was higher than in the MSS group (64%). The primary lesion in the only MSI-L patient in our study was located in the proximal colon.

Among all of the samples, 15 (68%) CRCs were classified as negative (for mucin production), 2 (9%) had focal mucin production, and 5 (23%) were predominantly mucin producing (Table 1). The percentage of MSI-H lesions (50%) that were mucin producing was higher than mucin-producing lesions in the MSS group (10%). The odds ratio of patients with mucin production having MSI-H was 16.5 times higher than that of patients without mucin production (P = 0.02).

Most of the tumors were moderately differentiated (77%). The percentage of moderately differentiated tumors was slightly lower in the MSI-H group (80%) when compared with the MSS group (82%). The single MSI-L lesion in our series was poorly differentiated.

Most patients in our series had tumors that were stage 3 or higher. Forty percent of MSI-H lesions were stage 2, 40% were stage 3, and the remaining 20% were stage 4. There was no statistically significant difference in the stage of the lesions between MSI and MSS groups. (P = 0.88; Table 1).

To determine whether sporadic MSI-H tumors were associated with methylation of p16, we determined the methylation status of the p16 gene promoter. Five patients in our series showed methylation of tumor suppressor gene p16. Three of these were in tumors from females and 2 in tumors from males. Of interest was the lack of MSI-H lesions in males with p16 promoter methylation, whereas all of the females with p16 promoter methylation showed MSI-H lesions. Three of the 10 MSI-H tumors demonstrated p16 promoter methylation, whereas 2 of 11 MSS tumors showed p16 promoter methylation (P = 0.62).

MSI is one of the molecular mechanisms leading to genomic instability. In recent years, this phenomenon has become better understood. Evidence suggests that patients with CRC manifesting MSI have a different prognosis and response to chemotherapeutic agents (8, 15, 16, 17). Several studies report that up to 86% of tumors in HNPCC patients exhibit MSI (6). Overall, 80–85% of sporadic CRC lack MSI, and the remaining tumors demonstrate microsatellite instability groups. In our series of AA patients, the incidence of MSI was 45%, a rate much higher than reported in the literature (12–17%; Refs. 18, 19). This may in part be related to the many right-sided cancers in our sample. This is consistent with a study using the Surveillance, Epidemiology, and End Results registry data from the National Cancer Institute demonstrating that proximal colon carcinoma rates in blacks were considerably higher than in whites (20). MSI is found more frequently in right-sided colon tumors; this may reflect the pathogenesis of CRC in the AA race and point to a higher incidence of MSI in this ethnic group compared with the general population.

Clinicopathologic features known to be associated with the presence of MSI include location of the primary tumor proximal to the splenic flexure, poorly differentiated cancers, predominance of mucin-producing cells in lesions with MSI, and peritumoral lymphocytic infiltration (18). The observations made in this study of tumor location and frequency of mucin production in AAs are in accordance with previous publications. However, one pathologic feature that was different in our series was the degree of differentiation of the cancers. Most of the tumors in the MSI-H group were moderately differentiated (80%). The MSS group in our analysis, like other studies, consisted predominantly of moderately differentiated cancers. This may represent a difference in expression of MSI-H in AAs, which needs additional investigation. Data regarding lymphocytic infiltration were unavailable for this series of patients.

In this study, we examined the frequency and significance of MSI in an AA population with apparently sporadic carcinoma. However, some individuals enrolled with apparent sporadic disease could harbor germ-line mutations. Novel germ-line mutations in MMR genes hMLH1 and hMSH2 have been demonstrated in AA CRC patients that include HNPCC families (21). At least 1 patient in our study fell into this germ-line mutation category. Two patients in our series satisfied the Bethesda criteria based on age (patients 8, 20, and 21; Table 2). One of these patients (patient 8) also met the requirements for HNPCC by the Amsterdam criteria. This patient showed MSI-H in malignant tissue consistent with an inherited mutation causing his cancer. But the tissue from patients 20 and 21 (Table 2) were microsatellite stable. This may be explained by the low positive predictive value (27%) of The Bethesda Guidelines for HNPCC demonstrated in previous studies.

Our results raise the question of how being female might influence the pathway for CRC development. As indicated by the odds ratio, the chance of female patients having MSI-H was 11.7 times higher than male patients (P < 0.03). Breivik et al. (22) also indicated a relationship between MSI in CRC and gender and age. These investigators found MSI most frequent among younger male and older female patients. In addition, epidemiological studies reported gender differences in the site distribution of CRC cancer, with proximal cancer most frequent among older women (23). One of the earliest hypotheses addressing the role of anatomical subsets and gender in the development of CRC postulated an influence of estrogen on bile acid secretion (6, 22, 24). According to this hypothesis, estrogen through influence on serum cholesterol levels alters the concentration of bile acids, which have toxic, trophic, and promoting effects on the colonic epithelium. Previous studies have shown a high incidence of methylation of the hMLH1 and p16 promoters in CRC arising in female patients (10, 24). In our series, 66% of patients with p16 methylation were female. Also, the high proportion of MSI-H tumors seen in female patients in our series may explain why some studies show that females with CRC have a better prognosis than males. Of note, survival rates are lower in AA patients with CRC. However, patients with high levels of MSI as a group have better outcomes. One possible explanation for this discrepancy is that AAs have decreased access to medical care and present at a latter stage of disease (25, 26). Methylation of hMLH1 MMR gene responsible for cases of sporadic MSI-H colon cancer will be analyzed to investigate whether MSI high prevalence in this population is because of hMLH1 methylation.

In conclusion, most of the clinicopathologic characteristics of MSI-H lesions in our study of AA patients are similar to those reported previously in Caucasians including the location of the primary tumor and extent of mucin production. However, most of the MSI-H lesions in our study group showed well-differentiated lesions, which may indicate a different pathologic expression of MSI-H lesions in AAs. Our results suggest a much higher proportion of CRC tumors with MSI in AAs compared with the general population, 45% versus <20%, respectively. This may have significant implications in the treatment of AA patients, because MSI-H lesions are often right-sided and may show a different response to chemotherapeutic agents like 5-fluorouracil (8). Additional studies with larger sample sizes are needed to confirm these data.

We thank Dr. Colin Steine (University of Maryland at Baltimore, Baltimore, MD) for critical reading of the MSI part of this paper.