Clinicopathological features and microsatellite instability (MSI) in colorectal cancers from African Americans

Abstract

African Americans (AAs) have a 1.5 times higher risk of colorectal carcinoma (CRC) than Caucasians. Gene silencing through CpG island hypermethylation has been associated with the genesis or progression of microsatellite instability (MSI) largely due to 1 target for hypermethylation being the DNA mismatch repair gene hMLH1; there is anecdotal evidence of an increased incidence of MSI among AAs. P16 and hMLH1 can be inactivated by hypermethylation of their respective promoter regions, abrogating the ability to regulate cell proliferation and repair processes. We studied such methylation, as well as hMHS2 expression in colorectal cancers from AA patients to determine if MSI is associated with epigenetic silencing. Experiments were conducted on matched normal and colon cancer tissues from AA patients (n = 51). A total of 5 microsatellite markers (D2S123, D5S346, D17S250, BAT25 and BAT26) were used to evaluate MSI status. P16 and hMLH1 promoter methylation status was determined following bisulfite modification of DNA and using methylation specific PCR, while immunohistochemistry (IHC) was used to examine expression of hMLH1 and hMSH2. A total of 22 (43%) cancers demonstrated microsatellite instability-high (MSI-H), while 27 were microsatellite stable (MSS) and 2 were microsatellite instability-low (MSH-L). Most of the MSI-H tumors were proximal, well differentiated and highly mucinous. Most patients in the MSI-H group were females (68%). The p16 promoter was methylated in 19 of 47 (40%) tumors. A total of 7 of these CRCs demonstrated MSI-H (33%). The hMLH1 promoter was methylated in 29 of 34 (85%) tumors, of which 13 CRCs demonstrated MSI-H (87%). hMLH1 and hMSH2 staining was observed in 66% and 38% of MSI-H tumors, respectively. Overall, the prevalence of MSI-H colorectal tumor was 2-3-fold higher, while the defect in the percentage expression of mismatch repair (MMR) genes (hMLH1 and hMSH2) was similar in AA patients compared to the U.S. Caucasian population. Similar numbers of AA MSS tumors with p16 and hMLH1 methylation likely indicate hemimethylation of genes that might reflect environmental or genetic influences that might be more common in the AA population.

Figure 1

MSP analysis of the promoter region of p16^INK4a and hMLH1 in CRC patients. The presence of visible PCR product in lanes “U” indicates the presence of unmethylated genes of p16^INK4a or hMLH1, the presence of product in lanes “M” indicates the presence of methylated genes. Water controls for PCR reaction are also shown. MSP of p16^INK4a in normal and tumors is shown. Patients 31 and 51 are methylated at p16^INK4a. Patients 31 and 40 are methylated at hMLH1. MSP of hMLH1 in normal and tumors is shown. Lymphocyte DNA was used as control for unmethylated at hMLH1. Semimethylated HT-29 DNA is used as positive control for methylation and unmethylation of hMLH1.

Figure 2

Immunohistochemical analysis of hMLH1 and hMSH2 expression in normal epithelial and tumor cells. (a) Normal colonic mucosa showing positive nuclear staining of hMLH1 protein in normal crypt epithelial cells. (b) Tumor showing partial or (c), no hMLH1 protein expression. Normal epithelial cells (c,d) and tumor (e) stained with anti h-hMSH2 antibody showing positive nuclear and negative staining for hMSH2, respectively.