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TGF beta/Smad signaling pathway members are potent tumor suppressors for many types of cancers. We hypothesize that breast tumors differentially express these genes and that this expression pattern plays a role in the proliferation of breast cancer. We examined the mRNA levels of TIEG, Smad7, Smad2, and Bard1 using real-time RT/PCR in 14 normal breast, five non-invasive, 57 invasive (including 29 with outcome data), and five metastatic breast tumor tissues. TIEG and Smad7 mRNA levels were lower in non-invasive tumors compared to normal breast tissues. TIEG, Bard1, and Smad2 mRNA levels were lower in invasive cancers compared to normal breast tissues. In addition, TIEG, Smad2, and Bard1, provided discriminatory ability to potentially distinguish between normal and tumor samples, N- and N+ tumors, and N-/good (no recurrence for at least 5 years) and N-/bad (recurrence within 3 years) outcome patients. TIEG mRNA levels accurately discriminated between normal breast tissue and primary tumors with a sensitivity and specificity of 96 and 93%, respectively. TIEG, in combination with Smad2, distinguished between N+ and N- primary tumors with a sensitivity and specificity of 75 and 85%, respectively. TIEG in combination with Bard1 discriminated between N-/bad outcome from N-/good tumors with a sensitivity and specificity of 83 and 82%, respectively. Our results support the hypothesis that the differential gene expression of TIEG, Smad2, and Bard1, which are tumor suppressor genes, plays a significant role in the proliferation of breast cancer. Further investigation is necessary to validate the ability of these genes to discriminate between different populations of breast cancer patients.

The role of microsatellite instability (MSI) in the pathogenesis of hepatocellular carcinoma (HCC) is incompletely defined. Although high-frequency MSI (MSI-H) is infrequently seen in HCC, some studies have suggested a role for MSI in HCC development. While MSI has been clearly defined for a subset of tumors, in particular colorectal, gastric and endometrial cancers, generally accepted criteria have not been developed for other tumors. Colorectal cancers (CRC) are classified as MSI-H if >30-40% of >5 microsatellite loci analyzed show instability. The MSI-H phenotype is associated with defective DNA mismatch repair (MMR) and is observed in the majority of tumors from patients with hereditary non-polyposis colon cancer (HNPCC) and also in 15% of sporadic CRCs. Inactivating mutations of the hMLH1 or hMSH2 genes lead to defects in MMR in HNPCC. In sporadic CRCs, MMR is usually due to hypermethylation of the hMLH-1 promoter. The role of defective MMR in hepatocellular carcinogenesis is controversial. Immunohistochemistry for hMLH1 and hMSH2 reliably indicates hMLH1 or hMSH2 loss in MSI-H CRC tumors. To investigate the role of defective MMR in HCC carcinogenesis, we performed immunohistochemistry for hMLH1 and hMSH2 on 36 HCCs. BAT26, a microsatellite marker that reliably predicts MSI-H was also examined. All 36 of the tumors stained positively for both hMLH1 and hMSH2, strongly suggesting an absence of either inactivating mutations of hMLH1 and hMSH2 or promoter hypermethylation of hMLH1. None of the tumors showed MSI at the BAT26 locus. These findings suggest that defective MMR does not contribute significantly to hepatocellular carcinogenesis.

Two microsatellite instability (MSI) phenotypes have been described in colorectal cancer (CRC): MSI-H (instability at >30% of the loci examined) and MSI-L (MSI at 1-30% of the loci examined). The MSI-H phenotype, observed in both hereditary nonpolyposis colon cancer-associated CRC and approximately 15% of sporadic CRC, generally results from mutational or epigenetic inactivation of the DNA mismatch repair (MMR) genes hMSH2 or hMLH1. The genetic basis for the MSI-L phenotype, however, is unknown. Several other proteins, including hMSH3 and hMSH6, also participate in DNA MMR. Inactivating mutations of MSH6 in yeast and human tumor cell lines are associated with an impaired ability to repair single-base mispairs and small insertion-deletion loops but not large insertion-deletion loops. This suggests that hMSH6 mutations are more likely to be associated with a MSI-L phenotype than a MSI-H phenotype in CRC. To explore this possibility, we screened tumors from 41 patients with MSI-L CRC for hMSH6 mutations with conformation-sensitive gel electrophoresis (CSGE) and for hMSH6 protein expression by immunohistochemistry. Alterations found with CSGE were confirmed by DNA sequencing of normal and tumor tissue. One somatic (Asp389Asn) and 15 germ-line changes were found. Of the 15 germ-line changes, 9 were found in an intron (none involving splice junctions), and 6 were found in an exon (Gly39Glu, Leu395Val, and 4 silent alterations). Immunohistochemical staining for hMSH6 performed on 34 of the 41 tumors revealed strong nuclear hMSH6 expression in all of the cases. Overall, our results suggest that hMSH6 mutations do not play a major role in the development of sporadic CRC with a MSI-L phenotype.

Recently, a novel tumor suppressor gene, p73, was isolated and mapped to chromosome 1p36, a region commonly associated with loss of heterozygosity in neuroblastoma and other human malignancies, including breast cancer. The p73 gene shares considerable homology with the common tumor suppressor gene p53, both in composition and function. This study examines the potential participation of p73 in the pathogenesis of sporadic and hereditary breast cancers. Mutation analysis of 29 hereditary breast cancer cases revealed five independent silent mutations in the hereditary cases that are unlikely to play a role in tumor development. Mutation analysis of 48 sporadic breast tumors did not identify any unique variants. Eleven common polymorphisms scattered throughout the gene were also detected. Thus, mutations in the p73 gene appear to play little if any role in hereditary or sporadic breast cancer.

Recent studies have demonstrated the presence of microsatellite instability (MSI) in tumors from patients with hereditary nonpolyposis colon cancer and in a subset of patients with sporadic colorectal cancer (CRC). In sporadic CRC, three tumor phenotypes have been defined: microsatellite stable (MSS), low-frequency MSI, and high-frequency MSI (MSI-H). Although defective mismatch repair, consisting primarily of alterations in hMSH2 and hMLH1, is believed to be responsible for the MSI phenotype in the majority of patients with hereditary nonpolyposis colon cancer, the genetic defect responsible for this phenotype in sporadic CRC has yet to be clearly delineated. Somatic or germ-line alterations in these two genes have been identified in only a minority of these cases. Analysis of the protein expression patterns of hMSH2 and hMLH1 in unselected CRC, however, suggests that alterations in hMLH1 may account for a majority of the MSI-H cases. In an effort to explore the underlying molecular basis for these findings, we have examined the methylation status of the presumptive hMLHI promoter region in 31 tumors that vary in regard to their MSI status (MSI-H or MSS), their hMLH1 protein expression (MLH- or MLH+), and their gene mutation (Mut+ or Mut-) status. Hypermethylation of the hMLH1 promoter occurred in all 13 MSI-H/ MLH- tumors that did not have a detectable mutation within the hMLH1 gene. Of those MSI-H tumors containing germ-line or somatic alterations in hMLH1 (n = 7, including 3 frameshift, 1 nonsense, 2 missense mutations, and 1 tumor containing multiple mutations: missense, splice-site alteration, and a frameshift), four had a normal methylation pattern, whereas three others demonstrated hypermethylation of the hMLH1 promoter region. Two of these cases had a missense alteration, the other a frameshift alteration. The single MSI-H/Mut+ tumor that had normal hMLH1 and hMSH2 expression, as well as 9 of the 10 MSS cases, lacked methylation of the hMLH1 promoter. Hypermethylation of the hMSH2 promoter was not observed for any of the cases. These results suggest that hypermethylation of the hMLH1 promoter may be the principal mechanism of gene inactivation in sporadic CRC characterized by widespread MSI.

Recent studies have demonstrated the presence of microsatellite instability (MSI) in tumors from patients with hereditary nonpolyposis colorectal cancer and in a large number of sporadic tumors. To further characterize the type of alterations at these loci and their frequency of involvement in colon cancer, we studied DNA extracted from paraffin-embedded tissue from 508 patients using 11 microsatellites localized to chromosomes 5, 8, 15, 17, and 18. Overall, MSI at each locus varied in character and frequency and was observed with at least one marker in 191 cases (37.6%). Based on the number of markers displaying instability per tumor, three groups of patients were defined: those with <30% of the markers showing instability (MSI-L,, n = 109, 21.5%); those with > or = 30% (MSI-H, n = 82, 16.1%); and those showing no instability (MSS, n = 317, 62.4%). These groups were tested for correlations with a number of clinical and pathological parameters, including age, sex, stage, ploidy status, and site of tumor. Comparing across the three groups and verified by pair-wise comparisons, the MSI-H group was associated with tumor site (proximal colon, P = 0.001), sex (females, P = 0.005), stage (Dukes' B, P = 0.01), and ploidy status (diploid, P = 0.03). No significant differences were noted between the MSI-L and MSS group for any of the parameters tested. An additional 188 consecutive surgical colorectal cancer cases were examined for the presence of MSI and for the immunohistochemical expression of hMLH1 and hMSH2 proteins. Of this group, 129 (68.6%) were classified as MSS, 17 (9.0%) as MSI-L, and 42 (22.3%) as MSI-H. None of the MSS and none of the MSI-L tumors had altered expression of either hMLH1 or hMSH2. However, the majority of MSI-H (40 of 42, 95%) cases demonstrated absence of staining for these proteins. The most frequently altered protein was hMLH1, occurring in 95% of the tumors with altered expression. Cumulatively, these data suggest that the tumor phenotype MSI-H is distinct from tumor phenotypes MSI-L and MSS, with no apparent differences between MSI-L and MSS. Furthermore, altered hMLH1 protein expression appears to be responsible for the mutator phenotype in the vast majority of MSI-H tumors.

To date, at least four genes involved in DNA mismatch repair (MMR) have been demonstrated to be altered in the germline of patients with hereditary nonpolyposis colon cancer: hMSH2, hMLH1, hPMS1, and hPMS2. Additionally, loss of MMR function has been demonstrated to lead to the phenomenon of microsatellite instability (MIN) in tumors from these patients. In this study, we have examined the protein expression pattern of hMSH2 and hMLH1 by immunohistochemistry in paraffin-embedded tumors from 7 patients with MIN+ sporadic cancer, 13 patients with familial colorectal cancer, and 12 patients meeting the strict Amsterdam criteria for hereditary nonpolyposis colon cancer. The relationship between the expression of these two gene products, the presence of germline or somatic mutations, and the presence of tumor MIN was examined. Nineteen of the 28 tumors studied demonstrated MIN, whereas mutations in hMLH1 and hMSH2 were detected in 6 and 2 patients, respectively. Of the eight MIN+/mutation+ cases, the absence of protein expression was observed for the corresponding gene product in all but one case (missense mutation in hMLH1). However, seven MIN+/mutation- cases also showed no expression of either hMLH1 (n = 5), hMSH2 (n = 1), or both (n = 1), whereas four MIN+/mutation- cases demonstrated normal expression for both. None of the MIN-/mutation- cases (n = 9) demonstrated an altered expression pattern for either protein. These data suggest that examination of protein expression by immunohistochemistry may be a rapid method for prescreening tumors for mutations in the MMR genes.