Most sporadic colorectal cancers develop through a mechanism that involves loss of relatively large chromosomal segments, which is thought to represent the deletion of wild-type tumor suppressor genes from the nucleus.102 A proportion of sporadic colorectal cancers (perhaps 15 percent) and nearly all HNPCC-related cancers progress through a different mechanism, with MSI. MSI is inversely correlated with loss of heterozygosity of chromosomes 5q, 17p, and 18q.6 In sporadic colorectal cancers, over half show mutations in the K-RAS oncogene.
What Growth-Controlling Genes are Altered in HNPCC Cancers?
One of the initial reports indicated that mutations at K-RAS, APC, and p53 were at least as common in HNPCC colon cancers as in sporadic ones.7 One of the other initial investigations in the area reported that K-RAS and p53 mutations were significantly less frequent in tumors with MSI but did not look for APC mutations.5 The third of these initial groups did not look for point mutations in APC, K-RAS, or p53 but noted an inverse relationship between MSI and loss of heterozygosity at 5q, 17p, and 18q.6 A failure to microdissect the neoplastic tissue from infiltrating stroma or inflammatory cells (which can be considerable in these tumors) could lead to a systematic underestimate of cancer-associated mutations. This concern notwithstanding, one group has reported an inverse relationship between MSI and p53 mutations in colon cancer cell lines, 103 and two groups reported a significant reduction in the incidence of mutations in APC, p53, and K-RAS-2 in HNPCC colon cancers.104,105 Additionally, immunohistochemical detection of the p53 protein in colorectal tissues is a surrogate for point mutations in the gene, since these mutations often stabilize the gene product. There is an inverse relationship between MSI and p53 immunostaining, which supports the contention that p53 mutations may be less common in colon cancers with MSI.106 One group has reported that the putative inverse relationship between MSI and p53 mutations may not obtain in distal colorectal cancers.107 This area still requires additional clarification.
An analysis of 101 colon cancers led the Vogelstein group to conclude that APC mutations were present in colorectal tumors with or without MSI, but that in MSI there was a significant predilection for frameshift mutations at repetitive sequences, particularly in polyadenine tracts.108 At this point in time, the data suggest that most colorectal neoplasia begins with inactivation of the APC gene whether or not the tumor has MSI. However, the genetic events after APC inactivation may diverge thereafter depending on the mechanism underlying the genomic instability, which will select for specific genes in multistep tumor progression (Fig. 32-4).
Dual pathways for tumor development. The initial pathway for multistep carcinogenesis is depicted as the “chromosomal instability” at the top and involves LOH at tumor suppressor genes. The lower pathway (MSI) or the “mutator pathway” involves a unique destabilizing mechanism and inactivation of different genes, although both result in cancer. Both pathways may begin with an inactivating mutation at the APC locus, but the mutations leading to the lower pathway are those which would be expected with the loss of DNA MMR activity. These very distinct mechanisms lead to a similar pathologic result.
The genomic instability seen at microsatellites in human colorectal neoplasms is an early event and can be found in the adenomatous polyps that serve as precursors to cancer in HNPCC.109 Although MSI has been described in phenotypically normal human lymphocytes in unusual instances, 65 the mucosa in the colons from most patients with HNPCC does not show this abnormality.110 The adenomatous polyps associated with sporadic tumors with MSI, or those in HNPCC, also have MSI, unlike sporadic adenomas, which rarely do.109,111 The proportion of microsatellite loci that are mutated increases with progression from adenoma to carcinoma in both instances.111,112
In one series of cancers from patients with HNPCC, 95 percent showed the MSI phenotype, regardless of stage. In contrast, this type of genomic instability was found in only 3 percent of early sporadic tumors (i.e., adenomas or intramucosal carcinomas) and 13 to 24 percent of sporadic invasive cancers. MSI has been reported in 35 percent of the liver metastases, suggesting that loss of the MMR system may play a role in tumor progression.104
One survey reported that 16 percent of sporadic colorectal cancers and 86 percent of colorectal cancers from HNPCC patients had MSI, including all patients in which a germ-line mutation in hMSH2 was found. This lesion (i.e., MSI) was present in only 3 percent of 33 sporadic colorectal adenomas but in 57 percent of 14 adenomas associated with HNPCC. In addition, MSI was present in all the extracolonic cancers derived from HNPCC patients.113 Although MSI has been described in phenotypically normal lymphocytes in patients with certain germ-line mutations in a DNA MMR gene, 65 the normal-appearing mucosa in the colons of patients with HNPCC does not show this abnormality.110
MSI was first described in colorectal cancers not selected on the basis of a suspicion for HNPCC. Depending on the criteria used, 12 to 28 percent of colorectal cancers have MSI.5-7 MSI is neither characteristic of colorectal cancer, limited to tumors of this organ, nor limited to HNPCC. MSI can be found in gastric cancers, endometrial cancers, ovarian tumors, urinary bladder tumors, non-small-cell lung cancers, small-cell lung cancers, breast cancers, and other tumors. In some instances, inactivation of a DNA MMR gene can be found, but this is not always the case. In some colon cancers, MSI can be found in association with loss of heterozygosity at one of the DNA MMR gene loci.66 In the overwhelming majority of instances, there are no germ-line mutations at any of the known HNPCC loci when MSI is found.
As mentioned, a sizable minority of all sporadic colorectal cancers have MSI, and most of these are unrelated to HNPCC. Most of these tumors do not express hMHL1 immunohistochemically.114,115 The mechanism responsible for silencing the hMLH1 gene is frequently hypermethylation of promoter sequences in its 5′ upstream regulatory region.116-119 Treating cultured tumor cells with 5-azacytidine can restore expression of hMLH1.117-118 Hypermethylation and silencing of the hMSH2 gene have not been found.117,119
Somatic Mutations Unique to Tumors with MSI
The finding of a type of hypermutability that was unrelated to the type of genomic instability seen in sporadic colorectal cancers (i.e., that manifested by widespread loss of heterozygosity) led to speculation that a distinct molecular pathway was responsible for HNPCC and related tumors.35 An increased rate of mutation at the hprt locus was found in colorectal cancer cell lines that had MSI.120 A recognition that MSI was associated with a disproportionate hypermutability at microsatellite sequences prompted several laboratories to look for repetitive sequences in the coding regions of genes that might be involved in growth control, with the speculation that these sequences would be at increased risk to experience a frameshift in a cell with MSI, resulting in a loss of function for the gene product.
The paradigm of this process was established on the identification of inactivating mutations in the type II transforming growth factor (TGF) β1 receptor (called RII) in colon cancer cells with MSI.121 TGFβ1 signaling results in an inhibition of growth in colorectal epithelium.122 Two repetitive sequences were found in coding regions of RII, a (CA)3 and an A8 sequence. Mutations in RII were concentrated on the A8 sequence in MSI tumors and not found in non-MSI tumors. In each instance, the mutation resulted in loss of the RII transcript expression and a failure of cells to bind or respond to the TGFβ ligand. This is of particular importance because the TGFβ system inhibits the growth of colonic epithelial cells, and loss of this system in tumors with MSI represents a critical escape from growth control. Similar inactivating mutations in the RII gene have been found in gastric cancer cell lines and resected gastric carcinoma specimens; the mutations usually occur in a coding polyadenine tract.123 It appears that inactivating mutations in growth-regulating genes may be a key mechanism by which tumors with MSI become neoplastic. These mutations are not commonly found in adenomas and may represent relatively late events in the multistep progressive neoplastic process in the evolution of a tumor in HNPCC.124,125
Additional genes that may be involved in growth control also contain repetitive sequences in coding regions, which puts them at risk for mutation when the DNA MMR system is inactivated. Several of these have been found to be mutated in some proportion of HNPCC tumors, including the insulin-like growth factor II (IGF-II) receptor (which acts “in series” with RII in TGFβ signaling), BAX, hMSH3, hMSH6, and E2F4; the genes and the hypermutable sequences are listed in Table 32-6.124,126-133 The requirement for involvement of any of these genes in the evolution of a tumor with MSI and the sequence of events involved remains to be determined. Of particular interest, two of the genes are themselves DNA MMR genes: hMSH3 and hMSH6. This raises the possibility of a cascade of events in which a partial loss of DNA MMR activity could lead to mutations in other MMR genes and amplify the genomic instability.134
Table 32-6: Growth Regulatory Genes Mutated in Colorectal Cancers with MSI (Somatic Mutations in HNPCC) |Favorite Table|Download (.pdf) Table 32-6: Growth Regulatory Genes Mutated in Colorectal Cancers with MSI (Somatic Mutations in HNPCC)
|Gene Target ||Repetitive Genetic Sequence ||Frequency of Mutation in Colorectal Cancers with MSI or HNPCC ||Reference |
| TGFβ1 RII ||A6 ||85–90%(MSI) ||121, 123, 124, 125 |
| IGF IIR ||G8 ||9% of MSI tumors, ||126 |
| || ||1/8 HNPCC tumors || |
| Bax ||G8 ||51–54% (MSI); ||127, 130, 131 |
| || ||52% in HNPCC || |
| MSH6 ||C8 ||33% in HNPCC ||131 |
| MSH3 ||A8 ||52% in HNPCC ||128, 129, 131, 132 |
| E2F4 ||(CAG)n ||65% (MSI) ||129, 133 |
| APC ||Several ||(See text) ||108 |
MSI and Resistance to Cytotoxic Drugs.
Inactivation of the DNA MMR system is associated with increased resistance to DNA alkylation, which is toxic to wild-type cells.135 Restoration of the DNA MMR system in a colon cancer cell line defective at the hMLH1 locus increases sensitivity to alkylation and restores the G2/M cell cycle checkpoint.136,137 The DNA MMR system also is required for transcription-coupled DNA repair.138 These findings imply additional growth advantages for tumor cells defective in DNA MMR, since the G2/M checkpoint may be bypassed in these cells, and an additional level of mutation repair is lost. Evidence is accumulating that DNA MMR-defective cells may be relatively resistant to cytotoxic chemotherapy used to treat cancer.136,137,139-143 Cell lines have been identified that are deficient for each of the major human DNA MMR genes, which provide valuable models for study.144 All these observations have come from experiments on cultured cells, and the clinical implications of this have not yet been tested on patients.
MSI and Neoplasia in Ulcerative Colitis.
MSI is commonly seen in cancers associated with ulcerative colitis, as well as the dysplasias that antedate these tumors.145 An intronic polymorphism in the hMSH2 gene is more common in patients who develop colitis-associated neoplasia than in control patients with or without colitis.146 Moreover, MSI may be found in the non-neoplastic colonic mucosa from patients with chronic ulcerative colitis.147 It has not yet been confirmed whether inactivation of the DNA MMR system is an essential part of tumor development in chronic inflammation.