Accumulated clonal genetic alterations in familial and sporadic colorectal carcinomas with widespread instability in microsatellite sequences

Abstract

A subset of hereditary and sporadic colorectal carcinomas is defined by microsatellite instability (MSI), but the spectra of gene mutations have not been characterized extensively. Thirty-nine hereditary nonpolyposis colorectal cancer syndrome carcinomas (HNPCCa) and 57 sporadic right-sided colonic carcinomas (SRSCCa) were evaluated. Of HNPCCa, 95% (37/39) were MSI-positive as contrasted with 31% (18/57) of SRSCCa (P < 0.000001), but instability tended to be more widespread in SRSCCa (P = 0.08). Absence of nuclear hMSH2 mismatch repair gene product by immunohistochemistry was associated with germline hMSH2 mutation (P = 0.0007). The prevalence of K-ras proto-oncogene mutations was similar in HNPCCa and SRSCCa (30% (11/37) and 30% (16/54)), but no HNPCCa from patients with germline hMSH2 mutation had codon 13 mutation (P = 0.02), and two other HNPCCa had multiple K-ras mutations attributable to subclones. 18q allelic deletion and p53 gene product overexpression were inversely related to MSI (P = 0.0004 and P = 0.0001, respectively). Frameshift mutation of the transforming growth factor beta type II receptor gene was frequent in all MSI-positive cancers (85%, 46/54), but mutation of the E2F-4 transcription factor gene was more common in HNPCCa of patients with germline hMSH2 mutation than in those with germline bMLH1 mutation (100% (8/8) versus 40% (2/5), P = 0.04), and mutation of the Bax proapoptotic gene was more frequent in HNPCCa than in MSI-positive SRSCCa (55% (17/31) versus 13% (2/15), P = 0.01). The most common combination of mutations occurred in only 23% (8/35) of evaluable MSI-positive cancers. Our findings suggest that the accumulation of specific genetic alterations in MSI-positive colorectal cancers is markedly heterogeneous, because the occurrence of some mutations (eg, ras, E2F-4, and Bax genes), but not others (eg, transforming growth factor beta type II receptor gene), depends on the underlying basis of the mismatch repair deficiency. This genetic heterogeneity may contribute to the heterogeneous clinical and pathological features of MSI-positive cancers.

Figure 1.

Illustration of MSI evaluation with two noncoding polymorphic CA dinucleotide repeat sequences on the long arm of chromosome 18 (D18S69 and D18S64). Alleles with shifts of size due to deletion of repeats in tumor DNA (T lanes) as compared with matched nonneoplastic mucosal DNA (N lanes) are indicated (arrowheads).

Figure 2.

Illustration of MSI evaluation with the noncoding nonpolymorphic BAT26 poly(A) tract in the fifth intron of the hMSH2 gene. Alleles with shifts of size due to deletion of nucleotides in tumor DNA are indicated (arrowheads). Note the consistent size of the wild-type allele (wt) in these tumor specimens.

Figure 3.

Immunohistochemistry for hMSH2 gene product expression. Nuclear staining is absent in the LoVo colon cancer cell line (A) with reported homozygous deletion of the hMSH2 gene and absence of expression of hMSH2 gene product by Western blotting. By contrast, nuclear staining is intense in the HCT116 colon cancer cell line with known hMHL1 mutation (B). Cytoplasmic staining is evident in both cell lines. Poorly differentiated colonic carcinomas and overlying mucosa from HNPCC patients with known germline hMSH2 mutation (C) and known germline hMLH1 mutation (D) show absence of nuclear staining of cancer cells (arrow) in C but intense staining (arrow) in D. Cytoplasmic staining is evident in both cancers. Nuclear staining of crypt epithelial cells in the proliferative compartment and of cells in germinal centers of lymphoid nodules (arrowheads) serve as internal positive controls.

Figure 4.

ras proto-oncogene sequence analysis in MSI-positive HNPCCa with three different ras mutations. Codons 12 and 13 are shown, and mutations are indicated by arrowheads. G-to-A transitions are evident in the first nucleotide of codon 12 and the second nucleotide of codon 13 in the initial tumor specimen. In microdissected specimens of approximately 1 mm , G-to-A transitions are evident in the second nucleotide positions of codons 12 and 13 in region 4 and in the second nucleotide position of codon 13 alone in region 6. Other microdissected regions showed no ras mutation (not illustrated). The findings indicate the presence of heterogeneous subclones in the MSI-positive cancer.

Figure 5.

Labeling indices for p53 gene product overexpression by immunohistochemistry and computerized image analysis. A bimodal distribution is evident with all indices above 70% or below 45%. High p53 labeling index of the type associated with p53 gene mutation is inversely related to MSI positivity (P = 0.0001).

Figure 6.

Analysis of tumor DNA for frameshift mutation in the nonpolymorphic poly(A) tract in the fourth exon of the TGFβ RII gene. Alleles with shifts of size due to deletion of nucleotides are indicated by arrowheads. Note the consistent size of the wild-type allele (wt) in these tumors. The microdissected tumor in the far right lane has two mutations with loss of 1 bp (upper arrowhead) and 2 bp (lower arrowhead), respectively, and presence of the wild-type allele.

Figure 7.

Analysis for mutation in the CAG trinucleotide repeat sequence in the E2F-4 transcription factor gene. Alleles with shifts of size in tumor DNA (T lanes) as compared with matched nonneoplastic mucosal DNA (N lanes) are indicated by arrowheads. Note the consistent size of the wild-type allele (wt) in neoplastic as well as nonneoplastic specimens. The MSI-positive LoVo colon cancer cell line (LoVo lane) has mutations in both alleles, one with insertion of a trinucleotide (upper arrowhead) and the other with deletion (lower arrowhead).

Figure 8.

Sequence analysis of the E2F-4 transcription factor gene in an SRSCCa specimen with allelic shift to a size 3 bp smaller in one allele (fourth lane in Figure 7 ▶ ) and available frozen tissue. Shortening of the length of the CAG trinucleotide repeat from 13 to 12 was found: wild type, (CAG)₁₂ CAG CAA CAG TAA CAG CAG CAG TTC GTC; tumor, (CAG)₁₂ CAa CAg tAa cAg CAG CAG ttc gTC. Nucleotides with detectably shifted position (lowercase letters) in tumor DNA are indicated by arrowheads on the photograph.

Figure 9.

Analysis of tumor DNA for frameshift mutation in the nonpolymorphic polydeoxyguanosine tract in the third exon of the proapoptotic Bax gene. Shifts of allelic size due to deletions are indicated by arrowheads. Note the consistent size of the wild-type allele (wt) in these tumor specimens.

Figure 10.

Mutational spectra of MSI-positive HNPCCa and SRSCCa arranged according to clinical setting and frequency of genetic alterations. Solid and open squares indicate that the feature was present or not found, respectively, and slashed squares indicate that the feature was not evaluated. Striking heterogeneity of the accumulated alterations is evident, including in members of the same HNPCC family who shared identical germline mutations (indicated by letters). The most common pattern of mutations was found in only 23% of the MSI-positive cancers (8 of 35 evaluable tumors; patients 11, 12, and 40 to 45). An inverse association between E2F-4 mutation and mutation in codon 13 of K-ras in HNPCCa from patients with germline hMSH2 mutation is evident in patients 1 to 17. Mutational status of TGFβ RII and E2F-4 had the most frequent concordance (71%, 22 of 31 evaluable cancers).