Some colorectal tumors with wild-type adenomatous polyposis coli gene have activating mutations in β-catenin (encoded by CTNNB1) that result in decreased phosphorylation by GSK-3β and increased signaling through the Tcf/Lef transcription factors. To investigate the relationship between CTNNB1 mutations and underlying pathways of genomic instability, we examined 80 colorectal cancers stratified by the presence or absence of microsatellite instability (MSI). CTNNB1 mutations were identified in 13 (25%) of 53 cancers with high frequency MSI (MSI-H), including 12 point mutations at exon 3 phosphorylation sites (codons 41 and 45) and one deletion of the entire exon 3 degradation box. No CTNNB1 mutations were identified in 27 microsatellite stable or low frequency MSI (MSI-L) colorectal cancers (P < 0.01). In contrast, CTNNB1 mutations were identified in 3 of 9 (33%) MSI-H and 10 of 20 (50%) MSS/MSI-L endometrial carcinomas, suggesting a more generalized involvement in these tumors. Only six (46%) of the endometrial carcinoma CTNNB1 mutations occurred at residues directly phosphorylated by GSK-3β, and only one of these was at either codon 41 or 45. All point mutations in MSI-H cancers were transitions, whereas 64% of those in MSS/MSI-L cancers were transversions (P < 0.01). The differences in the mutation profiles suggest that there may be molecular fingerprints of CTNNB1 mutations, determined by biological factors related to both tumor type and underlying pathways of genomic instability.

The Wnt signal transduction pathway was first implicated in mammalian tumorigenesis when the Drosophila gene, wingless, was shown to be the homologue of the mouse mammary oncogene, int-1 (later renamed Wnt-1; Refs. 1 and 2). The generalized importance of this pathway was realized when APC3 was found to regulate Wnt signaling by acting in a complex with GSK-3β to control phosphorylation and degradation of β-catenin (3, 4, 5). Inactivating APC mutations occur in about 85% of colorectal carcinomas, resulting in β-catenin stabilization and increased signaling through the Tcf/Lef transcription factors (6, 7, 8). Recently, up to 50% of colorectal carcinomas without APC mutations were found to contain CTNNB1 mutations (9, 10, 11, 12). These mutations alter APC-dependent exon 3 serine/threonine phosphorylation residues, leading to β-catenin stabilization and activation of signaling (10, 13). Interestingly, about 50% of primary colorectal carcinomas with CTNNB1 mutations are MSI-H, suggesting that CTNNB1 mutations may be more common in the DNA MMR-deficient pathway of tumorigenesis (10, 12, 14, 15). CTNNB1 mutations are found in a number of other cancers, including about 13% of endometrial carcinomas; however, there has been no investigation of associations with underlying pathways of genomic instability (16, 17, 18, 19). In the present study, we examined the relationship between CTNNB1 mutations and DNA MMR deficiency and found that CTNNB1 mutations were significantly more common in MSI-H colorectal carcinoma as compared to MSS/MSI-L colorectal carcinoma. Surprisingly, we found a high frequency of CTNNB1 mutations in endometrial carcinoma, regardless of the presence or absence of microsatellite instability. Comparison of these cancers suggests that CTNNB1 mutation profiles may reflect different underlying carcinogenic pathways.

Tissue Samples.

Tumor samples were obtained from an ongoing, unlinked, population-based study of the frequency of MSI in human cancer. Ethics approval was obtained from The University of Toronto Human Ethics Committee prior to commencement of the study. Resected colorectal and endometrial carcinomas in patients <50 years of age were identified by The Ontario Cancer Registry, and paraffin blocks were retrieved from the treating hospitals. A blinded histopathological review was performed on all cases, and the results were entered into a coded database. For each case, normal and invasive carcinoma samples of at least 50% neoplastic cellularity were microdissected from two to three 10-μm unstained slides. Adjacent adenomas (colorectal carcinoma cases) or hyperplasias (endometrial carcinoma cases) were dissected separately when available. If cancers had more than one discreet morphological pattern, multiple tissue samples were also dissected separately. Genomic DNA was extracted as follows. Samples were incubated in 50–100 μl of lysis buffer [10 mm Tris-Cl (pH 8), 100 mm KCl, 2.5 mm MgCl2, and 0.45% Tween 20] for 10 min at 95°C. Proteinase K (15–35 μl, 20 mg/ml) was added, and samples were incubated overnight at 65°C.

MSI Testing.

For the colorectal carcinomas, MSI was tested using the Bethesda Consensus Conference reference panel of 5 markers (BAT-25, BAT-26, D2S123, D5S346, and D17S250; Ref. 20), with conditions as described previously (21). Colorectal carcinomas were classified as MSI-H if two or more loci displayed MSI, or as MSS, if no loci displayed MSI. If one locus had MSI, up to five additional loci were tested (BAT-40, BAT-RII, D18S58, D18S69, and D17S787); these reevaluated tumors were then classified as MSI-H if ≥40% of loci displayed MSI, or as MSI-L if ≤30% of loci displayed MSI (20). For the endometrial carcinomas, MSI was tested using at least five markers from the Bethesda reference panel and the Bethesda alternative marker list (BAT-26, BAT-40, BAT RII, D2S123, D3S1611, D5S346, D17S787, D18S59, and D18S69). Definitions for MSS, MSI-L, and MSI-H were the same as for the colorectal carcinomas.

CTNNB1 Mutation Detection.

For the CTNNB1 mutation analysis, we selected cases from the ongoing population-based study, including all available MSI-H cancers, all MSI-L cancers, and a sampling of MSS cancers. In total, we studied 80 colorectal carcinomas, including 53 MSI-H cancers, 19 MSI-L cancers, and 8 MSS cancers, and 29 endometrial carcinomas, including 9 MSI-H cancers, 2 MSI-L cancers, and 18 MSS cancers. Exon 3 of CTNNB1 was amplified by PCR in five separate reactions using six primers as shown in Fig. 1 A. The primer sequences were as follows: P1, 5′-AGTCACTGGCAGCAACAGTC-3′; P2, 5′ -TCTTCCTCAGGATTGCCTT-3′; P3, 5′ -GATTTGATGGAGTTGGACATGG-3′; P4, 5′ -TGTTCTTGAGTGAAGGACTGAG-3′; P5, 5′ -TACAACTGTTTTGAAAATCCAGCGTGGAC-3′; and P6, 5′ -TCGAGTCATTGCATACTGTCC-3′. Using this approach, a small product including codons 30–48 (product P1-2) was amplified from all tumors for direct sequence analysis. Remaining PCR products were designed to screen for intragenic deletions beginning outside of the P1-2 primer, and including the β-catenin regulatory region of exon 3 (codons 32–45). Fragments >250 bp are not reliably amplified from paraffin tissue; therefore, overlapping fragments were chosen to increase the likelihood of detecting deletions. Because the P5-4 and P5-6 PCR products are too large for successful amplification in the absence of an intragenic deletion, DNA extracted from peripheral blood lymphocytes was included as a separate control for PCR set-up. Although this approach would be expected to identify most of the deletions reported previously in CTNNB1(11, 12, 19), it is not possible to rule out deletions in the absence of a full-length PCR product spanning P5-6. Therefore, the results may be an underestimate of the actual CTNNB1 intragenic deletion frequency. For each PCR reaction, 2 μl of DNA was combined with 1 unit of Perkin-Elmer AmpliTaq DNA polymerase in a 15-μl PCR mixture, with PCR buffer, 1.5 mm MgCl2, 0.13 mm deoxynucleotide triphosphates, and 0.4 μm of each primer. Samples were heat denatured at 94°C for 2 min, followed by 35 PCR cycles as follows: 94°C for 15 s, annealing temperature for 15 s, and 72°C for 20 s (in a DNA Engine, model PTC-200; MJ Research, Watertown, MA). The annealing temperatures were as follows: P1-2, 52°C; P3-4, 62°C; P5-4, 52°C; P3-6, 56°C; and P5-6, 46°C. To screen for intragenic deletions, PCR products (P3-4, P5-4, P3-6, and P5-6) were electrophoresed on 2% agarose gels and visualized by ethidium bromide staining. To detect nucleotide sequence alterations, the 98-bp P1-2 PCR product was electrophoresed on a 2% agarose gel, visualized by ethidium bromide staining, and excised from the gel, and DNA was purified using QIAquick Gel extraction kit (Qiagen, Mississauga, Ontario, Canada) as per the manufacturer’s instructions. Purified PCR products were sequenced using ThermoSequenase Radiolabeled Terminator Cycle Sequencing kit (Amersham, Cleveland, OH) as per the manufacturer’s instructions. Reactions were run on a 6% sequencing gel, dried onto filter paper, and exposed to Kodak Biomax film.

Statistical Methods.

Mutation frequencies, mutation profiles, and pathological parameters were compared by χ2 or Fisher exact test.

Mutations in the CTNNB1 gene were identified in 13 (25%; 95% CI, 14–39%) of the MSI-H colorectal carcinomas and none (95% CI, 0–10%) of the MSS/MSI-L colorectal carcinomas (P < 0.01; Figs. 1,B and 2; Table 1). All point mutations were amino acid substitutions at the threonine and serine residues at codons 41 and 45 (Fig. 2). A single 653-bp intragenic deletion was identified (Fig. 1,C) that joined nucleotide 27 of intron 2 to nucleotide 5 of intron 3, resulting in complete deletion of exon 3. CTNNB1 mutations predicting amino acid substitutions were identified in 3 (33%; 95% CI, 8–53%) of the MSI-H endometrial carcinomas and 10 (50%; 95% CI, 27–73%) of the MSS/MSI-L endometrial carcinomas (Fig. 2; Table 1). Only six of the endometrial carcinoma amino acid substitutions involved the serine and threonine residues at codons 33, 37, and 41 (Table 1). The remaining seven substitutions were at codons 32 and 34, flanking the serine at codon 33 (Table 1). No intragenic deletions were identified in the endometrial carcinomas. Silent nucleotide substitutions were also identified in two endometrial carcinomas (one MSI-H and one MSS; Table 1). These changes were not present in the normal tissues from these cases or in a separately dissected neoplastic sample, indicating that they were focal somatic alterations. All 16 of the nucleotide substitutions present in MSI-H cancers were transitions, whereas only 4 (36%) of the substitutions in MSS/MSI-L cancers were transitions (P < 0.01).

Adjacent adenoma samples were available from five MSI-H colorectal carcinomas with CTNNB1 mutations, and three of these contained the identical mutation as was present in the invasive component (Table 1). A second distinct histopathological subtype of invasive carcinoma was available in three MSI-H colorectal carcinomas with CTNNB1 mutations, and all three contained the same mutation in both components. Four endometrial carcinomas with CTNNB1 mutations predicting amino acid substitutions had an adjacent complex hyperplasia sample available, and two of these had the same mutation as was present in the invasive component. Both cases with mutation-positive complex hyperplasia were MSS, whereas the two cases with mutation-negative complex hyperplasia were MSI-H. Adjacent simple hyperplasia was also present in one of the cases with mutation-positive complex hyperplasia; however, no mutation was identified in this sample.

Comparison of pathological features (Table 2) revealed that MSI-H colorectal carcinomas with CTNNB1 mutations were more likely to be T4 tumors, right-sided, and of unusual histological subtypes. None of these trends were statistically significant. Irrespective of CTNNB1 mutation status, MSI-H colorectal carcinomas were more likely to be associated with right-sided location, marked tumor-infiltrating lymphocytes, marked Crohn’s-like lymphoid reactions, and nonmetastatic disease (Table 2). Although mutations had no apparent associations with pathological features in the endometrial carcinomas, the number of cases was too small for a comprehensive analysis (data not shown).

The CTNNB1 mutation frequency in MSI-H colorectal cancers (25%; 95% CI, 14–39%) compared with MSS/MSI-L colorectal cancers (0%; 95% CI, 0–10%) suggests that CTNNB1 mutations are relatively specific to the MMR-deficient genomic instability pathway. Although this difference is quite striking, it is perhaps not entirely unexpected. Activating β-catenin mutations are present in up to 50% of colorectal carcinomas with wild-type APC (9, 11, 12), and several studies have suggested that MSI-H colorectal cancers are less likely to have APC mutations (6, 22, 23). Furthermore, the frequency identified in our study is similar to previous observations of CTNNB1 mutations in selected MSI-H colorectal carcinoma cell lines (12). This contrasts the findings in primary melanomas, where CTNNB1 mutation frequencies are much lower than predicted from mutation screens of melanoma cell lines and suggests that this latter discrepancy could reflect differences in underlying genomic instability pathways (13, 24). The existence of MSI-L colorectal carcinomas as a distinct pathogenetic entity is controversial (20), and the absence of activating β-catenin mutations suggests that the pathogenesis of this group of tumors is quite unlike MSI-H cancers.

Endometrial carcinoma is the second most common cancer in individuals with germ-line MMR gene mutations, suggesting that the molecular pathogenesis has similarities with colorectal carcinoma. In endometrial cancers, however, we found that CTNNB1 mutations were not specific to the MMR-deficient pathway. Furthermore, the high mutation frequency raises the possibility that activation of Wnt signaling may be universally important in a subset of these neoplasms. Although previous studies have found a low frequency of loss of heterozygosity at 5q21 in endometrial carcinomas, we are not aware of any rigorous searches to identify APC mutations (25, 26).

CTNNB1 mutations were reported previously in only 13% of endometrial carcinomas (16). Most of our cancers were well to moderately differentiated endometrioid carcinomas, a subtype that had a higher CTNNB1 mutation frequency (18%) and a higher frequency of β-catenin stabilization (46%) in the previous study (16). Most CTNNB1 mutations reported in ovarian carcinomas are also found in endometrioid carcinomas, supporting a specific morphogenetic association (17). Our cases were also selected for early age of onset, which could be associated with differences in tumor genetic profiles. Finally, the mutation frequencies could reflect differences in carcinogenic influences between Japan and North America. For instance, intragenic deletions of CTNNB1 were the predominant mutations reported in one series of Japanese colorectal cancers (11).

The difference in the spectrum of missense mutations between colorectal and endometrial carcinomas was unexpected. All of the colorectal carcinoma amino acid substitutions were at known phosphorylation sites (codons 41 and 45), and three of the four mutations have been reported previously (10, 12, 13). Although the final substitution, T41I, has not been described in colorectal carcinoma, it was present in one endometrial cancer (16) and in carcinogen-induced rodent tumors (27, 28). Several mutations at codons 41 and 45 have been shown to lead to β-catenin stabilization (9, 10, 13, 16, 17). In contrast, only 1 of 13 substitutions in the endometrial cancers (T41S; previously unreported) occurred at either codons 41 or 45. The remaining 12 substitutions occurred at codons 32, 33, 34, and 37 and included three novel mutations (D32H, S33C, and G34R). Although codons 32 and 34 are not known to be phosphorylated, mutations at these sites are observed frequently in human and carcinogen-induced rat tumors (16, 17, 18, 19, 28, 29) and may affect phosphorylation by altering recognition sequences or tertiary protein structure. Furthermore, these two residues are important for β -catenin ubiquitination and proteasome-dependent degradation (30).

It is possible that the DNA sequence surrounding codons 41 to 45 of CTNNB1 is specifically hypermutable in the setting of MMR deficiency. The predominance of transition mutations supports this possibility and parallels the mutation profile of some MMR-deficient cell lines (31). Although codon 45 lies within a homocopolymer tract, which is known to be hypermutable (32), the specific sequences that predispose to transitions in the setting of MMR are not known (33). The presence of CTNNB1 mutations at codons 41 and 45 in small MSS colorectal adenomas (34) and the differences in the mutation profile of MSI-H endometrial cancers, however, suggest that sequence susceptibility is not the only factor. The presence of two silent CTNNB1 exon 3 alterations in the endometrial carcinomas also raises the possibility of a tissue-specific hypermutability or carcinogenic influence. An alternative explanation for the MSI pathway specificity of CTNNB1 mutations in colorectal cancer is that APC mutations and CTNNB1 mutations may not be biologically equivalent in tumorigenesis. Both genes may have functions outside of Wnt signaling that are important for clonal selection, and differences in the genetic targets of neoplastic progression between MSI-H and MSS colorectal cancers and between MSI-H colorectal and endometrial cancers are well established (6, 23, 35, 36). The recent finding that CTNNB1 mutations are frequent in small MSS adenomas suggests these neoplasms are less likely to progress and supports tumorigenic differences compared with APC inactivation (34).

Adjacent adenomas contained the same CTNNB1 mutation as the invasive carcinomas in three of five cases. Although this is generally consistent with the hypothesized role of the APC pathway as a neoplasia gatekeeper (6), at least two of the CTNNB1 mutations occurred during neoplastic progression from adenoma to carcinoma. In the endometrial carcinomas, we also found that two of the adjacent complex hyperplasias did not contain CTNNB1 mutations, suggesting that these alterations are not necessarily gatekeeper-type events. When multiple paired tumor samples were examined, however, the uniform presence of CTNNB1 mutations in both colorectal and endometrial cancers suggests that all have occurred during the early stages of malignant transformation. This contrasts findings in prostate cancer, where focal mutations suggest a late event during the advanced progression of subclones (18).

If activating β-catenin mutations are associated with specific biological attributes not present in tumors with APC inactivation, differences in the pathological features of these tumors might be expected. Although there was a tendency for MSI-H colorectal carcinomas with CTNNB1 mutations to be right-sided, higher stage, and of unusual histological subtypes, the number of cancers analyzed does not allow for definitive conclusions to be made. In comparison, many of the features known to be associated with MSI-H colorectal carcinomas (37) were uniformly present in tumors with and without CTNNB1 mutations. Although no associations between CTNNB1 mutations and pathological features of endometrial carcinomas were apparent, our series was very homogeneous in terms of the spectrum of grade, stage, and histological subtypes.

In summary, our findings confirm that CTNNB1 mutations are particularly common in MSI-H colorectal carcinomas. These mutations consist almost entirely of transitions at codons 41 and 45, revealing a relatively specific molecular fingerprint compared with CTNNB1 mutation profiles in other cancers. We also found that CTNNB1 mutations are very common in endometrial carcinomas; however, there is no association with the presence or absence of underlying microsatellite instability. Additional studies will be required to determine whether functional differences between β -catenin activation and APC inactivation may explain some of these findings.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

      
1

This research was supported in part by the National Cancer Institute of Canada (to E. H., B. B., S. G., and M. R.) with funds provided by the Canadian Cancer Society. R. G. is a Research Fellow of the National Cancer Institute of Canada with funds provided by the Terry Fox Run.

            
3

The abbreviations used are: APC, adenomatous polyposis coli; MMR, mismatch repair; MSI, microsatellite instability; MSI-L, low frequency MSI; MSI-H, high frequency MSI; CI, confidence interval.

Fig. 1.

Identification of CTNNB1 mutations. A, PCR strategy for CTNNB1 mutation detection. Primer pairs used to generate each of the five PCR products are as indicated. B, representative sequencing gel revealing point mutations in CTNNB1 exon 3. Colorectal carcinoma sample 54 (Lane 5) has an A to G substitution (S45P), and colorectal carcinoma sample 56 (Lane 6) has a G to A substitution (S45F). Sequencing reactions were run using the reverse primer P2. C, representative 2% agarose gel revealing intragenic deletion of CTNNB1 in PCR fragment P5-6. Carcinoma sample 128 (Lane 4, Del) has a 653-bp deletion. The deletion is not present in the adjacent adenoma sample (Lane 3). The full-length normal P5–6 PCR product is not successfully amplified from either of the paraffin DNA samples (Lanes 3 and 4). Lanes 1 and 6, 123-bp ladder; Lane 2, negative control; Lane 5 (Norm), full-length P5-6 PCR fragment from DNA extracted from peripheral blood lymphocytes.

Fig. 1.

Identification of CTNNB1 mutations. A, PCR strategy for CTNNB1 mutation detection. Primer pairs used to generate each of the five PCR products are as indicated. B, representative sequencing gel revealing point mutations in CTNNB1 exon 3. Colorectal carcinoma sample 54 (Lane 5) has an A to G substitution (S45P), and colorectal carcinoma sample 56 (Lane 6) has a G to A substitution (S45F). Sequencing reactions were run using the reverse primer P2. C, representative 2% agarose gel revealing intragenic deletion of CTNNB1 in PCR fragment P5-6. Carcinoma sample 128 (Lane 4, Del) has a 653-bp deletion. The deletion is not present in the adjacent adenoma sample (Lane 3). The full-length normal P5–6 PCR product is not successfully amplified from either of the paraffin DNA samples (Lanes 3 and 4). Lanes 1 and 6, 123-bp ladder; Lane 2, negative control; Lane 5 (Norm), full-length P5-6 PCR fragment from DNA extracted from peripheral blood lymphocytes.

Close modal
Fig. 2.

Summary of mutations stratified by underlying genomic instability pathway (left panel). Upper half of figure, endometrial carcinomas; lower half of figure, colorectal carcinomas. Right panel, distribution of β-catenin amino acid substitutions. Upper half, endometrial carcinomas; lower half, colorectal carcinoma mutations. β-Catenin is given in one-letter amino acid codes; serine and threonine phosphorylation residues are indicated by codon number. Underlined letters, mutations found in MSS/MSI-L cancers. Silent mutations are italicized.

Fig. 2.

Summary of mutations stratified by underlying genomic instability pathway (left panel). Upper half of figure, endometrial carcinomas; lower half of figure, colorectal carcinomas. Right panel, distribution of β-catenin amino acid substitutions. Upper half, endometrial carcinomas; lower half, colorectal carcinoma mutations. β-Catenin is given in one-letter amino acid codes; serine and threonine phosphorylation residues are indicated by codon number. Underlined letters, mutations found in MSS/MSI-L cancers. Silent mutations are italicized.

Close modal
Table 1

CTNNB1 mutationsa

Case NumberMorphologyMSI LocibMutationAmino AcidType
Colorectal neoplasms      
 54 CA, glandular 4 of 5 TCT-CCT S45P Transition 
 56 CA, glandular 2 of 5 TCT-TTT S45F Transition 
 ″ Adenoma, tubulovillous nt TCT-TTT S45F Transition 
 131 CA, mucinous 5 of 5 TCT-CCT S45P Transition 
 147 CA, cribriform 5 of 5 TCT-CCT S45P Transition 
 ″ Adenoma, villous nt TCT-CCT S45P Transition 
 175 CA, signet ring 5 of 5 ACC-GCC T41A Transition 
 ″ CA, undifferentiated 5 of 5 ACC-GCC T41A Transition 
 ″ Adenoma, tubulovillous nt ACC WT  
 195 CA, glandular 5 of 5 TCT-CCT S45P Transition 
 200 CA, glandular 4 of 5 ACC-ATC T41I Transition 
 ″ Adenoma, tubulovillous nt ACC-ATC T41I Transition 
 218 CA, undifferentiated 5 of 5 ACC-GCC T41A Transition 
 222 CA, glandular 3 of 5 ACC-GCC T41A Transition 
 ″ CA, mucinous 3 of 5 ACC-GCC T41A Transition 
 295 CA, undifferentiated 3 of 5 TCT-TTT S45F Transition 
 307 CA, mucinous 2 of 5 ACC-GCC T41A Transition 
 ″ CA, cribriform 2 of 5 ACC-GCC T41A Transition 
 257 CA, glandular 2 of 5 TCT-TTT S45F Transition 
 128 CA, glandular 5 of 5 653 bp deletion deletion Deletion 
 ″ Adenoma, tubulovillous nt WT WT  
Endometrial neoplasms      
 15 CA, undifferentiated 5 of 5 GAC-AAC D32N Transition 
 ″ Hyperplasia, complex nt GAC WT  
 30 CA, endometrioid 3 of 5 ACC-ACT T41T silent Transition 
 ″ Hyperplasia, complex nt ACC WT  
 57 CA, endometrioid 2 of 5 TCT-CCT S33P Transition 
 62 CA, endometrioid 3 of 5 GAC-AAC D32N Transition 
 ″ Hyperplasia, complex nt GAC WT  
 13 CA, endometrioid 0 of 5 GGA-AGA G34R Transition 
 16 CA, endometrioidc 0 of 5 ACC-TCC T41S Transversion 
  0 of 5 CTG-CTT L31L silent Transversion 
 ″ CA, endometrioidd nt ACC-TCC T41S Transversion 
 18 CA, endometrioid 0 of 5 TCT-TGT S33C Transversion 
 27 CA, endometrioid 0 of 5 GAC-CAC D32H Transversion 
 38 CA, endometrioid 0 of 5 GGA-GTA G34V Transversion 
 43 CA, endometrioid 0 of 5 TCT-TTT S37F Transition 
 45 CA, endometrioid 0 of 5 TCT-TTT S37F Transition 
 48 CA, endometrioid 0 of 5 GAC-TAC D32Y Transversion 
 ″ CA, squamous nt GAC-TAC D32Y Transversion 
 ″ Hyperplasia, complex nt GAC-TAC D32Y Transversion 
 49 CA, endometrioid 0 of 5 GGA-GAA G34E Transition 
 ″ Hyperplasia, complex nt GGA-GAA G34E Transition 
 ″ Hyperplasia, simple nt GGA WT  
 54 CA, endometrioid 0 of 5 TCT-TGT S37C Transversion 
Case NumberMorphologyMSI LocibMutationAmino AcidType
Colorectal neoplasms      
 54 CA, glandular 4 of 5 TCT-CCT S45P Transition 
 56 CA, glandular 2 of 5 TCT-TTT S45F Transition 
 ″ Adenoma, tubulovillous nt TCT-TTT S45F Transition 
 131 CA, mucinous 5 of 5 TCT-CCT S45P Transition 
 147 CA, cribriform 5 of 5 TCT-CCT S45P Transition 
 ″ Adenoma, villous nt TCT-CCT S45P Transition 
 175 CA, signet ring 5 of 5 ACC-GCC T41A Transition 
 ″ CA, undifferentiated 5 of 5 ACC-GCC T41A Transition 
 ″ Adenoma, tubulovillous nt ACC WT  
 195 CA, glandular 5 of 5 TCT-CCT S45P Transition 
 200 CA, glandular 4 of 5 ACC-ATC T41I Transition 
 ″ Adenoma, tubulovillous nt ACC-ATC T41I Transition 
 218 CA, undifferentiated 5 of 5 ACC-GCC T41A Transition 
 222 CA, glandular 3 of 5 ACC-GCC T41A Transition 
 ″ CA, mucinous 3 of 5 ACC-GCC T41A Transition 
 295 CA, undifferentiated 3 of 5 TCT-TTT S45F Transition 
 307 CA, mucinous 2 of 5 ACC-GCC T41A Transition 
 ″ CA, cribriform 2 of 5 ACC-GCC T41A Transition 
 257 CA, glandular 2 of 5 TCT-TTT S45F Transition 
 128 CA, glandular 5 of 5 653 bp deletion deletion Deletion 
 ″ Adenoma, tubulovillous nt WT WT  
Endometrial neoplasms      
 15 CA, undifferentiated 5 of 5 GAC-AAC D32N Transition 
 ″ Hyperplasia, complex nt GAC WT  
 30 CA, endometrioid 3 of 5 ACC-ACT T41T silent Transition 
 ″ Hyperplasia, complex nt ACC WT  
 57 CA, endometrioid 2 of 5 TCT-CCT S33P Transition 
 62 CA, endometrioid 3 of 5 GAC-AAC D32N Transition 
 ″ Hyperplasia, complex nt GAC WT  
 13 CA, endometrioid 0 of 5 GGA-AGA G34R Transition 
 16 CA, endometrioidc 0 of 5 ACC-TCC T41S Transversion 
  0 of 5 CTG-CTT L31L silent Transversion 
 ″ CA, endometrioidd nt ACC-TCC T41S Transversion 
 18 CA, endometrioid 0 of 5 TCT-TGT S33C Transversion 
 27 CA, endometrioid 0 of 5 GAC-CAC D32H Transversion 
 38 CA, endometrioid 0 of 5 GGA-GTA G34V Transversion 
 43 CA, endometrioid 0 of 5 TCT-TTT S37F Transition 
 45 CA, endometrioid 0 of 5 TCT-TTT S37F Transition 
 48 CA, endometrioid 0 of 5 GAC-TAC D32Y Transversion 
 ″ CA, squamous nt GAC-TAC D32Y Transversion 
 ″ Hyperplasia, complex nt GAC-TAC D32Y Transversion 
 49 CA, endometrioid 0 of 5 GGA-GAA G34E Transition 
 ″ Hyperplasia, complex nt GGA-GAA G34E Transition 
 ″ Hyperplasia, simple nt GGA WT  
 54 CA, endometrioid 0 of 5 TCT-TGT S37C Transversion 
a

CA, carcinoma; nt, not tested; WT, wild type; ", additional sample from same tumor, either discreet focus with different morphology or adjacent adenoma/hyperplasia.

b

Number of loci with MSI of the total number tested.

c

Two different CTNNB1 mutations were identified in this tumor.

d

Tumor sample dissected from a separate block.

Table 2

Pathological characteristics of colorectal neoplasms

MSS/MSI-LMSI-H
CTNNB1 wild-typeCTNNB1 mutated
Site (right side) 8 (30) 25 (63)a 11 (85)a 
Tumor stage    
 T1– 2 1 (4) 6 (15) 
 T3 25 (93) 31 (78) 9 (69) 
 T4 1 (4) 3 (8) 4 (31) 
Histology    
 Glandular 19 (70) 26 (65) 6 (46) 
 Mucinous 6 (22) 8 (20) 4 (31) 
 Signet ring 2 (5) 1 (8) 
 Undifferentiated 2 (7) 4 (10) 2 (15) 
Grade    
 Well differentiated 6 (22) 4 (10) 1 (8) 
 Moderately differentiated 10 (37) 16 (40) 6 (46) 
 Poorly differentiated 11 (41) 20 (50) 6 (46) 
Tumor-infiltrating lymphocytes 3 (11) 20 (50)a 9 (69)a 
Crohn’s-like lymphoid reaction 9 (33) 26 (67)b,c 8 (62) 
Adjacent adenoma (present) 5 (19) 13 (33) 6 (46) 
Lymph node metastasis (present) 17 (63) 14 (35)b 3 (25)b,d 
Distant metastasis (present) 8 (30) 4 (10)b 1 (8) 
MSS/MSI-LMSI-H
CTNNB1 wild-typeCTNNB1 mutated
Site (right side) 8 (30) 25 (63)a 11 (85)a 
Tumor stage    
 T1– 2 1 (4) 6 (15) 
 T3 25 (93) 31 (78) 9 (69) 
 T4 1 (4) 3 (8) 4 (31) 
Histology    
 Glandular 19 (70) 26 (65) 6 (46) 
 Mucinous 6 (22) 8 (20) 4 (31) 
 Signet ring 2 (5) 1 (8) 
 Undifferentiated 2 (7) 4 (10) 2 (15) 
Grade    
 Well differentiated 6 (22) 4 (10) 1 (8) 
 Moderately differentiated 10 (37) 16 (40) 6 (46) 
 Poorly differentiated 11 (41) 20 (50) 6 (46) 
Tumor-infiltrating lymphocytes 3 (11) 20 (50)a 9 (69)a 
Crohn’s-like lymphoid reaction 9 (33) 26 (67)b,c 8 (62) 
Adjacent adenoma (present) 5 (19) 13 (33) 6 (46) 
Lymph node metastasis (present) 17 (63) 14 (35)b 3 (25)b,d 
Distant metastasis (present) 8 (30) 4 (10)b 1 (8) 
a

Versus MSS/MSI-L, P < 0.01.

b

Versus MSS/MSI-L, P < 0.05.

c

Only 39 cases were available for this analysis.

d

Only 12 cases were available for this analysis.

We thank Kazy Hay for assistance in acquiring paraffin blocks and Eugene Hsieh for assistance in analysis of data and preparation of the manuscript.

1
Nusse R., Varmus H. E. Many tumors induced by the mouse mammary tumor virus contain a provirus integrated in the same region of the host genome.
Cell
,
31
:
99
-109,  
1982
.
2
Rijsewijk F., Schuermann M., Wagenaar E., Parren P., Weigel D., Nusse R. The Drosophila homolog of the mouse mammary oncogene int-1 is identical to the segment polarity gene wingless.
Cell
,
50
:
649
-657,  
1987
.
3
Rubinfeld B., Souza B., Albert I., Muller O., Chamberlain S. H., Masiarz F. R., Munemitsu S., Polakis P. Association of the APC gene product with β -catenin.
Science (Washington DC)
,
262
:
1731
-1734,  
1993
.
4
Su L. K., Vogelstein B., Kinzler K. W. Association of the APC tumor suppressor protein with catenins.
Science (Washington DC)
,
262
:
1734
-1737,  
1993
.
5
Rubinfeld B., Albert I., Porfiri E., Fiol C., Munemitsu S., Polakis P. Binding of GSK3β to the APC-β-catenin complex and regulation of complex assembly.
Science (Washington DC)
,
272
:
1023
-1026,  
1996
.
6
Kinzler K. W., Vogelstein B. Lessons from hereditary colorectal cancer.
Cell
,
87
:
159
-170,  
1996
.
7
Korinek V., Barker N., Morin P. J., van Wichen D., de Weger R., Kinzler K. W., Vogelstein B., Clevers H. Constitutive transcriptional activation by a β-catenin-Tcf complex in APC−/− colon carcinoma.
Science (Washington DC)
,
275
:
1784
-1787,  
1997
.
8
Rubinfeld B., Albert I., Porfiri E., Munemitsu S., Polakis P. Loss of β-catenin regulation by the APC tumor suppressor protein correlates with loss of structure due to common somatic mutations of the gene.
Cancer Res.
,
57
:
4624
-4630,  
1997
.
9
Ilyas M., Tomlinson I. P., Rowan A., Pignatelli M., Bodmer W. F. β-Catenin mutations in cell lines established from human colorectal cancers.
Proc. Natl. Acad. Sci. USA
,
94
:
10330
-10334,  
1997
.
10
Morin P. J., Sparks A. B., Korinek V., Barker N., Clevers H., Vogelstein B., Kinzler K. W. Activation of β-catenin-Tcf signaling in colon cancer by mutations in β-catenin or APC.
Science (Washington DC)
,
275
:
1787
-1790,  
1997
.
11
Iwao K., Nakamori S., Kameyama M., Imaoka S., Kinoshita M., Fukui T., Ishiguro S., Nakamura Y., Miyoshi Y. Activation of the β-catenin gene by interstitial deletions involving exon 3 in primary colorectal carcinomas without adenomatous polyposis coli mutations.
Cancer Res.
,
58
:
1021
-1026,  
1998
.
12
Sparks A. B., Morin P. J., Vogelstein B., Kinzler K. W. Mutational analysis of the APC/β-catenin/Tcf pathway in colorectal cancer.
Cancer Res.
,
58
:
1130
-1134,  
1998
.
13
Rubinfeld B., Robbins P., El-Gamil M., Albert I., Porfiri E., Polakis P. Stabilization of β-catenin by genetic defects in melanoma cell lines.
Science (Washington DC)
,
275
:
1790
-1792,  
1997
.
14
Kitaeva M. N., Grogan L., Williams J. P., Dimond E., Nakahara K., Hausner P., DeNobile J. W., Soballe P. W., Kirsch I. R. Mutations in β-catenin are uncommon in colorectal cancer occurring in occasional replication error-positive tumors.
Cancer Res.
,
57
:
4478
-4481,  
1997
.
15
Muller O., Nimmrich I., Finke U., Friedl W., Hoffmann I. A β-catenin mutation in a sporadic colorectal tumor of the RER phenotype and absence of β-catenin germline mutations in FAP patients.
Genes Chromosomes Cancer
,
22
:
37
-41,  
1998
.
16
Fukuchi T., Sakamoto M., Tsuda H., Maruyama K., Nozawa S., Hirohashi S. β-Catenin mutation in carcinoma of the uterine endometrium.
Cancer Res.
,
58
:
3526
-3528,  
1998
.
17
Palacios J., Gamallo C. Mutations in the β-catenin gene (CTNNB1) in endometrioid ovarian carcinomas.
Cancer Res.
,
58
:
1344
-1347,  
1998
.
18
Voeller H. J., Truica C. I., Gelmann E. P. β-Catenin mutations in human prostate cancer.
Cancer Res.
,
58
:
2520
-2523,  
1998
.
19
Koch A., Denkhaus D., Albrecht S., Leuschner I., von Schweinitz D., Pietsch T. Childhood hepatoblastomas frequently carry a mutated degradation targeting box of the β-catenin gene.
Cancer Res.
,
59
:
269
-273,  
1999
.
20
Boland C. R., Thibodeau S. N., Hamilton S. R., Sidransky D., Eshleman J. R., Burt R. W., Meltzer S. J., Rodriguez-Bigas M. A., Fodde R., Ranzani G. N., Srivastava S. A National Cancer Institute Workshop on Microsatellite Instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer.
Cancer Res.
,
58
:
5248
-5257,  
1998
.
21
Bapat B. V., Madlensky L., Temple L. K. F., Hiruki T., Redston M., Baron D. L., Xia L., Marcus V. A., Soravia C., Mitri A., Shen W., Gryfe R., Berk T., Chodirker B. N., Cohen Z., Gallinger S. Family history characteristics, tumor microsatellite instability and germline MSH2 and MLH1 mutations in hereditary colorectal cancer.
Hum. Genet.
,
104
:
167
-176,  
1999
.
22
Heinen C. D., Richardson D., White R., Groden J. Microsatellite instability in colorectal adenocarcinoma cell lines that have full-length adenomatous polyposis coli protein.
Cancer Res.
,
55
:
4797
-4799,  
1995
.
23
Olschwang S., Hamelin R., Laurent-Puig P., Thuille B., De Rycke Y., Li Y. J., Muzeau F., Girodet J., Salmon R. J., Thomas G. Alternative genetic pathways in colorectal carcinogenesis.
Proc. Natl. Acad. Sci. USA
,
94
:
12122
-12127,  
1997
.
24
Rimm D. L., Caca K., Hu G., Harrison F. B., Fearon E. R. Frequent nuclear/cytoplasmic localization of β-catenin without exon 3 mutations in malignant melanoma.
Am. J. Pathol.
,
154
:
325
-329,  
1999
.
25
Jones M. H., Koi S., Fujimoto I., Hasumi K., Kato K., Nakamura Y. Allelotype of uterine cancer by analysis of RFLP and microsatellite polymorphisms: frequent loss of heterozygosity on chromosome arms 3p, 9q, 10q, and 17p.
Genes Chromosomes Cancer
,
9
:
119
-123,  
1994
.
26
Berchuck A., Boyd J. Molecular basis of endometrial cancer.
Cancer (Phila.)
,
76
:
2034
-2040,  
1995
.
27
de La Coste A., Romagnolo B., Billuart P., Renard C. A., Buendia M. A., Soubrane O., Fabre M., Chelly J., Beldjord C., Kahn A., Perret C. Somatic mutations of the β-catenin gene are frequent in mouse and human hepatocellular carcinomas.
Proc. Natl. Acad. Sci. USA
,
95
:
8847
-8851,  
1998
.
28
Takahashi M., Fukuda K., Sugimura T., Wakabayashi K. β-Catenin is frequently mutated and demonstrates altered cellular location in azoxymethane-induced rat colon tumors.
Cancer Res.
,
58
:
42
-46,  
1998
.
29
Dashwood R. H., Suzui M., Nakagama H., Sugimura T., Nagao M. High frequency of β-catenin (ctnnb1) mutations in the colon tumors induced by two heterocyclic amines in the F344 rat.
Cancer Res.
,
58
:
1127
-1129,  
1998
.
30
Aberle H., Bauer A., Stappert J., Kispert A., Kemler R. β-Catenin is a target for the ubiquitin-proteasome pathway.
EMBO J.
,
16
:
3797
-3804,  
1997
.
31
Malkhosyan S., McCarty A., Sawai H., Perucho M. Differences in the spectrum of spontaneous mutations in the hprt gene between tumor cells of the microsatellite mutator phenotype.
Mutat. Res.
,
316
:
249
-259,  
1996
.
32
Redston M. S., Caldas C., Seymour A. B., Hruban R. H., da Costa L., Yeo C. J., Kern S. E. p53 mutations in pancreatic carcinoma and evidence of common involvement of homocopolymer tracts in DNA microdeletions.
Cancer Res.
,
54
:
3025
-3033,  
1994
.
33
Greene C. N., Jinks-Robertson S. Frameshift intermediates in homopolymer runs are removed efficiently by yeast mismatch repair proteins.
Mol. Cell. Biol.
,
17
:
2844
-2850,  
1997
.
34
Samowitz W. S., Powers M. D., Spirio L. N., Nollet F., van Roy F., Slattery M. L. β-catenin mutations are more frequent in small colorectal adenomas than in larger adenomas and invasive carcinomas.
Cancer Res.
,
59
:
1442
-1444,  
1999
.
35
Lengauer C., Kinzler K. W., Vogelstein B. Genetic instabilities in human cancers.
Nature (Lond.)
,
396
:
643
-649,  
1998
.
36
Gurin C. C., Federici M. G., Kang L., Boyd J. Causes and consequences of microsatellite instability in endometrial carcinoma.
Cancer Res.
,
59
:
462
-466,  
1999
.
37
Kim H., Jen J., Vogelstein B., Hamilton S. R. Clinical and pathological characteristics of sporadic colorectal carcinomas with DNA replication errors in microsatellite sequences.
Am. J. Pathol.
,
145
:
148
-156,  
1994
.