Clinicopathological and molecular differences between right-sided and left-sided colorectal cancer in Japanese patients.

BACKGROUND: The aim of this study was to clarify clinicopathological features, frequencies of molecular biomarkers, and prognoses in Japanese colorectal cancer patients and compare them with right-sided colon cancer (RCC) and left-sided colorectal cancer (LCRC). METHODS: We consecutively selected 575 colorectal cancer patients who underwent surgical resection from 2008 to 2011. RCC was located from the cecum to the transverse colon, and LCRC was located from the splenic flexure to the rectum. Frequencies of KRAS gene mutation, BRAF gene mutation, microsatellite instability (MSI), l18qLOH and CpG island methylator phenotype (CIMP) were statistically analyzed between groups. RESULTS: Tumors were located in the RCC in 26.3% of patients and in the LCRC in 73.7%. Elderly patients, females and advanced diseases were significantly more frequent in the RCC group than in the LCRC group. However, venous invasion was significantly more frequent in LCRC than in RCC. Between groups, BRAF mutant type, KRAS mutant type, MSI and CIMP+ were significantly more frequent in RCC, whereas 18qLOH was significantly more frequent in LCRC. In overall survival, RCC demonstrated poor prognosis compared with LCRC; however, age, gender, stage, lymphatic invasion, KRAS status and BRAF status rather than tumor location were independent prognostic factors. In addition, the independent prognostic factors in RCC were different from those in LCRC in each stage. However, the consistency between OS and DFS was not observed in this study, excluding lymphatic invasion in LCRC. CONCLUSION: Comparing RCC with LCRC, RCC is different from LCRC in clinicopathological features, molecular biomarkers and prognostic factors in Japanese colorectal cancer patients. Since the proportions of molecular biomarkers of CRC in this study are different from Western CRCs, further studies are required to clarify the clinicopathological differences between Japanese CRCs and Western CRCs.

Colorectal Cancer with BRAF D594G Mutation Is Not Associated with Microsatellite Instability or Poor Prognosis.

OBJECTIVE: BRAF D594G mutations in colorectal cancer patients are not clearly understood. We retrospectively investigated the clinicopathological features of colorectal cancers with BRAF D594G mutations. METHODS: We selected 908 colorectal cancer patients who underwent surgical resection from January 2008 to January 2013, and assessed BRAF, KRAS, microsatellite instability, and CpG island methylator phenotype (CIMP). RESULTS: We detected BRAF D594G in 7 patients and BRAF V600E in 45 patients. The clinicopathological features of cancers with BRAF D594G mutation were similar to those with BRAF wild-type, but differed from those with BRAF V600E mutations. Regarding microsatellite instability status, 44.4% of cases with BRAF V600E mutations exhibited high microsatellite instability, compared to 14.3% of those with BRAF D594G mutations and 4.4% of those with BRAF wild-type. There were no CIMP-positive tumors in cancers with BRAF D594G mutations, whereas 67.8% of tumors with BRAF V600E mutations were CIMP-positive. In stage IV cancers, the survival rates of patients at 2 years were 8.5, 50.0, and 68.2% in the BRAF V600E mutation, BRAF D594G mutation, and BRAF wild-type groups, respectively. CONCLUSION: Colorectal cancers with BRAF D594G mutations exhibit similar clinicopathological features, microsatellite instability status, and prognosis as those with BRAF wild-type.

Underexpression of miR-126 and miR-20b in hereditary and nonhereditary colorectal tumors.

OBJECTIVES: The aim of the study was to determine the significance of miR-126 and miR-20b in colorectal carcinogenesis. METHODS: We analyzed the expressions of miR-126 and miR-20b in 136 colorectal tumors from 39 microsatellite stable (MSS) tumors, 23 high microsatellite instability (MSI-H) tumors, 16 Lynch syndrome, and 58 familial adenomatous polyposis (FAP) tumors including adenoma, intramucosal carcinoma, and invasive carcinoma. RESULTS: All four kinds of tumors showed underexpression of both miR-126 and miR-20b. The frequency of miR-126 downregulation was 100.0% in FAP adenomas, 85.7% in FAP intramucosal carcinomas, 78.9% in invasive carcinomas, 81.3% in Lynch syndrome tumors, 68.4% in MSS tumors, and 65.4% in MSI-H tumors. The frequency of miR-20b downregulation was 64.0% in FAP adenomas, 50.0% in FAP intramucosal carcinomas, 73.3% in invasive carcinomas, 62.5% in Lynch syndrome tumors, 79.5% in MSS tumors, and 91.3% in MSI-H tumors. The current study demonstrated underexpression of miR-126 and miR-20b in various types of colorectal cancer. These findings support the hypothesis that angiogenesis results from underexpressions of miR-126 and miR-20b and occurs as an early event in colorectal carcinogenesis. CONCLUSIONS: Underexpression of miR-126 and miR-20b was observed in various types of colorectal cancer, and occurs as an early event of colorectal carcinogenesis in FAP tumors.

A large deletion of chromosome 5q22.1-22.2 associated with sparse type of familial adenomatous polyposis: report of a case.

The proband was a 32-year-old man with sparse type of familial adenomatous polyposis with fundic gland and duodenal polyps and congenital hypertrophy of the retinal pigment epithelium without osteoma, dental abnormalities and desmoid tumors. Direct DNA sequencing did not detect germline mutations in any APC exon. However, using the multiplex ligation-dependent probe amplification method, we detected germline deletions of all APC exons. Using dual-color fluorescence in situ hybridization, we identified germline deletion of locus 5q22.1-22.2 that includes APC. Analysis of colorectal tumors identified somatic APC mutations in the cluster region in all polyps, but no loss of heterozygosity was detected in any polyp.

Somatic mutations of the CDC4 (FBXW7) gene in hereditary colorectal tumors.

OBJECTIVES: Cdc4 (Fbxw7) is a potential tumor suppressor that regulates ubiquitination and proteolysis of multiple targets such as cyclin E, c-Myc, c-Jun and Notch. CDC4 mutations were investigated in 194 colorectal carcinomas and adenomas for comparison between sporadic and hereditary cancers. METHODS: Mutations of the CDC4 gene were analyzed by PCR-SSCP and sequencing, and loss of heterozygosity (LOH) was analyzed by microsatellite marker analysis. RESULTS: Somatic CDC4 mutations were detected in 9% (3 of 33) of hereditary nonpolyposis colorectal cancer (HNPCC), 9% (3 of 33) of familial adenomatous polyposis (FAP) carcinomas, and 10% (7 of 73) of sporadic carcinomas. CDC4 mutations were also detected in adenomas at frequencies of 6% (2 of 31) and 4% (1 of 24) in FAP and sporadic cases, respectively. Frameshift mutations were observed in HNPCC tumors, while single-base substitutions predominantly occurred in FAP and sporadic tumors. LOH at the chromosome 4q region was rarely detected in tumors with CDC4 mutations. CONCLUSIONS: The results indicate that the frequency of CDC4 mutations in colorectal tumors is similar in patients with HNPCC and FAP compared to patients with sporadic carcinomas. Moreover, infrequent LOH suggests that the CDC4 gene does not follow the general 2-hit model.

Microsatellite instability is inversely associated with type 2 diabetes mellitus in colorectal cancer.

BACKGROUND: Microsatellite instability (MSI) is a clonal change in the number of repeated DNA nucleotide units in microsatellites. High-frequency MSI (MSI-H) colorectal cancers (CRCs) are known to have different clinicopathological features compared with microsatellite stable (MSS) CRCs. In addition, previous studies have shown that type2 diabetes mellitus (T2DM) is a risk factor for malignant tumors including CRCs. The aim of this study was to investigate the relationship between T2DM and MSI-H colorectal cancer. METHODS: The study design is a single center, cross-sectional study. Data from a series of 936 patients with CRCs were collected and MSI status was assessed. RESULTS: In total, 29 (3.1%) and 907 (96.9%) tumors were classified as having MSI-H and low-frequency microsatellite instability or being MSS (MSS), respectively. Of the 936 patients, 275 (29.6%) were associated with T2DM. One (3.4%) of the 29 MSI-H patients and 274 (30.2%) of the 907 MSS patients had T2DM. Thus, the incidence of T2DM was significantly less frequent in MSI-H compared with MSS patients (Fisher's exact test: p = 0.0007). CONCLUSIONS: We conclude that MSS tumors are significantly more common than MSI-H tumors among individuals with T2DM.

Difference in characteristics of APC mutations between colonic and extracolonic tumors of FAP patients: variations with phenotype.

To clarify the differences in characteristics of adenomatous polyposis coli (APC) mutations between colorectal tumors from various phenotypes of familial adenomatous polyposis (FAP) and between colorectal and extracolonic tumors, we analyzed APC mutations in 86 colorectal tumors from FAP patients including profuse, sparse and attenuated types, 23 sporadic colorectal tumors and 40 FAP extracolonic tumors including duodenal, gastric and desmoid tumors. In all tumors, 1 allele of the truncated APC gene retained armadillo repeats, 15-amino-acid (aa) repeats and various numbers of 20-aa repeats, but lacked SAMP repeats, as a result of germline and somatic mutations. Regarding 20-aa repeats, the truncated APC gene retained 1 repeat due to allele loss in 96% (27/28) of colorectal tumors from profuse-type FAP, 69% (36/52) of sparse-type retained 2 repeats due to somatic mutation, and 100% (6/6) of attenuated-type retained 2 or 3 repeats due to the third or second hit. In sporadic colorectal tumors 74% (17/23) retained 1 or 2 repeats. The truncated APC gene retained 3 repeats in 88% (7/8) of FAP duodenal tumors, 100% (26/26) of gastric tumors retained 2 or 3 repeats and 83% (5/6) of desmoid tumors retained 2 repeats. These data suggest that the number of beta-catenin downregulating 20-aa repeats in truncated APC gene associated with colorectal tumorigenesis is different in profuse, sparse and attenuated types of FAP, and that the association with tumorigenesis is also different between colorectal and extracolonic tumors.

High incidence of protein-truncating mutations of the p53 gene in liver metastases of colorectal carcinomas.

To clarify the significance of p53 mutations in liver metastasis of colorectal carcinogenesis, the characteristics of p53 mutations from 51 liver metastases and 76 primary invasive carcinomas without liver metastasis (Dukes' A, B and C) were compared. The frequency of tumors with p53 mutations was 61% (31 out of 51) in the liver metastases, and 51% (39 out of 76) in the primary carcinomas without liver metastasis. Approximately 90% of the informative cases having p53 mutation showed 17pLOH. Mutations detected within exons 4-10 of the p53 gene included missense, nonsense, frameshift, inframe deletion, and inframe insertion mutations. Out of the tumors with p53 mutations, we found that the percentage of tumors with protein-truncating mutations (nonsense and frameshift mutations) was extremely higher in liver metastases (16 out of 31, 52%) than in primary carcinomas without liver metastasis (5 out of 39, 13%) (P=0.0005). The present results suggest that protein-truncating mutations of the p53 gene are more relevant than missense mutations as one of the prognostic factors in liver metastasis of colorectal carcinomas.

Germline mutations of the MYH gene in Japanese patients with multiple colorectal adenomas.

Germline mutations of the MYH gene have been revealed to associate with the recessive inheritance of multiple colorectal adenomas in Caucasian population. However, MYH mutations in Japanese patients have not yet been clarified. In an assessment of MYH mutations, we examined 35 Japanese patients with multiple colorectal adenomas who had neither dominant inheritance of colorectal tumors, nor germline APC mutations. One patient had a homozygous biallelic MYH mutation, R231C and three independent patients had monoallelic MYH mutations at a splice-site on exon 11 (IVS10-2 A to G). These four patients had 21 to around 100 colorectal adenomas and 1-3 synchronous colorectal carcinomas. The most common mutations in Caucasian patients, Y165C and G382D, were not detected in our Japanese cases. The MYH mutations detected in Japanese patients were novel and different from those detected among Caucasian, Indian and Pakistani patients, which suggests the existence of ethnic differentiation in MYH mutations.

Both BRAF and KRAS mutations are rare in colorectal carcinomas from patients with hereditary nonpolyposis colorectal cancer.

The BRAF mutations have been suggested to be linked with defective mismatch repair in colorectal carcinomas. To clarify the extent of BRAF mutations in HNPCC colorectal carcinomas, which are typical mismatch repair deficient carcinomas, we compared the frequency of BRAF mutations between HNPCC, familial adenomatous polyposis (FAP) and sporadic cases. The frequency of KRAS mutations was also compared between these three syndromes. No BRAF mutations were detected in 33 HNPCC colorectal carcinomas, while they were detected in 3 of 26 (12%) FAP carcinomas and 2 of 53 (4%) microsatellite stable sporadic carcinomas. KRAS mutations were detected in 2 of 33 (6%) HNPCC, 9 of 26 (35%) FAP and 18 of 53 (34%) sporadic carcinomas. Such extremely low frequencies of BRAF and KRAS mutations in HNPCC colorectal carcinomas suggest that the participation of RAS-RAF signaling is minor in HNPCC, and that the previously suggested high frequency of BRAF mutations in mismatch repair deficient colorectal carcinomas is not due to mutations of mismatch repair genes.

Extended lymphadenectomy and preoperative radiotherapy for lower rectal cancers.

BACKGROUND: Extended lymphadenectomy including lateral node dissection (EXT-L) contributes to a low incidence of local recurrence of lower rectal cancer. However, EXT-L is frequently associated with impairment of sexual and urinary function. We therefore compared the effectiveness of preoperative radiotherapy with that of EXT-L. METHODS: One hundred fifteen patients were studied. Seventy-eight patients underwent preoperative radiotherapy with a total dose of 50 Gy (Rad[+] group), and 37 did not (Rad[-] group). Seventy-five patients received EXT-L (EXT-L[+] group), and 40 did not (EXT-L[-] group). Patients were further divided into 4 subgroups (Rad[+]&EXT-L[-], Rad(+)&EXT-L[+], Rad[-]&EXT-L(+), and Rad[-]&EXT-L[-]), and clinicopathologic features were examined. In the Rad(+) group, the relation between the p53 gene and survival was also examined. RESULTS: There was a significant difference in disease-free survival between the Rad(+) and Rad(-) groups (5-year disease-free survival rate, 74.6% vs 45.9%; P =.006). However, there was no significant difference between the Rad(+)&EXT-L[-] and Rad[-]&EXT-L(+) groups. The p53 gene status did not affect survival in the Rad(+) group. CONCLUSIONS: This study suggests that in terms of curative effect, preoperative radiotherapy can be one alternative therapy in place of EXT-L for patients with lower rectal cancer.

APC gene mutations in a jejunal adenoma causing intussusception in a patient with familial adenomatous polyposis.

Adenomatous polyps of the jejunum/ileum in patients with familial adenomatous polyposis (FAP) are usually small (<5 mm) and are considered to be of little clinical importance. Genetic alterations in these polyps have not previously been analyzed. We herein report an extremely rare case of FAP presenting with intussusception caused by jejunal adenomas. Both somatic and germline mutations of the APC gene were detected in one of the polyps. A 40-year-old man with FAP was admitted for closure of an ileostomy that had been created because of an anastomotic leak after subtotal proctocolectomy with ileo-anal-canal anastomosis. During the follow-up after that surgery, he had occasionally complained of colicky abdominal pain, but it had quickly subsided. At the second laparotomy, for closure of the ileostomy, jejuno-jejunal intussusception was incidentally found, and segmental resection of the jejunum, including the leading point of the intussusception, was performed. There were five polyps clustered in the resected jejunum. Histologically, the polyps, ranging from 5 to 26 mm in diameter, were adenomas with moderate to severe atypia. Genetic examinations of one of the largest polyps, using polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) and direct sequencing methods, revealed somatic (T insertion at codon 1557) and germline mutations (4 base-pair deletion at codons 181-182) of the APC gene. This is the first evidence that the coexistence of somatic and germline alterations in the APC gene is involved in the development of a jejunal adenoma causing small-bowel intussusception.

Association of K-ras mutations with liver metastases from colorectal carcinoma.

BACKGROUND: Colorectal carcinomas were studied regarding early proof of liver metastases through determination of K-ras mutations. PATIENTS AND METHODS: Seventy-seven colorectal carcinomas were investigated for the presence of point mutations in codon 12 and 13 of the K-ras gene, using single-strand conformation polymorphism (SSCP) analysis and direct sequencing. RESULTS: Twenty-six carcinomas were positive for K-ras mutations, of which 21 had codon 12 and 5 had codon 13 mutations. Twenty patients with K-ras-positive tumor (20 out of 26: 77%) developed liver metastases, of which 13 had simultaneous metastases and 7 had metachronous metastases. There was a significant association between K-ras mutations and liver metastases (p=0.03). A multivariate logistic regression model demonstrated that the involvement of lymph node (p<0.01) and K-ras mutations (p=0.02) were predictive factors for liver metastases from colorectal carcinoma. Sequencing in carcinomas without liver metastases showed the base change in the first position of codon 12, whereas with liver metastases it showed significantly frequent base change in the second position of codon 12 (p<0.01). CONCLUSION: It is suggested that the presence of K-ras mutation, especially base change in the second position of codon 12, may predict liver metastases from colorectal carcinoma.

Novel germline hMSH2 genomic deletion and somatic hMSH2 mutations in a hereditary nonpolyposis colorectal cancer family.

Germline and somatic mutations of the hMSH2 gene were determined in a Japanese hereditary nonpolyposis colorectal cancer (HNPCC) family fulfilling the Amsterdam criteria. PCR-SSCP-sequencing of genomic DNA detected a somatic hMSH2 mutation of an A deletion at codon 227-229 in a duodenal carcinoma and a somatic hMSH2 mutation of an A insertion at codon 21 in a gastric carcinoma from affected family members, both carcinomas exhibiting high microsatellite instability. However, no germline hMSH2 mutation was detected by the PCR-SSCP-sequencing method. Genomic DNA was then analyzed by Southern blot hybridization using three hMSH2 cDNA probes (probe A involving exons 1-5, probe B involving exons 4-11 and probe C involving exons 9-16) after digestion by restriction enzymes, EcoRI, HindIII and NsiI. The NsiI digest of DNA from normal tissues of affected members exhibited an aberrant 8.6 kb restriction fragment, in addition to the normal 10.6 kb fragment, when hybridized to probes A and B. This suggested the presence of a heterozygous 2kb genomic deletion encompassing exon 4, 5 or 6. RT-PCR-sequencing revealed that the deleted region encompassed exon 5. This novel genomic deletion of the hMSH2 gene was confirmed to be pathogenic, and the Southern hybridization pattern was applied to the pre-symptomatic diagnosis.

Immunohistochemistry and c-kit gene analysis in determining malignancy in gastrointestinal stromal tumors.

BACKGROUND/AIMS: Usefulness of immunohistochemistry and c-kit proto-oncogene mutation was examined for determining malignancy in 24 cases of gastrointestinal stromal tumors. METHODOLOGY: Cases were histologically diagnosed and subjected to immunohistochemical staining and c-kit gene analysis. All parameters were compared to prognosis. RESULTS: There were significant differences in tumor size, central necrosis, mitotic activity and histological diagnosis between recurrent and non-recurrent cases. Positivity to KIT staining was 100% in recurrent and 87.5% in non-recurrent cases. Positivity to Ki-67 and p53 staining were significantly higher in recurrent cases than in non-recurrent cases. Mutations in exon 11 of the c-kit gene were significantly more frequent in recurrent cases than in non-recurrent cases. CONCLUSIONS: Histological diagnosis, tumor size, central necrosis, and mitotic activity were reconfirmed to be indicators for recurrence of gastrointestinal stromal tumors. Furthermore, it is suggested that positivity of Ki-67 and p53 immunostaining and c-kit gene mutation also need to be done for prediction of recurrence.

Mutations of the PIK3CA gene in hereditary colorectal cancers.

Somatic mutations of the PIK3CA gene have recently been detected in various human cancers, including sporadic colorectal cancer. However, mutations of the PIK3CA gene in hereditary colorectal cancers have not been clarified. To elucidate the mutation status in familial adenomatous polyposis (FAP) and hereditary nonpolyposis colorectal cancer (HNPCC), which are the most common hereditary colorectal cancers, we investigated PIK3CA mutations in 163 colorectal tumors, including adenomas, intramucosal carcinomas and invasive carcinomas. For comparison, we also analyzed mutations of the same gene in 160 sporadic colorectal tumors at various histopathological stages. Analysis at exons 1, 7, 9 and 20 of the PIK3CA gene revealed somatic mutations in 21% (8 of 39) of FAP invasive carcinomas, 21% (7 of 34) of HNPCC invasive carcinomas, 15% (8 of 52) of sporadic invasive carcinomas, and 14% (7 of 50) of sporadic colorectal metastases in the liver. Mutations in FAP and HNPCC carcinomas predominantly occurred in the kinase domain (exon 20), while the majority of mutations in sporadic cases occurred in the helical domain (exon 9). Adenomas and intramucosal carcinomas from all patients exhibited no mutations (0 of 148). Our data suggest that PIK3CA mutations contribute to the invasion step from intramucosal carcinoma to invasive carcinoma in colorectal carcinogenesis in FAP and HNPCC patients at a similar extent to that seen in sporadic patients.

Difference in the role of loss of heterozygosity at 10p15 (KLF6 locus) in colorectal carcinogenesis between sporadic and familial adenomatous polyposis and hereditary nonpolyposis colorectal cancer patients.

OBJECTIVES: To clarify the role of the KLF6 (Kruppel-like factor 6) locus in multistep colorectal carcinogenesis, we analyzed loss of heterozygosity (LOH) at 10p15 (KLF6 locus) and mutations of the KLF6gene in 298 colorectal tumors at various pathological stages of sporadic and familial adenomatous polyposis (FAP) and hereditary nonpolyposis colorectal cancer (HNPCC) patients. METHODS: 10p15 LOH was analyzed using KLF6M1 and KLF6M2, and KLF6 gene mutation was analyzed using PCR-SSCP and sequencing. RESULTS: It was found that the frequencies of LOH (sum of M1 and M2) were 4% in adenomas, 0% in intramucosal carcinomas, 35% in invasive carcinomas, and 33% in liver metastases in sporadic cases. Invasive carcinomas from FAP patients showed only 6% LOH, and invasive carcinomas from HNPCC patients exhibited 0% LOH. Mutation analysis of the KLF6 gene in 298 colorectal tumors detected no somatic mutations. CONCLUSIONS: The present data suggest that LOH of the KLF6 locus at chromosome 10p15 contributes to the invasion step from an intramucosal carcinoma to an invasive carcinoma specifically in sporadic colorectal carcinogenesis, but is rarely involved in the carcinogenesis of FAP and HNPCC cases. Moreover, the absence of somatic mutations suggests the uncertainty of the KLF6 gene as a classical tumor suppressor gene at the lost 10p15 region in colorectal carcinogenesis.

Accumulation profile of frameshift mutations during development and progression of colorectal cancer from patients with hereditary nonpolyposis colorectal cancer.

PURPOSE: Role and timing of frameshift mutations during carcinogenesis in hereditary nonpolyposis colorectal cancer have not been examined. This study was designed to clarify the relationship between frameshift mutations and clinicopathologic features in colorectal cancer from patients with hereditary nonpolyposis colorectal cancer. METHODS: Thirty-one colorectal cancers from patients with hereditary nonpolyposis colorectal cancer at different clinicopathologic stages were analyzed for frameshift mutation in 18 genes. RESULTS: The frameshift mutations of the ACVR2 and PTHLH genes were found to have an extremely high frequency (94-100 percent) in all pathologic stages, and mutation of the MARCKS gene also was high (94 percent) in Dukes B and C cancers. These frequencies were higher than the frequency of TGFbetaRII gene inactivation (64-88 percent). Mutations of the hMSH3, TCF4, CASP5, RIZ, RAD50, and MBD4 genes were comparatively frequent (>35 percent) in all stages. Frequencies of inactivation of the MARCKS, BAX, IGFIIR, and PTEN genes were significantly higher in Dukes B and C cancers than in Dukes A cancer (P < 0.05). The number of accumulated frameshift mutations was larger in Dukes B and C cancers (9.4) than in Dukes A cancer (6.8) (P = 0.003). CONCLUSIONS: The present data suggest that the disruption of the transforming growth factor-beta super-family signaling pathway by the alteration of the ACVR2 and/or TGFbetaRII genes and the disruption of antiproliferative function by the PTHLH gene alteration contribute to the development of early colorectal cancer. Moreover, the further accumulation of alterations in the MARCKS, BAX, IGFIIR, and PTEN genes seem to be associated with progression from early to advanced colorectal cancer from patients with hereditary nonpolyposis colorectal cancer.

Role of Smad4 (DPC4) inactivation in human cancer.

The tumor suppressor gene Smad4 (DPC4) at chromosome 18q21.1 belongs to the Smad family, which mediates the TGFbeta signaling pathway suppressing epithelial cell growth. This review summarizes the mutational events of the Smad4 gene in human cancer. The Smad4 gene is genetically responsible for familial juvenile polyposis, an autosomal dominant disease characterized by predisposition to gastrointestinal polyps and cancer. In this syndrome, polyps are formed by inactivation of the Smad4 gene through germline mutation and loss of the unaffected wild-type allele. In pancreatic and colorectal cancer, inactivation of the Smad4 gene through homozygous deletion or intragenic mutation occurs frequently in association with malignant progression. However, mutation of this gene is seen only occasionally in the rest of human cancers. The majority of Smad4 gene mutations in human cancer are missense, nonsense, and frameshift mutations at the mad homology 2 region (MH2), which interfere with the homo-oligomer formation of Smad4 protein and the hetero-oligomer formation between Smad4 and Smad2 proteins, resulting in disruption of TGFbeta signaling. Supporting evidence for the above observation was provided by genetically manipulated mice carrying either a heterozygote of the Smad4 gene or a compound heterozygote of the Smad4 and APC genes, which develop either gastrointestinal polyps/cancer mimicking familial juvenile polyposis or progressed colorectal cancer, respectively.