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We have previously observed that all human hepatocellular carcinomas (HCCs) from HBV carriers examined had the integrated X region. In this study, HBV DNA was isolated from an integration site in one HCC that had a single, very small integrated viral DNA including the X region, but it had no expression of X gene as poly(A)RNA. It was found that HBV DNA was present between alphoid repetitive sequences, and it included Enhancer and X regions, encompassing the adr sequence from 910 to 1811. Nucleotides for 8 amino acids at the 3' end, a stop codon of X gene and a poly(A) signal downstream of X gene were lost by integration, and nucleotides for 7 amino acids and a stop codon were substituted by a connected alphoid sequence. When this cloned HBV DNA was transfected with an expression vector to an immortalized mouse liver epithelial cell line, MLE-10, malignant transformation occurred. Transformants having expressed poly(A)RNA of the X gene showed anchorage-independent growth in soft agar and tumor formation in the subcutis of nude mice. The mRNA level of EGFR was found to be remarkably enhanced in X-transformed cells, in contrast with the absence of this mRNA in parental and ras-transformed MLE-10. Our data provide evidence that the Enhancer-X region alone is the key contributor to the malignant change of pre-malignant liver cells in HBV carriers through activation of some specific genes, such as EGFR.

Hereditary non-polyposis colorectal cancer (HNPCC) is characterized by familial clustering and early onset. It is unclear, however, whether the early onset of colorectal cancer necessarily represents HNPCC. A 13-year-old patient had rectal cancer and underwent curative surgery. DNA from this patient was examined for replication errors (RER) and genes related to familial colorectal cancer (APC, hMSH2, and hMLH1). The patient had a negative family history of colorectal cancer, did not show the RER phenotype, and had no germline mutation of the APC, hMSH2, and hMLH1 genes. The present case suggests that an unusually young patient with colorectal cancer is not always an HNPCC proband. Observation over time, however, will be needed, as a first mutator of familial colorectal cancer could be missed.

We have previously detected an increased frequency of loss of heterozygosity (LOH) on chromosome 18q during progression of colorectal carcinomas. To clarify the target of 18qLOH, mutation of Smad4 and Smad2 genes was analysed in 176 colorectal tumors with different stages, including liver metastasis, from 111 sporadic, 52 familial adenomatous polyposis (FAP) and nine hereditary nonpolyposis colorectal cancer (HNPCC) patients. Mutation of other Smad gene families in the TGF-beta signaling pathway was also examined. Twenty-one Smad4 mutations and one Smad2 mutation were detected, whereas mutation of Smad3, 6 and 7 genes was not detected. Smad4 mutations included seven frameshift, one inframe deletion, four nonsense and nine missense mutations, 95% of which resulted in alteration of Smad4 protein regions included in homo-oligomer and hetero-oligomer formation. Frequencies of tumors with Smad4 mutation were 0/40 (0%) in adenoma, 4/39 (10%) in intramucosal carcinoma, 3/44 (7%) in primary invasive carcinoma without distant metastasis, 6/17 (35%) in primary invasive carcinoma with distant metastasis, and 11/36 (31%) in distant metastasis (metastatic/non-metastatic: P=0.006 approximately 0.01). Loss of the other allele was observed in 19 of 20 (95%) invasive and metastasized carcinomas with Smad4 mutations. In four cases both primary and metastasized carcinomas in the same patients showed the same mutations. The present results suggest that Smad4 gene is one of true targets of 18qLOH, and that its inactivation is involved in advanced stages, such as distant metastasis, in human colorectal carcinogenesis.

We report the existence of both germline and somatic mutations of the APC gene in thyroid carcinomas from familial adenomatous polyposis (FAP) patients. One papillary thyroid carcinoma from a 210-year-old woman, with germline mutation of the APC gene (TCA to TGA at codon 1110), showed a somatic mutation of AAAAC deletion between codons 1060 and 1063. Another somatic mutation of CAG to TAG at codon 886 was also found in one of multiple thyroid carcinomas from a 26-year-old woman with attenuated FAP and germline mutation at codon 175 (C deletion). This is the first evidence that total absence of the normal function of the APC gene is involved in development of thyroid carcinomas in FAP.

BACKGROUND The authors examined somatic mutations of the adenomatous polyposis coli (APC) gene in 84 human aberrant crypt foci (ACF) to determine whether APC gene mutations were involved in the histologic progression of ACF. METHODS Mutation cluster regions of the APC gene were subjected to polymerase chain reaction single-strand conformation polymorphism analysis and direct sequencing. RESULTS Four kinds of deletion were detected in the mutation cluster regions of APC gene in five ACF. APC mutation was detected in 1 of 18 ACF with Stage I abnormalities (6%). Four of 10 adenomatous ACF (40%) harbored the mutation. There were no mutations in 56 hyperplastic ACF. CONCLUSIONS These results suggest that APC mutations may be involved initially in only a limited number of adenomas in ACF. Cancer 1998;83:896-900. © 1998 American Cancer Society.

BACKGROUND DNA replication errors (RERs) are closely associated with hereditary nonpolyposis colorectal carcinoma (HNPCC). Recently, alterations in DNA mismatch repair genes, including hMSH2, hMLH1, and hPMS2, have been implicated in the pathogenesis of HNPCC. Several studies have demonstrated RER in 13-17% of nonfamilial colorectal carcinomas. It is unclear, however, as to whether or not these RER positive nonfamilial colorectal carcinomas are incomplete forms of HNPCC or are caused by incidental alterations of DNA mismatch repair genes. Consequently, the authors studied the characteristics of RER positive nonfamilial colorectal carcinomas, placing particular emphasis on hMSH2 and hMLH1 gene mutations. METHODS Fresh or frozen samples of 103 nonfamilial colorectal carcinomas were examined for RERs using the polymerase chain reaction (PCR) and specific microsatellite primers. The authors also identified mutations of the hMSH2 and hMLH1 genes in RER positive samples by a PCR single strand conformational polymorphism analysis followed by direct nucleotide sequencing. RESULTS The incidence of RER was 15.7% (17/103) in nonfamilial colorectal carcinomas, and only 1 case, which was found in the ascending colon, showed a somatic mutation at exon 12 in the hMSH2 gene. Neither germline nor somatic mutations of the hMSH2 or hMLH1 genes could be found in any of the remaining RER positive tumors. RER positive nonfamilial carcinomas tended to be located more frequently in the right colon. There was no increased prevalence in young patients, and the clinicopathologic characteristics of HNPCC were absent in the patients with RER positive nonfamilial colorectal carcinoma. CONCLUSIONS Based on these findings, the carcinogenesis of RER positive nonfamilial colorectal carcinoma is considered different from that of HNPCC. Cancer 1998;82:279-85. © 1998 American Cancer Society.

Turcot syndrome is characterized by an association of malignant brain tumors and colon cancer developing in the patient's teens. Since the mechanism of carcinogenesis in Turcot syndrome is still unclear, we analysed genetic changes in tumors from a Turcot patient with no family history of the condition. All tumors, including one astrocytoma, three colon carcinomas, and two colon adenomas, exhibited severe replication error (RER), and all colon tumors showed somatic mutations at repeated regions of TGFbetaRII, E2F-4, hMSH3, and/or hMSH6 genes. Somatic APC mutations were detected in three of three colon carcinomas, and somatic p53 mutations were detected in the astrocytoma and two of three colon carcinomas, both of which showed two mutations without allele loss. We also found that normal colon mucosa, normal skin fibroblasts and normal brain tissue from this patient showed respective high frequencies of RER, in contrast to usual HNPCC patients in which RER was very rare in normal tissues. These results suggest that extreme DNA instability in normal tissues causes the early development of multiple cancer in Turcot syndrome. A missense mutation (GAG to AAG) at codon 705 of hPMS2 gene was detected in one allele of this patient, which was inherited from his mother without tumors. Additional unknown germline mutation may contribute to the genetic instability in normal tissues.

BACKGROUND & AIMS: Microsatellite instability (replication error [RER]) is a characteristic of tumors in hereditary nonpolyposis colon cancer (HNPCC), but the mechanism of HNPCC carcinogenesis is not yet understood. To clarify the nature of HNPCC tumors, RER and genetic changes were compared between HNPCC and non-HNPCC tumors. METHODS: RER and genetic changes were analyzed in 21 HNPCC, 389 familial adenomatous polyposis, and 206 sporadic tumors using polymerase chain reaction, single-strand conformation polymorphism, sequencing, and Southern hybridization. RESULTS. in HNPCC, 95% tumors at all stages showed RER positivity (altered loci, 4.3 of 5). In familial adenomatous polyposis and sporadic tumors, RER positivity (1.7 of 5) was 3% in adenoma and intramucosal carcinoma, 13%-24% in invasive carcinoma, and 35% in carcinoma metastasized to liver. Fifty percent of RER-positive HNPCC tumors had both germline and somatic mutations of hMSH2 or hMLH1 gene, whereas 6% of RER-positive non-HNPCC had somatic mutation. APC, p53, and K-ras-2 mutations and loss of heterozygosity of tumor-suppressor genes were significantly less frequent (P = 0.03 to 0.0006) but transforming growth factor beta type II receptor mutation was significantly more frequent (P = 0.000001) in HNPCC than in non-HNPCC. CONCLUSIONS: RER positivity occurs from an early stage of carcinogenesis in HNPCC but in later stages in non-HNPCC. Most HNPCC tumors may develop through different genetic changes from those in the adenoma-carcinoma sequence, although a certain percentage develops through APC mutation. (Gastroenterology 1996 Aug;111(2):307-17)

Familial adenomatous polyposis (FAP) is a dominantly inherited disease characterized by the development of numerous adenomatous polyps and a high risk of colorectal cancer. Affected individuals have multiple adenomas at various intermediate stages between benign and malignant, so FAP is a supportive model for the adenoma-carcinoma sequence of colorectal carcinogenesis. The APC (adenomatous polyposis coli) gene, which is a tumor suppressor gene, was discovered in 1991, and mutation of this gene was found to be the cause of FAP. To date, more than 150 germ-line mutations have been determined in FAP patients, and more than 300 somatic mutations of the APC gene have been detected in colorectal adenomas and carcinomas from both FAP and non-FAP patients - almost all of these resulting in truncated APC protein. The nature of germline and somatic mutations is described in detail herein, including the finding of several codons in which no germ-line, but only somatic mutation, frequently occurs. Existence of somatic APC mutations in adenomas from both FAP and non-FAP patients suggests that inactivation of the APC gene by two mutations is generally involved in the development of adenoma. Further development of adenoma to advanced carcinoma is associated with loss of heterozygosity (LOH) of the APC gene and additional inactivation of multiple tumor suppressor genes (possibly through mutation and LOH) that include the p53 gene, DCC gene and tumor suppresor genes on chromosomes 1p, 8p and 22q in both FAP and non-FAP patients. Recent findings on the correlation of these genetic changes with the adenomacarcinoma sequence are also described. In vitro models for carcinogenesis, and experiments of suppression of tumorigenicity in advanced colon carcinoma cells by introduction of a normal single chromosome or a normal suppressor gene are also referred to. Furthermore, evidence of inactivation of the APC gene in extracolonic tumors, in FAP patients, including desmoid and adrenocortical tumors, is demonstrated. This confirms the pleiotropic effect of the mutant APC gene on both colonic and extracolonic manifestations in FAP patients, and that the normal APC gene acts as a growth control gene throughout the body

Loss of heterozygosity (LOH) on chromosomes 1p, 4q, 5q, 8p, 13q, 16q, 17p, and 22q, and mutation of the p53 gene were simultaneously analyzed in 63 hepatocellular carcinomas (HCCs) with distinct histopathological grades, 80% of the tumors being from patients who had been exposed to hepatitis B virus (HBV) or hepatitis C virus (HCV). The frequencies of LOH on 8 chromosomes were 0–25% in 10 well differentiated HCCs, LOH being observed on 4q, 5q and 17p, 21–53% in 26 moderately differentiated HCCs, LOH on 8p and 17p being high, and 29–75% in 27 poorly differentiated HCCs, LOH on 17p, 4q and 8p heing the most frequent. p53 gene mutation was detected in moderately and poorly differentiated HCCs at 15% and 52%, respectively, but not at all in well differentiated HCCs. Of the mutations detected, 42% were transition mutation and only 5% were CpG transition, in contrast to the high frequencies of these types of mutations in colon tumors (78% and 54%, respectively). LOH on every chromosome and p53 mutation were more frequent in more advanced tumors, and accumulation of genetic changes increased with increase of the histopathological grade. Frequency of genetic changes in HCCs from HBV‐positive patients was comparable to that from HCV‐positive patients. The present results suggest that accumulation of genetic changes in multiple tumor suppressor genes, especially LOH on 17p, 4q and 8p, and mutation in p53 gene, are involved in the progression of liver cancer, and LOH on 17p and 4q precedes other genetic changes. Differences in the direction of p53 mutation between HCC and colon carcinoma suggest that liver carcinogens are distinct from colon carcinogens. Furthermore, mechanisms affecting the frequency of LOH in HCCs in HBV‐infected patients may be similar to those in HCV‐infected patients.

Background: Familial adenomatous polyposis (FAP) patients develop various extracolonic lesions; however, the relationship between germline mutation of the APC gene and extracolonic manifestations is mostly unknown. To examine the genotype–phenotype relationship, we compared the APC mutation and clinical data. Methods: Germline mutations from codon 157 to 1465 of the APC gene were identified in 39 families of FAP and clinical data were collected from 80 patients of these families. Germline mutations were classified into two groups: mutations from exon 4 to 9 (codon 157 to 416, Group 1) and those from exon 10 to 15H (codon 564 to 1465, Group 2). The complication rates of extracolonic manifestations were compared between these two groups. Results: Frequencies of duodenal polyps and gastric adenomas in Group 2 were higher than those in Group 1 (p < 0.0001 and p < 0.0004, respectively) and development of osteoma was more frequent in Group 2 (p = 0.01). The number of colorectal polyps and retinal pigments also correlated with the germline mutation, which was consistent with previous reports. However, such correlations were less obvious with regard to gastric fundic polyps, desmoid tumors, soft tissue tumors and colorectal cancer. Conclusion: There are two types with regard to extracolonic manifestations of FAP: one is more severely affected according to the position of germline mutation of the APC gene and the other is not affected.

Mutations in hMSH2 and hMLH1 genes were analyzed in patients from 11 Japanese families that had been diagnosed as carrying hereditary nonpolyposis colorectal cancer (HNPCC) by clinical examination. Germ line mutations of hMSH2 gene were identified in 5 independent families in which colorectal (87% of patients), endometrial (30%), ovarian (17%), gastric (14%), and other cancers existed. Five mutations detected between codons 136 and 811 included single-base substitutions (C--T and T--G), a T deletion, and an A insertion, all of which produced stop codons resulting in truncated proteins, and an A--T substitution at splice donor site of exon 5 which resulted in deletion of this exon. Moreover, one HNPCC family was presumed to have germ line mutation of hMSH2 gene because a somatic mutation of hMSH2 gene was detected in a cancer from a patient in this family. In addition to these 11 families already diagnosed with HNPCC, 3 new families with germ line mutations of hMSH2 gene and hMLH1 gene were found through analysis of DNA from patients who had multiple cancers with alteration in microsatellite DNA. These mutations included an AG deletion at codons 877-878 of hMSH2 gene, an AAG deletion at codons 616-618 of hMLH1 gene, and a C--T single-base substitution at codon 217 of hMLH1 gene. Seven of eight germ line mutations found in this study are new mutations that have not been reported previously. In families in which germ line mutations were identified presymptomatic examination was then carried out using polymerase chain reaction single-strand conformation polymorphism analysis of DNA from peripheral blood, and the result was the detection of family members predisposed to HNPCC who did not yet show signs of cancer. These results indicate the value of DNA analysis in the screening and diagnosis of HNPCC patients and families.

Endocrine neoplasms have been reported occasionally in patients with familial adenomatous polyposis (FAP). An adrenocorotical carcinoma was studied in a patient with a family history of FAP. Loss of heterozygosity (LOH) in the region close to the adenomatous polyposis coli (APC) gene was detected in this carcinoma, and evidence was obtained that there was a loss of the normal allele of the APC gene. This is the first demonstration of LOH at the APC locus in adrenocortical tumors. The present results and our previous data on LOH in a recurring desmoid tumor suggest that the heterozygous mutant/wild-type condition of the APC gene may give rise to benign tumors, and that functional loss of this gene leads to development of tumors not only in the colon but also in other various parts of the body in FAP patients.

The patients with typical beriberi symptoms and with primary Perleche (fissure and erosion at the angle of the mouth) served as thiamine deficient and riboflavin deficient patients for this study. The serum protein pattern was examined by paperchromatography. The following results were obtained.1) The serum proteins of the beriberi patients were of various grades, from normal level to high degree of hypoproteinemia. Edema, which is the main symptom of beriberi, is not necessarily related with the serum protein contents, and it can often been noticed even in patients with normal serum protein contents. In the treatment of edema, injection of thiamine as well as its derivatives (oral administration is the same) was very effective. In the cases of beriberi, edema with low serum protein level was not cured unless given large amounts of protein or amino acids together with thiamine.2) Among riboflavin deficient patients including those who seemed to be healthy in general condition, their serum protein patterns were of various grades from normal level to hypoproteinemia. The serum protein patterns of patients who repeatedly suffered from riboflavin deficiency for many years were worse. The patients with normal protein pattern cannot be cured with riboflavin alone. Only large amount of good quality protein or amino acids togetherwith FAD injections were sufficiently effective.