Analysis of Ki-ras gene mutations associated with DNA diploid, aneuploid, and multiploid colorectal carcinomas using a crypt isolation technique.

BACKGROUND AND AIM: Current evidence suggests a possible relationship between DNA ploidy status and Ki-ras gene mutations in human cancers. However, the conventional method does not enable accurate determination of DNA ploidy status of a tumor cell. The present study attempts to clarify whether Ki-ras gene mutations are associated with DNA ploidy status in sporadic colorectal carcinomas using a crypt isolation technique coupled with DNA cytometric sorting. METHODS: Polymerase chain reaction and single-strand conformation polymorphism and direct sequencing were used to analyze Ki-ras gene mutations in 82 sporadic colorectal carcinomas: 21 diploid, 12 aneuploid, and 49 multiploid. In addition, microsatellite instability (MSI) was assessed using seven microsatellite markers to study the relationship to Ki-ras mutations. RESULTS: Ki-ras mutations were found in 12 of 21 diploid carcinomas and in 8 of 12 aneuploid carcinomas. In contrast, Ki-ras gene mutations were detected infrequently in the 34 multiploid carcinomas examined, 8 of which were seen in diploid populations and 10 in aneuploid populations. On the other hand, Ki-ras gene mutations were inversely correlated with MSI, which was found in diploid carcinomas only. CONCLUSIONS: The low frequency of Ki-ras gene mutations that we observed in multiclonal colorectal carcinomas suggests that development of multiclonal colorectal carcinoma may involve a mechanism different from that involved in the development of diploid or aneuploid colorectal carcinomas.

Frequent allelic imbalance at the ATM locus in DNA multiploid colorectal carcinomas.

DNA multiploidy may involve specific DNA ploidy states with respect to genetic alterations such as oncogenes, tumor suppressor gene mutation and microsatellite instability. To clarify the role of DNA multiploidy in colorectal cancer, we analysed allelic imbalance involving the ATM gene, localized to chromosome 11q22-23 and thought to be involved in genetic stability, in a series of multiploid colorectal carcinomas. In addition, p53 gene mutation (exons 5-8) and allelic imbalance at 11q24 loci distal to the ATM locus were also examined. The crypt isolation technique coupled with DNA cytometric sorting and polymerase chain reaction assay using 10 microsatellite markers tightly linked to the ATM gene were used to study ATM allelic imbalance in 55 colorectal carcinomas (15 diploid, 13 aneuploid, 27 multiploid). While allelic imbalance at the ATM locus was rarely observed in diploid and aneuploid carcinomas, multiploid carcinomas exhibited a high frequency of ATM allelic imbalance. In multiploid carcinoma samples, diploid subpopulations showed a smaller range of allelic imbalance at the loci tested compared to aneuploid subpopulations that demonstrated allelic imbalance over a relatively large region. Also, the frequency of AI at 11q24 showed a similar tendency to that at the ATM locus for each DNA ploidy state. An association between p53 gene mutation and ATM allelic imbalance in multiploid carcinoma was also observed. Our results suggest that ATM allelic imbalance and p53 gene mutations occur during the progression from diploid to aneuploid cell populations in multiploid colorectal carcinomas.

Infrequent microsatellite instability in biliary tract cancer.

BACKGROUND AND OBJECTIVES: Microsatellite instability (MSI) has been reported in several tumors. However, few reports are available concerning MSI in biliary tract cancers. We investigated MSI and allelic loss at the hMLH1 and hMSH2 gene loci in biliary tract cancers. METHODS: We analyzed microsatellite alterations using 7 microsatellite markers in 38 cases of extrahepatic bile duct (EHBD) cancer and 16 cases of ampullary cancer using polymerase chain reaction and an automated fluorescent DNA sequencer. RESULTS: A MSI prevalence of 13.2% (5/38) was observed for EHBD cancer and a prevalence of 12.5% (2/16) was observed for ampullary cancer. Loss of heterozygosity at the hMLH1 and hMSH2 gene loci were observed in 4% (1/25 informative cases) and 6.1% (2/33) of EHBD cancer cases, respectively; and in 11.1% (1/9) and 8.3% (1/12) of ampullary cancer cases, respectively. The cumulative survival rate of patients with MSI was significantly better than that of patients without MSI in EHBD cancer. However, MSI was not an independent prognostic factor. CONCLUSIONS: Our results suggest that genetic defects in the DNA mismatch repair system and MSI do not play an important role in the majority of biliary tract cancers.

Correlation between C677T MTHFR gene polymorphism, plasma homocysteine levels and the incidence of CAD.

The lesions of coronary atherosclerosis represent the result of a complex, multicellular, inflammatory-healing response in the coronary arterial wall. In vivo and in vitro cellular and molecular studies have suggested a role for tissue homocysteine in endothelial cell injury and adverse extra-cellular matrix remodeling. Gene polymorphisms in relation with numerous risk factors might increase the incidence of coronary artery disease (CAD). In this review we have focused on the correlations between plasma homocysteine levels, the incidence of cardiovascular disease and the cytosine-to-thymidine substitution at nucleotide 677 (C677T) of the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene, coding for a key enzyme in methionine-homocysteine metabolism. The role of the C677T MTHFR gene polymorphism in the causation of CAD is controversial. We reviewed 12 recent case-control studies comprising 5370 genotyped patients with CAD and 4961 genotyped participants without CAD. There was no significant difference between those with and without CAD in the frequency of the C677T polymorphism (34.9 vs 33.6%). The frequency of homozygous C677T polymorphism in these groups was 10.9 versus 12.8%, respectively, although there were some ethnic differences in the C677T MTHFR polymorphism. In the analysis of the 12 studies, the odds ratio of CAD associated with the TT genotype (homozygous C677T polymorphism) was 1.18. Only slightly higher plasma homocysteine levels were observed in participants with the val/val (TT) genotype (14.4+/-2.9 micro mol/L in TT genotype vs 11.1+/-1.9 and 11.9+/-2 micro mol/L in CC and CT genotype, respectively). In addition, the relation between homocysteine increase after methionine loading and MTHFR genotypes is also controversial. However, hyperhomocysteinemia because of the C677T MTHFR allele may be corrected with oral folic acid therapy. Further investigations on the relationships between MTHFR genotypes and the incidence of CAD should be based on larger samples, paying attention to the differences between various ethnic populations. Individual therapeutic strategies based on single nucleotide polymorphism may become increasingly important for preventive treatment against polygenic CAD.

Genetic alterations in DNA diploid, aneuploid and multiploid colorectal carcinomas identified by the crypt isolation technique.

Loss of heterozygosity (LOH) and microsatellite instability (MSI) commonly occur in colorectal carcinomas. However, the role of these genetic alterations in determining DNA ploidy status of tumors (diploid, aneuploid and multiploid) remains unclear. In the present study, we attempted to clarify the relationship between genetic alterations and DNA ploidy status. Crypt isolation coupled with DNA cytometric sorting and polymerase chain reaction assay (17 microsatellite markers) were used to study allelic losses and MSI in 59 colorectal carcinomas (diploid, 15; aneuploid, 10 and multiploid, 34). Of the 15 diploid carcinomas, 6 exhibited MSI in which allelic losses were rarely found. The other 9 diploid tumors mostly exhibited allelic losses, but none displayed MSI status. Whereas allelic losses frequently occurred in the aneuploid carcinomas and the aneuploid populations of multiploid carcinomas, they were rarely detected in the diploid populations of multiploid carcinomas. MSI status was not observed in aneuploid carcinomas nor in either population of multiploid carcinomas. Although multiploid carcinomas genetically resemble aneuploid carcinomas in the expression of the severe LOH phenotype, the genetic alterations seen in the diploid populations of multiploid carcinomas may differ from those of diploid carcinomas. Furthermore, all diploid, aneuploid and both the diploid and aneuploid fractions of the multiploid tumors that were non-MSI exhibited a high rate of LOH, suggesting that LOH is independent of the tumor's ploidy status.

Microsatellite instability in gallbladder carcinoma: two independent genetic pathways of gallbladder carcinogenesis.

Although the genetic basis for gallbladder carcinogenesis has not been clarified, considerable evidence has shown that genetic alterations play an important role in the development and progression of human cancers. In this study, we analyzed 30 gallbladder carcinomas to investigate the role of genetic alterations in their tumorigenesis, and to study correlations with their clinicopathological features. Tissue samples were obtained from 30 patients with gallbladder carcinoma (11 men and 19 women; mean age, 62 years; age range, 38-80 years). Genomic DNAs were extracted from fresh tumor tissue. We examined loss of heterozygosity (LOH) in the p53, APC, DCC, RB, and NM23-H1 gene regions by polymerase chain reaction (PCR)-LOH assay using an automated fluorescent DNA sequencer employing four microsatellite markers (p53, APC, DCC, NM23-H1). Five additional microsatellite markers were used for the determination of microsatellite instability (MSI). LOH was found at p53 in 9 of 15 informative cases (60%), at DCC in 10 of 22 (45%), at APC in 5 of 15 (33%), at RB in 1 of 8 (13%), and at NM23-H1 in 1 of 15 (7%). MSI was observed in 5 of 30 cases (17%) in at least one chromosomal loci of these nine microsatellite markers. None of the patients with MSI-positive tumors showed lymph node metastasis, and there was an inverse correlation between MSI and the presence of LOH in gallbladder carcinoma. These results suggest that there are two independent genetic pathways in gallbladder carcinogenesis; that is, an MSI pathway and an LOH pathway.

DNA mapping of gastric cancers using flow cytometric analysis.

Although numerous studies of gastric cancers on DNA ploidy have been reported, differences in the degree of aneuploidy (DNA index, DI) during progression have not been identified. We attempted to chart the differences in DIs during progression to clarify the role of aneuploidy in gastric cancers. We classified the gastric cancers examined into intestinal (n = 88) and diffuse (n = 48) types, and then analyzed 136 gastric cancers (intramucosal cancer, 42; submucosal cancer, 39; advanced cancer, 55) by flow cytometry using multiple sampling. In addition, we examined the DNA ploidy pattern of mucosal and submucosal lesions using the same submucosal cancers to study the tumor progression in individual cancers. Intratumoral DNA differences in DNA ploidy were observed in both types of gastric cancers. In intestinal-type cancers, multiple subclones indicated by a different DI occurred during the early stage of gastric cancers, whereas in diffuse-type cancers, multiple subclones were found primarily in advanced cancers. Although the DI varied widely in early intestinal-type cancers between 1.0 and 2.0, in early diffuse-type cancers, the DI tended to be less than 1.2. However, in advanced stage gastric cancers, the DI distribution was similar for both histological types. In intestinal-type cancers, high DI (>1.3) aneuploidy was frequently found in mucosal lesions. In contrast, only low DI (<1.2) aneuploid clones were observed in mucosal lesions of diffuse-type cancers. The present results suggest that high DI aneuploid tumor clones in intramucosal cancers acquire invasive ability when they progress to submucosal cancers, whereas DNA aneuploidy itself plays an important role in submucosal invasion of diffuse-type cancers.

Allelic losses of 17p, 5q, and 18q loci in diploid and aneuploid populations of multiploid colorectal carcinomas.

17p, 5q, and 18q allelic losses are involved in the pathogenesis and progression of colorectal carcinoma, and DNA aneuploidy in this type of cancer is thought to result from alterations of these chromosomal loci. However, genetic differences between diploid and aneuploid populations of multiploid carcinoma, defined as the coexistence of diploid and aneuploid populations in the same area, remain unclear. The differences in 17p, 5q, and 18q allelic losses between the diploid and aneuploid populations in 24 sporadic DNA multiploid colorectal carcinomas were analyzed by use of crypt isolation coupled with DNA cytometric sorting and polymerase chain reaction assay. 17p Allelic loss was observed in 7 of 22 diploid populations excluding 1 case of microsatellite instability but was found in 21 of 23 aneuploid populations. Although 5q allelic loss was detected in only 3 of 22 diploid populations, 13 of 22 aneuploid populations had 5q allelic loss. Losses of the 18q allele were frequently found in aneuploid populations (15 of 20), although no 18q allelic loss was detected in corresponding diploid populations. 17p Allelic losses may play an important role in the progression from a diploid status to an aneuploid status in a specific subset of colorectal cancer. However, 18q or 5q allelic losses do not appear to precede nor to facilitate the aneuploid clonal divergence of cancer cells. Multiploidy is a useful model to study genetic alterations between diploid and aneuploid populations.

Esophageal squamous cell carcinoma characterized by extensive chondroid differentiation.

A rare case of carcinoma characterized by extensive chondroid elements at a site of primary esophageal and metastatic lesion is reported. The patient was a 67-year-old man complaining of dysphagia due to an ulcerative lesion at the lower middle esophagus. He underwent irradiation treatment prior to surgery. Histologically, the tumor consisted of both carcinomatous and chondroid elements and had invaded deeply into the esophageal wall. The carcinomatous cells had gradually become chondroid cells embedded within an extensive extracellular matrix. In addition, the metastatic lesion showed findings similar to those of the primary lesion. Immunohistochemistry revealed that both carcinomatous and chondroid elements were immunostained with cytokeratin and epithelial membrane antigen, suggesting an epithelial nature to the chondroid cells. Conversely, only chondroid cells were positively stained for S-100 protein. Furthermore, bone morphogenetic proteins (BMP) were positive for chondroid cells and their surrounding carcinomatous cells. Given the apparent transition between carcinomatous and chondroid cells based on microscopy and immunohistochemical findings in the present case, we concluded that the chondroid cells were derived from carcinomatous cells. In addition, our findings suggest that BMP produced by carcinomatous cells lead to chondroid differentiation of the carcinoma cells.

Microsatellite instability and mutation of mitochondrial and nuclear DNA in gastric carcinoma.

BACKGROUND & AIMS: Microsatellite instability (MSI) in mitochondrial DNA (mtDNA) is observed in some colorectal carcinomas. We attempted to determine if mitochondrial MSI (mtMSI) and mutations occur in gastric carcinomas and if the mtMSI phenotype underlies specific clinicopathologic profiles. METHODS: Sixty-two gastric carcinomas (34 intestinal and 28 diffuse types) were investigated. Coding mutations in 8 different mitochondrial genes, mtMSI in a noncoding (C)n tract, and p53 gene mutations were examined by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis. MSI in nuclear DNA (nMSI) and loss of the p53 gene were examined using microsatellite markers. RESULTS: Ten of 62 (16%) carcinomas showed the mtMSI phenotype. Mitochondrial gene mutation was detected in 5 carcinomas, 4 of which also showed the mtMSI phenotype. There was a positive correlation between mtMSI and nMSI status. In intestinal carcinomas, mtMSI, nMSI, and p53 gene alterations were frequently detected from early to advanced stages. In diffuse carcinomas, both kinds of MSI were found in only advanced (subserosal or serosal invasion) carcinomas. Six of 7 carcinomas with the nMSI phenotype and all 5 carcinomas with mitochondrial coding mutations had a considerable intestinal-type tumor cell component. CONCLUSIONS: Mitochondrial gene mutations, which are associated with the mtMSI phenotype, may play a specific role in the tumorigenesis of intestinal-type gastric carcinomas.

Aberrations of the K-ras, p53, and APC genes in extrahepatic bile duct cancer.

BACKGROUND AND OBJECTIVES: The genetic alterations involved in extrahepatic bile duct (EHBD) cancer are poorly understood. Our aim was to identify aberrations of the K-ras, p53, and APC genes in EHBD cancer. METHODS: We investigated aberrations of these genes in 52 EHBD cancers using polymerase chain reaction (PCR) single-strand conformation polymorphism analysis, followed by direct sequence determination and a PCR restriction fragment length polymorphism assay. RESULTS: The K-ras, p53, and APC genes were mutated in 9.6%, 32.7%, and 0% of EHBD cancers, respectively. Loss of heterozygosity at the p53 and APC gene loci was identified in 15.6% and 38.5% of EHBD cancers, respectively. CONCLUSIONS: Our results suggest that an unknown suppressor gene on 5q other than the APC gene may be responsible for EHBD cancer.

A unique method for mutation analysis of tumor suppressor genes in colorectal carcinomas using a crypt isolation technique.

BACKGROUND: Contamination of nontumor tissue makes genetic analysis difficult. For this reason, it is important to obtain pure tumor tissue to ensure accurate genetic analysis. OBJECTIVE: To accurately assess the incidence of mutation of tumor suppressor genes (p53: exon 5-8; APC: mutated cluster region; NF-2 gene: all exons) in 45 colorectal carcinomas. METHODS: We developed an application of the polymerase chain reaction-single-strand conformation polymorphism and DNA sequence by coupling them with crypt isolation. RESULTS: Mutations of p53 and APC genes were found in 24 and 22 of 45 colorectal carcinomas, respectively. No mutation of the NF-2 gene was observed in this cancer. Single-strand conformation polymorphism using a crypt isolation technique showed a clear migrating band and no false-positive data. CONCLUSIONS: The crypt isolation technique is a useful method for accurately analyzing genetic alterations. Furthermore, our proposed method confirmed the morphological findings obtained before the genetic analysis.

Use of crypt isolation to determine loss of heterozygosity of multiple tumor suppressor genes in colorectal carcinoma.

Analysis of loss of heterozygosity (LOH) is very important in the study of tumor suppressor genes. However, accurate LOH analysis of tumor suppressor genes is difficult because of dilution by contaminating non-tumor DNA. Thus, enrichment of tumor DNA is required to accurately determine LOH of the tumor. We developed a new application of the fluorescent polymerase chain reaction by coupling it with crypt isolation to accurately assess the incidence of LOH of tumor suppressor genes in 45 colorectal carcinomas. LOH was observed at p53 in 26 of 37 tumors (70.3%), at APC in 13 of 35 (37.1%), at DCC in 16 of 25 (64.0%), at NF-2 in 5 of 23 (21.7%), and at nm23 H-1 in 7 of 30 (23.3%). We could clearly determine LOH of these genes because the crypt isolation technique was used. Although the incidence of LOH at each of these loci, as determined by using this technique, was similar to that obtained in previous studies using conventional methods, this method provides a simpler, more accurate way to assess LOH. In addition, the morphology of the samples can be analyzed before genetic analysis.

Mitochondrial gene mutation, but not large-scale deletion, is a feature of colorectal carcinomas with mitochondrial microsatellite instability.

We have shown that microsatellite instability (MSI) occurs in mitochondrial DNA (mtDNA) of colorectal carcinomas. To determine whether such mitochondrial microsatellite instability (mtMSI) is associated with certain forms of mitochondrial gene alterations, we extended the screening in the same series of 45 carcinomas. Analysis by whole mtDNA amplification (16.5 kb) and digestion revealed no detectable large-scale change in these carcinomas. In contrast, single-strand conformation polymorphism (SSCP) analysis demonstrated NADH dehydrogense (ND) gene alterations in 7 carcinomas (16%), including 3 mononucleotide repeat alterations, 2 missense mutations and 1 small (15 bp) deletion. Six of these 7 carcinomas also exhibited mtMSI of the (C)n sequence in the displacement-loop (D-loop) region. Thus, frameshift or missense mutations rather than large-scale changes in the mtDNA were more common features in colorectal carcinomas with mtMSI. By analogy to mutational features of nuclear MSI, mtMSI most likely results from certain repair deficiencies in the mtDNA and probably plays a role in the tumor development of certain colorectal carcinomas.

Usefulness of proliferative activity, DNA ploidy pattern and p53 products as diagnostic adjuncts in colorectal adenomas and intramucosal carcinomas.

Although numerous studies have assessed the biologic parameters of tumors, measurement of these parameters has had, to date, little impact on histologic diagnosis. Furthermore, analysis of a single parameter is insufficient to evaluate tumor malignant potential. In the present study, cell proliferation, DNA ploidy and p53 product were analyzed to objectify the tumor malignant potential in colorectal adenomas and intramucosal carcinomas. Sixty-one adenomas and 49 intramucosal carcinomas were studied using immunohistochemical analysis of Ki-67 and p53, silver-staining nucleolar organizer region (AgNOR) stain and DNA ploidy in fresh samples. Intramucosal carcinoma exhibited a greater Ki-67-positive rate and AgNOR count than the adenomas, although these parameters varied widely among samples. The incidence of aneuploidy and p53 over-expression in colorectal intramucosal carcinomas was significantly higher than in colorectal adenomas. These results indicate that DNA aneuploidy and p53 accumulation are the most reliable parameters for distinguishing benign and malignant lesions.

Role of DNA aneuploidy, overexpression of p53 gene product, and cellular proliferation in the progression of gastric cancer.

DNA aneuploidy, p53 overexpression, and high cell proliferation frequently occur in gastric cancer. However, little is known about the time of their appearance throughout cancer progression. Therefore, the objective of the present study was to determine when such abnormalities occur during gastric cancer progression. We classified the gastric cancers examined into intestinal (n = 65) and diffuse (n = 34) types. DNA ploidy was examined using flow cytometry and expression of MIB-1 and p53 immunoreactivity were studied using the avidin-biotin complex method in three stages of gastric cancer (mucosal, submucosal, deeply invasive cancer, i.e., advanced cancer). The incidence of DNA aneuploidy in intestinal-type mucosal cancers (15/27, 55.6%) was lower than that of submucosal invasive cancers (14/16, 87.5%) or advanced cancers (19/22, 86.4%), while a low incidence of DNA aneuploidy was observed in each diffuse-type cancer group (mucosal, 1/12, 8.3%; submucosal invasive, 3/9, 33.3%; advanced, 8/14, 57.1%). Although overexpression of the p53 gene in intestinal-type cancer was found in early stage, that in diffuse-type cancer was observed in advanced stage. Among the intestinal-type mucosal cancers, the MIB-1 percent positive was higher in aneuploid tumors than diploid ones. DNA aneuploidy and overexpression of the p53 gene may play an important role in the early tumorigenesis of intestinal-type gastric cancer and in the late event of tumorigenesis of diffuse-type gastric cancer.

Analysis of subclonal expansion of colorectal carcinomas by flow cytometry.

DNA heterogeneity of colorectal carcinomas has been investigated by flow cytometry, most studies have focused on the clinical usefulness of DNA ploidy analysis. Since cancers consist of predominant subclones with proliferative advantage due to clonal expansion, we attempted to analyse the clonal expansion of colorectal carcinomas within a tumour by measuring DNA ploidy. The DNA ploidy and heterogeneity of multiple fresh samples obtained from 164 colorectal adenocarcinomas were analysed by flow cytometry. Each tumour was divided into an average of six specimens, which were analysed separately. For 146 of the tumours (89%) at least one DNA aneuploid population was found within the cancer tissue examined. DNA multiploidy was detected in 26 cases (17.8%) among the cancers with aneuploidy. Based on the DNA index (DI), hypertriploid aneuploidy (1.7

Polymorphism of the apolipoprotein E and angiotensin I converting enzyme genes in Japanese patients with myocardial infarction.

OBJECTIVE: To investigate the genetic contribution for myocardial infarction. METHODS: We investigated common polymorphisms of apolipoprotein E gene and angiotensin converting enzyme (ACE) gene in Japanese population. Subjects were 422 healthy people and 254 patients with myocardial infarction. We evaluated the 287 base pair (bp) insertion (I)/deletion (D) polymorphism in intron 16 of the ACE gene and a polymorphism in the apolipoprotein E gene by using the polymerase chain reaction. RESULTS: The ACE genotype prevalences for II, ID, and DD were 36.2, 46.1, and 17.7%, respectively, among the myocardial infarction patients. The prevalence of the D allele of the ACE gene among the myocardial infarction patients (0.593) exceeded that among the healthy controls (0.407). The prevalences of the epsilon 2, epsilon 3, and epsilon 4 alleles of the apolipoprotein E genotype among healthy controls were 0.024, 0.882, and 0.094, and those among survivors of myocardial infarction were 0.024, 0.834, and 0.142, respectively. Myocardial infarction patients had an excessive prevalence of the apolipoprotein E epsilon 4 allele (P < 0.05). Multiple regression analysis demonstrated that the independent risk factors for developing myocardial infarction were age, DD genotype of ACE gene, and apolipoprotein E epsilon 4 allele. Stenotic coronary vessels in myocardial infarction patients did not differ significantly among the patients with various ACE and apolipoprotein E genotypes in the present study. CONCLUSIONS: Among the Japanese, apolipoprotein E epsilon 4 carriers and subjects with ACE DD genotype are at an increased risk of myocardial infarction.

Microsatellite instability in the mitochondrial DNA of colorectal carcinomas: evidence for mismatch repair systems in mitochondrial genome.

The role, if any, that mitochondrial (mt) DNA alterations play in the carcinogenic process remains unclear. To determine whether mtDNA instability occurs in cancers, nine microsatellite sequences in the mtDNA were examined in 45 sporadic colorectal carcinomas. Alteration in a polycytidine (C)n tract within a non-coding displacement-loop (D-loop) region was detected in 20 carcinomas (44%), three of which also exhibited frameshift mutations in a polyadenosine (A)8 or polycytidine (C)6 tract within NADH dehydrogenase (ND) genes. Interestingly, all three mutant genes were predicted to encode truncated ND proteins, which lacked a large portion of the C-terminus. These results suggested that certain repair systems, like the mismatch repair systems in the nuclear genome, are required for mtDNA maintenance and that defects in these systems can lead to target mitochondrial gene mutations in colorectal carcinomas.

Mismatch repair deficiency leads to a unique mode of colorectal tumorigenesis characterized by intratumoral heterogeneity.

In order to determine the effects of mismatch repair (MMR) deficiencies in sporadic colorectal carcinomas, 45 such cancers were examined using a sensitive method called crypt isolation technique. Loss of heterozygosity (LOH) in the MSH2 or MLH1 gene was more frequently observed in replication error (RER) (+) carcinomas than in RER (-) carcinomas, which implied that loss of one normal allele could partly affect repair capacity. MSH2 gene defects at both alleles were observed in two carcinomas, which showed severe repair deficiencies. Interestingly, unlike the situation observed in the p53 gene, the MSH2 and MLH1 genes did not show complete LOH. Novel crypt isolation-based subpopulation (CISP) analysis demonstrated that at least two distinct carcinoma subpopulations existed in most carcinomas that showed incomplete LOH; one with and one without LOH. In one carcinoma that had germline mutation and somatic incomplete LOH of the MSH2 gene, the mutator phenotype was only observed in populations affected in both alleles. Thus, the MSH2 gene appears to possess the two hits mechanism of tumor suppressor genes. However, unlike the tumor suppressor genes, MMR gene defects lead to a unique mode of colorectal tumorigenesis characterized by intratumoral heterogeneity.