Hiroshi Kumimoto

Frequent somatic mutations of mitochondrial DNA in esophageal squamous cell carcinoma

Recent studies of various cancers, such as those of the breast, head and neck, bladder and lung, reported that 46–64% of somatic mutations in the D‐loop region of mitochondrial DNA (mtDNA) are observed. However, in esophageal cancer, only a low rate (5%) of somatic mutations has so far been reported in one article (Hibi, K. et al., Int J Cancer 2001;92:319–321). Thus, to confirm this we analyzed the somatic mutations for hypervariable regions (HVR‐I and HVR‐II) in the D‐loop of mtDNA to reevaluate the possibility of mitochondrial genetic instability in this cancer. We amplified both HVRs by PCR and DNA samples obtained from 38 esophageal tumors and matched normal tissues, and then sequenced them. Comparing the sequences of tumors to those of normal tissues, we found 14 somatic mutations in 13 patients (34.2%). Eleven mutations were at the C consecutive stretch from position 303 to 309 of MITOMAP in the mitochondria databank (http://www.mitomap.org/), 1 at position 215 in HVR‐II and 2 at positions 16,304 and 16,324 in HVR‐I. There were 41 types of germ line variations in HVR‐I including 2 not so far recorded in the mtDNA databank and 17 in HVR‐II including 1 not yet recorded. We also determined nuclear genome instability of these 38 specimens by analyzing 3 independent microsatellite sequences. While 4 specimens showed a single microsatellite change, which is tumor specific, we did not find any co‐relation between a somatic mtDNA mutation and microsatellite instability of nuclear genome DNA. These results suggest that mtDNA mutations might show a genetic instability in esophageal cancer independently from a nuclear genome instability.

Mismatch repair and microsatellite instability in esophageal cancer cells

Using in vitro mismatch repair (MMR) assay, we have identified 3 of 22 esophageal cancer cell lines exhibiting reduced MMR activity. By means of gel‐shift assay, decreased binding ability to GT mismatch and CA loop was observed in these 3 cell lines. However, we could not find any mutations in the hMSH2, hMSH3 and hMSH6 genes, the protein products of which exhibit mismatch binding activity in human cells. In addition, when using antibodies against 5 MMR‐related proteins (hMSH2, hMSH3, hMSH6, hPMS2 and hMLH1), no aberrant expression was detected in any of them. When we examined 9 microsatellite loci in endogenous genomic DNA, these 3 esophageal cancer cell lines, deficient in MMR, did not exhibit microsatellite instability. However, when we examined the repetitious sequence on exogenous plasmid DNA which was introduced into these 3 esophageal cancer cells, the results suggested that MMR deficiency in esophageal cancer cells could result in moderate instability of the exogenous sequence.

Different susceptibility of each L-myc genotype to esophageal cancer risk factors.

To understand the relationship between the L‐myc genotypes and esophageal cancer risk, a polynierase chain reaction‐based restriction fragment length polymorphism analysis was performed on 91 Japanese patients with esophageal cancer and 241 non‐cancer outpatients. No significant difference in the distribution of genotypes was observed between patients and controls; 18.7% LL genotype, 56.0% LS and 25.3% SS among patients, and 24.5%, 55.6% and 19.9%, respectively, among controls. Frequency of the s‐allele in patients (0.533) was slightly higher than in controls (0.477), but the difference was not statistically significant. However, the odds ratios (ORs) for smoking or heavy drinking were markedly higher in SS and LS genotypes than in LL genotype; age‐sex‐adjusted ORs for smoking was 7.57 in the SS genotype, 6.40 in the LS genotype and 1.77 in the LL genotype. Age‐sex‐adjusted ORs for heavy drinking were 19.78, 18.20 and 7.40, respectively. The age‐sex‐adjusted ORs for both factors combined were 12.77, 18.45 and 1.44, respectively. These results suggested that the L‐myc polymorphism might modify the effects of lifestyle factors on esophageal cancer risk.

Opposite impact of NKG2D genotype by lifestyle exposure to risk of aerodigestive tract cancer among Japanese

It was reported that there are 2 haplotypes in natural killer complex (NKC) region. One of them could be divided by NKG2D polymorphism into 2 haplotype alleles (high and low natural killer (NK) cell activity) and were associated with overall cancer risks. However, its impact on a specific cancer is unclear. Therefore, by a case‐control study, we analyzed the association between NKG2D genotype and aerodigestive tract cancer risk. Subjects were 502 aerodigestive tract cancer patients (276 with head and neck, 226 with esophageal) and 1,004 sex‐age matched noncancer controls. Exposures to 2 lifestyle factors, smoking and drinking, were evaluated by a self‐administered questionnaire. The genotype of NKG2D was determined by the TaqMan method, and its impact was assessed by multivariable logistic regression models. Association strength was measured by the odds ratio (OR) and its confidence intervals (CI). An overall analysis revealed no statistically significant association between NKG2D genotype and the risk of aerodigestive tract cancer. However, we found protective effects of G allele among never smokers (OR 0.35; 95% CI 0.15–0.84) and never drinkers (0.42; 0.19–0.94). In contrary, increased risks were observed for G allele among heavy smokers (5.92; 3.23–10.85) and heavy drinkers (4.13; 2.29–7.47). Interactions between NKG2D genotype and lifestyle exposure were statistically significant (interaction p = 0.001 for smoking, 0.005 for drinking). The same trends were observed in both sexes, and in head and neck cancer and esophageal cancer independently. These results suggest an opposite impact of NKG2D genotype by lifestyle exposure to the risk of aerodigestive tract cancer among a Japanese population.

L‐myc Genotype is Associated with Different Susceptibility to Lung Cancer in Smokers

We have shown that L‐myc genotype is associated with the risk of esophageal cancer from smoking and heavy drinking. In this study, we have analyzed the relationship between the L‐myc genotypes and lung cancer risk from smoking in 191 Japanese lung‐cancer patients and 241 non‐cancer controls. The odds ratios (ORs) were markedly higher in SS and LS genotypes than in LL genotype; age‐sex‐adjusted ORs were 3.19, 2.30 and 0.92, respectively. This result suggests that the L‐myc polymorphism may affect the induction of lung cancer by smoking. The OR for smoking in SS‐genotype patients diagnosed within 2 years was higher than that in other SS patients, suggesting that smoking‐related lung cancer in SS genotype might exhibit a poorer prognosis.