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A 42‑year‑old woman presented with ~30 adenomatous polyps of the left sided‑colon with early rectosigmoid cancer. The patient had no previous medical history and no familial history of inherited colorectal disease. No germline gene mutations associated with colorectal adenomatous polyposis, including APC regulator of WNT signaling pathway, mutY DNA glycosylase, DNA polymerase‑ε, catalytic subunit, DNA polymerase δ1, catalytic subunit, and mismatch repair genes, were detected via germline genetic testing. A heterozygous germline mutation in methyl‑CpG binding domain 4, DNA glycosylase (MBD4), c.217C>T/p.Gln73*, which resulted in the generation of a stop codon, was identified by genetic analyses including whole‑exome sequencing. Immunohistochemical staining analysis revealed that the expression of MBD4 protein was absent in the cancer tissue, while it was expressed in the normal epithelium. Sequencing and copy‑number analyses demonstrated the loss of the remaining allele of MBD4 in the cancer tissue. Furthermore, somatic mutation signature analysis showed preferential transition of cytosine to thymine residues at CpG dinucleotides in cancer tissues. Although it has been previously reported that germline missense mutations and somatic mutations of MBD4 are associated with the development of colorectal cancer, this is the first report, to the best of our knowledge, in which a germline nonsense mutation of the MBD4 gene has been identified in an early‑onset colorectal cancer patient with oligopolyposis.

Genetic testing is needed for the treatment of colorectal cancer (CRC), especially molecular-targeted therapy. The effects of anti-EGFR therapy and prognosis are affected by the presence of KRAS mutations. However, whether primary CRC or metastatic tissues are appropriate in the analysis is still unclear. In the present study, we assessed the concordance of KRAS/BRAF mutation status and microsatellite instability (MSI) in primary CRC and corresponding metastases. This study enrolled 457 patients with surgically resected primary and corresponding metastatic CRC (499 synchronous metastases and 57 metachronous metastases) and seven local recurrences, and KRAS/BRAF mutation and MSI status were analysed for these tumours. The concordance rates of KRAS mutation, BRAF mutation, wild-type, MSI-H and MSS between primary CRC and corresponding metastases were 93.9% (214/228), 100% (30/30), 99.3% (304/306), 87.5% (21/24) and 100% (137/137), respectively. These high concordance rates were not different between synchronous and metachronous metastases. In conclusion, a high concordance of KRAS/BRAF mutation status and MSI status was observed between primary CRC and corresponding metastases in this study. Either primary CRC or metastatic tissues can be used for testing KRAS/BRAF mutation status and MSI status.

At present, molecular markers of colorectal cancer (CRC), including KRAS, NRAS and BRAF mutations, and the microsatellite status are evaluated for the development of personalized treatments. However, clinicopathological and molecular characteristics and the prognostic role of NRAS mutations remain unclear. In the present study, a total of 1,304 consecutive stage 0-IV CRC tumor samples were analyzed for KRAS (exon 2, 3 and 4), NRAS (exon 2 and 3) and BRAF (exon 15) mutations. Multivariate analysis was performed to assess the prognostic impact of NRAS mutations. KRAS, NRAS and BRAF mutations were identified in 553 (42.4%), 35 (2.7%), and 59 (4.5%) of 1,304 CRC cases, respectively. Tumors with NRAS mutations were more frequently located in the distal colorectum compared with those with KRAS or BRAF mutations. Multivariate analysis indicated that KRAS and BRAF mutations were found to be associated with poor prognosis [hazard ratio (HR)=1.44, 95% confidence interval (CI), 1.18-1.76 and HR=2.09; 95% CI, 1.33-3.28, respectively], whereas NRAS mutations were associated with a trend toward favorable prognosis (HR=0.53; 95% CI, 0.27-1.03). Characteristics and prognosis of CRC with NRAS mutations are different from those with KRAS or BRAF mutations.

The MSH6 gene is one of the mismatch repair genes involved in Lynch syndrome and its mutations account for 10-20% of Lynch syndrome. Although previous studies suggested that the difference of the geographical region affects the clinical phenotype of Lynch syndrome, there has been no report on the detailed features of Japanese Lynch syndrome patients carrying an MSH6 mutation. The aim of the present study was to investigate the clinical and molecular features of MSH6 mutation carriers in Japan. Surgically resected 1720 colorectal carcinoma specimens were screened by microsatellite instability (MSI) testing and the MSI-high cases were subjected to a germline mutation analysis of the mismatch repair genes MLH1, MSH2 and MSH6. We investigated the clinical and molecular features of the MSH6 variants, such as the family cancer history, pathological findings, immunohistochemistry, methylation status of the MLH1 promoter and BRAF mutation in the colorectal tumor. Furthermore, the impact of the missense variants on MSH6 protein was predicted by using in silico tools. We identified nine novel pathogenic mutations and eight unclassified missense variants. Among the eight missense variants, three were suspected pathogenic by in silico analysis. We also found that most colorectal cancers in the MSH6 mutation carrier were diagnosed after the age of 50 and were localized distally. Furthermore, the mean age at diagnosis of endometrial cancer in Japanese MSH6 mutation carriers (49.2 years) was earlier than previous reports from Western countries (56.5 years). These results may improve the surveillance program for Japanese MSH6 mutation carriers.

Antitumor effects of hyperthermia are enhanced by lowering the pH in the tumor tissue with administration of glucose. This decreased pH in the tumor tissue with glucose administration was determined using mouse experimental tumors. 31P-MRS microelectrodes were used for the measurement of pH. By using these two measurement methods, time course change in the tumor tissue was determined in the controls and the groups treated with 6 g/kg of intraperitoneal glucose. The determination of pH with 31P-MRS was calculated from the chemical shift of the peak of creatine phosphate (Pcr) and that of inorganic phosphate (Pi). Following glucose administration, the tumor tissue showed a decrease of 0.3 pH units with the microelectrode method, but did not show any significant decrease in pH with the MRS determination. This finding suggested that 31P-MRS showed intracellular pH (pHi) due to the localization of Pi and that the microelectrode indicated interstitial or extracellular pH (pHe). The ATP/Pi ratio obtained in tumor tissue 24 h after heat treatment (with, without glucose) was correlated with tumor inhibition.

The efficacy of temporary arterial embolization using degradable starch microspheres combined with hyperthermia was investigated in rabbits bearing VX2 tumors. Microsphere injection caused a marked decrease of tumor blood flow and pH. During heating, there was a marked increase of the maximum temperature in tumor tissue compared with normal muscle. Tumor growth was suppressed 330% times at 3 weeks after hyperthermia alone and 270% times following combined treatment with microspheres and hyperthermia. Damage to normal muscle tissue was mild. In conclusion, this combination therapy may be useful for causing selective tumor damage and reducing the effect on normal tissues.

Arterial ligation was combined with hyperthermia in rabbits with VX2 tumors implanted in the leg. For seven days after arterial ligation, blood flow was decreased and the pH was low in both normal muscle and tumor tissue. The temperature of normal muscle and tumor tissue increased faster and reached a higher level on heating immediately after ligation than without ligation. The antitumor effect of hyperthermia was stronger immediately after ligation than two or seven days afterwards. However, damage to normal muscle was severe with this combination therapy, so a better method of therapeutic arterial blockade is needed.

Arterial chemoembolization using degradable starch microspheres and adriamycin was combined with hyperthermia to treat advanced liver cancer. The prolonged peak adriamycin level in hepatic venous blood suggested that the drug persisted for longer in the liver after injection containing microspheres. Heating efficiency was increased more in tumor tissue than in normal liver tissue after embolization. This combined therapy was performed in eight patients with advanced liver cancer and was effective in three (complete or partial remission). The mean survival time was 25 weeks and there were no severe side effects. This combined therapy may be useful for liver cancer.

Irradiation, dose-dependently, increases the percentage of cells in the G2 + M phase and the duration of cell cycle arrest, reflected by changes in cell kinetics in the first 48 h. Cells in the G2 + M phase are considered radiosensitive but little is known about their proliferative ability. We studied proliferative ability of irradiated G2 phase-arrested cells following flow cytometric cell sorting using fluorescent Hoechst 33342 staining. Although proliferative ability of non-irradiated cells was not different between G1 and G2 + M phases, cells arrested in G2 + M after irradiation, especially at higher doses, showed less ability than cells in G1. Proliferative ability also correlated well with of G2 block duration.

The intracellular calcium ion concentration of SCK cells was measured after the cells were heated to determine the relationship between thermotolerance and intracellular calcium concentration. SCK cells, which are breast cancer cells that spontaneously arise in A/J mice, were used. Exponentially growing cells were trypsinized and incubated for 60 min with fura-2/AM at a concentration of 10 M. The incubation was carried out at a density of 1x106 cells/ml in serum-free RPMI medium. Thermotolerance was induced by incubating cells for 5 h at 37yC after heating them at 43yC for 30 min. When heated at 44yC in a calcium containing buffer, the intracellular calcium concentration of non-thermotolerant and thermotolerant cells increased continuously as the cells were heated for longer periods of time. The same increase in intracellular calcium concentration was seen in these two cell lines when heated at 43yC in a calcium-free buffer. Thus, our data indicated that intracellular calcium ion increased during heating due to an influx into the cytoplasm from both internal stores and the extracellular medium.

We investigated the effect of angiogenesis inhibitors (FR118487 and TNP-470) on two murine tumors (FM3A and SCC-VII) with and without hyperthermia. When angiogenesis inhibitors were administered immediately after tumor implantations there was no tumor growth. As the tumor diameter increased, the antitumor effect of the inhibitors decreased. When FR118487 was combined with hyperthermia, the antitumor effect was potentiated. This study suggested that angiogenesis inhibitors show their maximal effect against small tumors and have increased activity when combined with hyperthermia.