Alterations of repeated sequences in 5' upstream and coding regions in colorectal tumors from patients with hereditary nonpolyposis colorectal cancer and Turcot syndrome.

One of the characteristics of tumors from patients with germline mutations of DNA mismatch repair genes is instability at microsatellite regions (MSI). We analysed alterations at repeated sequences of coding regions, as well as those of 5' upstream regions, in 29 MSI-High colorectal tumors from patients with hereditary nonpolyposis colorectal cancer (HNPCC) and Turcot syndrome. We found that repeated sequences in 5' upstream regions were altered in these tumors, at considerable frequencies. The (A)10 repeat in the promoter region (position -178 to approximately -169) of the GAPDH gene was altered in 17% of the tumors. The (A)10(TA)9 in the 5' upstream region (position -318 to approximately -291) of the mitochondrial isoleucyl tRNA synthetase gene (IleRS-A), coded in nuclear DNA, was altered in 59% of the tumors, whereas (A)9 in the 5' upstream region (position -859 to approximately -851) of cytoplasmic isoleucyl tRNA synthetase gene (IleRS-B) was not altered. Alteration at repeated sequences in the coding regions were 72% at TGFbetaRII(A)10, 24% at IGFIIR(G)8, 45% at BAX(G)8, 55% at E2F4(CAG)13, 66% at caspase-5 (A)10, 31% at MBD4(A)10, 55% at hMSH3(A)8 and 34% at hMSH6(C)8. The number of altered genes increased with the advancement of carcinoma according to Dukes categories: mean numbers of altered genes within these 10 genes were 2.6 for Dukes A, 4.7 for Dukes B and 7.8 for Dukes C. The mean number for adenomas was 2.0. These results suggest that the MSI phenotype also causes alteration of 5' upstream regions which may affect apoptosis and some mitochondrial functions in HNPCC and Turcot tumors, and that accumulation of altered genes with repeated sequences is associated with the progression of HNPCC and Turcot colorectal tumors.

[Two cases of alpha-fetoprotein producing gastric cancer, showing marked response to continuous hepatic arterial infusion (HAI) chemotherapy with adriamycin].

We report two cases of alpha-fetoprotein producing gastric cancer (AFPGC) with multiple liver metastases showing marked response to continuous HAI chemotherapy with adriamycin (ADM). In both cases, 5-FU 500 mg/body/day and Leucovorin (LV) 30 mg/body/day was infused continuously for 7 days and ADM 30 or 60 mg/body/day was infused continuously for 4 hours on day 7 as preoperative HAI chemotherapy. The primary gastric lesions were reduced and became resectable. After gastrectomy, they were treated with 4-hour continuous HAI of ADM 30 or 60 mg/body with or without 5-FU and LV once a week several times in our outpatient clinic. After these treatments, the multiple liver metastases were reduced remarkably, with a marked decrease of serum AFP levels. During these treatments, neither patient showed remarkable side effects, so they could work as before. This frequently low-dose ADM administration resulted in a high local response without severe side effects.

Detection of dichromate (VI)-induced DNA strand breaks and formation of paramagnetic chromium in multiple mouse organs.

DNA single-strand breaks (and/or alkali-labile sites) induced by Cr(VI) were evaluated with the alkaline single cell gel electrophoresis (SCG) (Comet) assay in five organs (liver, kidney, spleen, lung, and brain) of male mice dosed with K(2)Cr(2)O(7) (20 mg Cr/kg) by a single ip injection in vivo, and the formation of paramagnetic Cr(V) in these organs was investigated by electron spin resonance (ESR) spectrometry. Furthermore, the in vivo effects of deferoxamine (DFO), an iron chelator, and dimethylthiourea (DMTU), a hydroxyl radical scavenger, on the formation of Cr(V) and DNA strand breaks induced by the metal in the liver and kidney were examined. SCG assay detected DNA strand breaks were detected in the liver and kidney at 15 min and showed that they were being repaired at 3 h after Cr(VI) injection. The ESR spectra of paramagnetic Cr(V) were also observed in the liver and kidney for 15 min to 24 h after Cr(VI) injection. In contrast, there were no significant levels of DNA strand breaks and Cr(V) in the spleen, lung, or brain. The pretreatment of mice with DFO reduced the formation of Cr(VI)-induced DNA strand breaks and Cr(V) complexes as well as the total contents of Cr in the liver and kidney at 15 min after the metal injection. In the case of the pretreatment with DMTU, DNA strand breaks induced by Cr(VI) were suppressed in the liver and kidney at 15 min, without any influence on the levels of Cr(V) complexes and total Cr contents in the organs. The in vitro study showed that DFO decreased the levels of Cr(V)-GSH complexes and Cr(V)-mediated hydroxyl radicals, while DMTU reduced only the levels of Cr(V)-mediated hydroxyl radicals without affecting the formation of Cr(V)-GSH complexes. These results demonstrated that the SCG assay may be useful for detecting DNA strand breaks and/or alkali-labile sites caused by Cr(VI) in vivo. The results also indicated that the in vivo formation of hydroxyl radicals during the reduction of Cr(VI) may play an important role in the induction of the DNA strand breaks caused by this metal and implied that the levels of Cr(V) inside the cells may not always be related to the induction of DNA strand breaks.

Somatic mutations of LKB1 and beta-catenin genes in gastrointestinal polyps from patients with Peutz-Jeghers syndrome.

Peutz-Jeghers syndrome (PJS) is characterized by multiple gastrointestinal hamartomatous polyps, mucocutaneous melanin deposition, and increased risk of cancer, mainly in the gastrointestinal tract. We examined mutations of the LKB1, beta-catenin, APC, K-ras, and p53 genes in 27 gastrointestinal hamartomatous polyps from 10 patients in nine PJS families. Of these hamartomatous polyps, one intestinal polyp had an adenomatous lesion, and one gastric polyp contained adenomatous and carcinomatous lesions. Germ-line mutations of the LKB1 gene were detected in six PJS families. Somatic mutations of the LKB1 gene were found in 5 polyps, whereas loss of heterozygosity (LOH) at the LKB1 locus at 19p was seen in 14 other polyps. In adenomatous lesions microdissected from hamartomatous polyps, both beta-catenin mutation and 19p LOH were detected. Furthermore, a carcinomatous lesion in a gastric hamartomatous polyp was found to contain a mutation of the p53 gene and LOH at the p53 locus in addition to LOH at the LKB1 locus and a beta-catenin mutation. K-ras mutations were detected in a few polyps, whereas no APC mutation or 5q LOH was detected in hamartomatous polyps. These results suggest that gastrointestinal hamartomatous polyps in PJS patients develop through inactivation of the LKB1 gene by germ-line mutation plus somatic mutation or LOH of the unaffected LKB1 allele, and that additional mutations of the beta-catenin gene and p53 gene convert hamartomatous polyps into adenomatous and carcinomatous lesions.

[Indication for hepatic resection after hepatic arterial infusion chemotherapy for multiple liver metastases of colorectal cancer].

The indications for hepatic resection after hepatic arterial infusion chemotherapy (HAI) for unresectable metastatic liver tumor of colorectal cancer were analyzed from the surgical outcome of hepatic resections in 23 cases of hepatic resection after HAI. The mean duration of HAI until hepatic resection was 7.4 months (5-14 months). The total dose of 5-FU was 25.7 +/- 8.0 g for a CR + PR group and 14.0 +/- 3.5 g for a NC + PD group. There was a significant difference between two groups (p < 0.01). The group in which serum CEA level normalized after HAI (the normal CEA group) included 7 patients, and the group in which serum CEA level did not normalize (the high CEA level group) had 9 patients. The total dose of 5-FU was 30.0 +/- 7.6 g in the normal CEA level group and 19.1 +/- 6.9 g in the high CEA level group. There was a significant difference between the two groups. The 3-year survival rate was 40.0% in the group with the duration of HAI for longer than 8 months (n = 10) and 0% in the group with the duration of HAI for shorter than 8 months (n = 7). The 3-year survival rate was 66.7% in the normal CEA level group (n = 3) and 0% in the high CEA level group (n = 8). The surgical outcome was better in the HAI for longer than 8 month and normal CEA groups.

Histologic grade of metastatic lymph node and prognosis of rectal cancer.

PURPOSE: It is important to identify cases with a high risk of recurrence to improve the prognosis of colorectal cancer. In this study the difference between the histology of the primary lesion and that of the metastatic lymph node was investigated in an attempt to identify the cases with a high risk of recurrence. METHODS: One-hundred eighty-five patients with Dukes C rectal cancer who had undergone curative resection were investigated. The histologic grade of the metastatic lymph node was determined and compared with other clinicopathologic factors to determine its significance as a prognostic factor. RESULTS: The histologic grade was the same between the primary lesion and the metastatic lymph node in 46.2 percent of all cases, although in the group with well-differentiated adenocarcinoma at the primary lesion the concordance was only 29.5 percent. In the group with well-differentiated adenocarcinoma at the primary lesion, the five-year survival rate was 75.3, 64, and 25 percent in the groups with well-differentiated, moderately differentiated, and poorly differentiated adenocarcinoma at the metastatic lymph node, respectively. The differences between the survival rates of well-differentiated and poorly differentiated adenocarcinoma at the metastatic lymph node were statistically significant (P < 0.05). According to multivariate analysis the histologic grade of primary lesion was the most significant prognostic factor (hazard ratio: 2.2801, P = 0.0008). However, in well-differentiated adenocarcinoma of patients with Dukes C rectal cancer at the primary lesion, the histology of metastatic lymph node was also an important prognostic factor. CONCLUSIONS: It is clear that the histologic grade between the primary lesion and metastatic lymph node was frequently different, especially in the group with well-differentiated adenocarcinoma at the primary lesion. The analysis of the metastatic lymph node was considered to have additional importance for the prediction of prognosis.

[The prospects of the outpatient clinic or home anti-cancer chemotherapy for unresectable recurrence of colorectal cancer].

In this study, we analyzed the effectiveness of anti-cancer chemotherapy at the outpatient clinic. We administered CDDP 5 mg/day every 5 days for a week with continuous systemic chemotherapy by 5-FU 500 mg/day for 3 weeks in the hospital, and in the outpatient clinic CDDP 5 mg/day every two days for a week with UFT 300 mg/day orally every day to 145 patients with unresectable recurrence of colorectal cancer. The 50% survival duration after beginning of this chemotherapy was 310.0 days. One-year and 2-year survival rates were 44.4% and 11.7%. One hundred and nine cases were evaluated. The total response rate was 19.3%, and the highest response rate was seen in metastatic lymph nodes (37.5%). The mean duration of continuation of this chemotherapy and the mean duration of staying at home was 188.2 days and 237.8 days. The mean rate of home stays was 68.6%. The group of performance status (PS) 0 or improving PS, accounted for 74.5% of all cases. The occurrence rate of adverse effects was as follows; stomatitis: 17.9%, diarrhea: 11.7% and bone marrow suppression: 13.1%. However, the occurrence rate for cases of grade 3 or 4 was only 1-4%. According to these results, this was an excellent chemotherapy to maintain the patient's QOL from the outpatient clinic.

[Evaluation of the non-effective cases of irresectable multiple liver metastasis of colorectal cancer receiving hepatic arterial infusion chemotherapy].

PURPOSE: Hepatic arterial infusion (HAI) chemotherapy is one of the suitable therapies for irresectable multiple liver metastasis from colorectal cancer, but in nearly half of such cases the therapy does not prove effective. Our goal is to clarify the characteristics of non-effective cases. METHODS: 84 cases with irresectable multiple liver metastasis from colorectal cancer were investigated clinicopathologically, and were divided into two groups; non-effective cases (N = 38) and effective cases (N = 46). All cases received continuous arterial infusion chemotherapy using 5-FU according to the following regimen; 5-FU (500 mg/day) was infused in the hepatic artery over 7 or 10 days for induction, and the infusion was maintained (250 mg/day) to the hepatic artery for 7 days every other week after the induction therapy. We evaluated the efficacy of HAI chemotherapy by Computed Tomography. RESULTS: There were statistically significant differences among these two groups in histological types. Rates of the histological type of non-effective cases were well (31.6%), mod (57.9%), por (7.9%), and muc (2.6%), respectively. Those of the effective cases were well (63.0%), mod (34.8%), por (0%), and muc (2.2%), respectively. In non-effective cases, 16 out of 38 cases (42.1%) had extra-hepatic metastasis. On the other hand, only 3 out of 46 cases (6.5%) had such metastasis in effective cases. CONCLUSION: There were non-well type cancers and extra-hepatic metastasis in a large number of non-effective cases. We thought that those cases were basically high-grade malignancies, so these were the limits of HAI chemotherapy for irresectable multiple liver metastasis of colorectal cancers.

Frequent mutation of beta-catenin and APC genes in primary colorectal tumors from patients with hereditary nonpolyposis colorectal cancer.

Hereditary nonpolyposis colorectal cancer (HNPCC) is characterized by defective DNA mismatch repair, which results in genetic instability of tumors; however, only a few target genes have been recognized. Our previous study detected a low frequency of APC gene mutation (21%) in colorectal tumors from HNPCC patients, in contrast to a high frequency of APC gene alteration (>70%) in non-HNPCC tumors. Because both beta-catenin and ACP gene mutations have recently been shown to activate the same signaling pathway, we analyzed beta-catenin mutation in HNPCC tumors. A notable frequency of beta-catenin gene mutation (43%, 12 of 28) was found to occur in HNPCC colorectal tumors. Beta-catenin mutations were not detected in tumors with APC mutations. All beta-catenin mutations detected in HNPCC tumors existed within the regulatory domain of beta-catenin. Immunohistochemical staining of tumors with this mutation showed accumulation of beta-catenin protein in nuclei. These and previous data from our laboratory suggest that activation of the beta-catenin-Tcf signaling pathway, through either beta-catenin or APC mutation, contributes to HNPCC colorectal carcinogenesis in approximately 65% of cases.

Higher frequency of Smad4 gene mutation in human colorectal cancer with distant metastasis.

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.

Pseudomyxoma retroperitonei.

We report a rare case of pseudomyxoma retroperitonei in a 58-year-old woman with a past history of severe appendicitis. The imaging showed a multicystic mass similar to pseudomyxoma peritonei, but the tumor was located in the retroperitoneal space.

[Present and future of home anti-cancer chemotherapy for unresectable recurrence of colorectal cancer].

Continuous hepatic arterial infusion chemotherapy (HAI) was performed for 100 colorectal cancer patients with unresectable liver metastases in our outpatient clinic. The administration schedule was 5-FU 250 mg/day for 7 days every other week. In this study, 93 cases were evaluable. The average dose of 5-FU was 24 g (3.0-66.5 g). The response rate was 59.1% (CR: 4 cases, PR: 51 cases, NC: 12 cases and PD: 26 cases). The prognostic outcome was significantly prolonged in the effective cases (50% survival duration was as follows: CR: 764 days, PR: 620 days, NC: 255 days and PD: 152 days). The complication rate with HAI was 15.1%, but nobody died from these complications. Hepatectomy after HAI was performed in 19 cases (20.4%) because of the effect of HAI. The three-year survival rate was 35.3% for HAI with hepatectomy and 3.8% for only HAI. There was statistically significant difference (p < 0.0001) between these two groups. Hepatectomy after HAI is a new strategy for unresectable liver metastasis of colorectal cancer. Recently, we performed home systemic chemotherapy using 5-FU for 3 recurrent patients of colorectal cancer and were able to continue this therapy safely for more than 2 months.

Clinicopathological characteristics of skipping lymph node metastases in patients with colorectal cancer.

BACKGROUND: We have sometimes experienced cases of colorectal cancer with skipping lymph node metastasis in which distant nodes were positive but those closer to the tumor were negative. There have been few reports of this condition and its clinical characteristics have not been clarified. This study was conducted to clarify the status of skipping lymph node metastasis and its clinicopathological characteristics in colorectal cancer. METHODS: We analyzed 452 patients with colorectal cancer and nodal metastases (270 with colon cancer and 182 with rectal cancer). All the resected nodes were examined using histological procedures with a microscope and were classified by their location according to the General Rules for Clinical and Pathological Studies on Cancer of the Colon, Rectum and Anus. We studied the status of skipping nodal status and the correlation between the nodal status and clinicopathological findings, including the disease-free survival, depth of tumor, histological type, staging and recurrence. RESULTS: Twenty-eight (10.4%) of the colon cancer patients and 20 (11.0%) of the rectal cancer patients were found to have skipping nodal metastases. In rectal cancer patients with n2 (nodal metastases at the N2 site) in the direction of the main node, patients with skipping lymph node metastases had a significantly better prognosis than those without (p = 0.026). In all colon cancer patients and rectal cancer patients with lateral n3 (nodal metastases at the lateral N3 site), there were a tendency for those with skipping nodal metastases to have better disease-free survival rates (p = 0.1). Also, the mean number of positive nodes in skipping cases was significantly lower than that in non-skipping cases. In addition, skipping nodal metastases in rectal cancer suggested a possibility of bypass flow which was not generally recognized. CONCLUSION: These findings in colorectal cancer suggest the presence of previously unknown lymphatic tracts and that the cancers concerned have a better prognosis than those without skipping nodal metastases.

[Efficacy of intraoperative radiotherapy (IORT) for local recurrence of rectal cancer].

The efficacy of intraoperative radiotherapy (IORT) for the local recurrence of rectal cancer was evaluated. Operations were performed for a total of 43 patients with local recurrence of rectal cancer, and among them 13 cases received IORT. These patients were divided into two groups those with a suspicion of cancer remaining in the surgical margin macroscopically (EW+ group) or those with no such suspicion (EW- group). The length of pain relieving period and total survival between the IORT and no IORT groups in EW- patients showed no statistical difference. Though the same result was found in EW+ patients, in the patients whose area of positive surgical margin was less than 10 cm2 and given IORT, the pain relief period was prolonged compared with the no IORT patients such as 270 days v.s. 218 days. IORT did not show efficacy in terms of the survival period, but if the local remnant area was small, it was beneficial for the patients in terms of the pain period.

13th Meeting of the Scientific Group on Methodologies for the Safety Evaluation of Chemicals (SGOMSEC): alternative testing methodologies for ecotoxicity.

There is growing public pressure to minimize the use of vertebrates in ecotoxicity testing; therefore, effective alternatives to toxicity tests causing suffering are being sought. This report discusses alternatives and differs in some respects from the reports of the other three groups because the primary concern is with harmful effects of chemicals at the level of population and above rather than with harmful effects upon individuals. It is concluded that progress toward the objective of minimizing testing that causes suffering would be served by the following initiatives--a clearer definition of goals and strategies when undertaking testing procedures; development of alternative assays, including in vitro test systems, that are based on new technology; development of nondestructive assays for vertebrates (e.g., biomarkers) that do not cause suffering; selection of most appropriate species, strains, and developmental stages for testing procedures (but no additional species for basic testing); better integrated and more flexible testing procedures incorporating biomarker responses, ecophysiological concepts, and ecological end points (progress in this direction depends upon expert judgment). In general, testing procedures could be made more realistic, taking into account problems with mixtures, and with volatile or insoluble chemicals.

Radical resection with autonomic nerve preservation and lymph node dissection techniques in lower rectal cancer surgery and its results: the impact of lateral lymph node dissection.

Lateral lymph node metastases occur in 9% of rectal cancer patients. For cancers localized in the lower rectum below peritoneal reflection, the frequency increases to 13% of all cases and to 25.5% in those of Dukes' stage C. The most important technique in colorectal surgery for decreasing local failure in the pelvis is lateral lymph node dissection. Today, however, it is also regarded as crucial that with perfect lymph node dissection of the lateral area outside of the pelvic plexus, the postoperative functions of urination and sexual ability are preserved. Since 1988 we have performed autonomic nerve-preserving curative resection (ANP) with lateral dissection in most patients with advanced lower rectal cancer. The mean 5-year survival rate of patients with lateral lymph node metastasis from the lower rectum was 37.5%, improving from 32.1% to 43.4% during this period. In cases of ANP with lateral dissection the local recurrence rate was 4.8% overall and 7.4% in the Dukes' C group. Postoperative urinary function has been good or fair in all ANP patients. Sexual function remained problematic, especially regarding male ejaculation. We describe our method for preserving the autonomic nervous system in the pelvis and for achieving complete dissection in the lateral area.

[The outcome of the postoperative radiotherapy of autonomic nerve preserving operation for lower rectal cancer].

PURPOSE: It is unclear whether adjuvant radiotherapy before or after surgery is effective for locally advanced, resectable lower rectal cancer. This study consists of a prospective randomized trial of postoperative radiotherapy for locally advanced curatively resected lower rectal carcinoma. METHODS: We divided patients into two groups, one with postoperative 50 Gy radiation therapy to pelvic wall (N = 64), and the other with no radiation therapy (N = 46). RESULTS: The 5 year disease-free rate was 61.8% in the radiation group and 70.6% in the no radiation group. There was statistically no significant difference between these two groups. The local recurrence rate was 2.7% (radiation therapy group: 1.6%, no radiation therapy group: 4.3%). This local recurrence rate was very low. These results made us suspect that postoperative radiation therapy was not always necessary to prevent local recurrence. Postoperative complications had a higher incidence in the radiation therapy group than in the no-radiation therapy group. In the radiation therapy group, 35.5% of the patients suffered from diarrhea or frequent defecation, and 10.8% from severe abdominal pain. We operated on 4 cases of radiation-induced ileal stenosis and ileus. CONCLUSION: Postoperative radiation therapy did not help prevent local recurrence even though many complications resulted.

[The autonomic nerve preserving radical resection for the patients with lower rectal cancer].

From 1988, we have begun the autonomic nerve preserving radical lymph adenectomy for the patients with lower rectal advanced cancer. The most important point to perform this method is the correct and well acquainted anatomical knowledge with the lower pelvis especially for the lateral area and distal area of internal iliac artery. Although there is some possibility that the cancer cells remain around the nerve system in the case of advanced cancer with lymph node involving, the sharp dissection can be possible to achieve the radical lymphadenectomy with autonomic nerve preserving in lateral area. The 5 years survival rate of the total cases which was consisted with more than 40% of Dukes C cases was 83% and that of the patients with lateral lymph node metastasis was 56%. The local recurrence rate was 3.4% in all cases, and 7.2% in Dukes C cases. Concerning with post operative urinary function, the urinary function was excellent when the bi-lateral nerve system was preserved and fare in uni-lateral preservation. But as to the sexual function in male patients was not satisfactory in uni-lateral preservation, and fare in bi-lateral preservation.