Osamu Usuba

Distinct methylation patterns of two APC gene promoters in normal and cancerous gastric epithelia

The adenomatous polyposis coli (APC) tumor suppressor gene is mutationally inactivated in both familial and sporadic forms of colorectal cancers. In addition, hypermethylation of CpG islands in the upstream portion of APC, a potential alternative mechanism of tumor suppressor gene inactivation, has been described in colorectal cancer. Because a subset of both gastric and colorectal cancers display the CpG island methylator phenotype, we hypothesized that epigenetic inactivation of APC was likely to occur in at least some gastric cancers. APC exhibits two forms of transcripts from exons 1A and 1B in the stomach. Therefore, we investigated CpG island methylation in the sequences upstream of exons 1A and 1B, i.e., promoters 1A and 1B, respectively. We evaluated DNAs from 10 gastric cancer cell lines, 40 primary gastric cancers, and 40 matching non-cancerous gastric mucosae. Methylated alleles of promoter 1A were present in 10 (100%) of 10 gastric cancer cell lines, 33 (82.5%) of 40 primary gastric cancers, and 39 (97.5%) of 40 non-cancerous gastric mucosae. In contrast, promoter 1B was unmethylated in all of these same samples. APC transcripts from exon 1A were not expressed in nine of the 10 methylated gastric cancer cell lines, whereas APC transcripts were expressed from exon 1B. Thus, expression from a given promoter correlated well with its methylation status. We conclude that in contrast to the colon, methylation of promoter 1A is a normal event in the stomach; moreover, promoter 1B is protected from methylation in the stomach and thus probably does not participate in this form of epigenetic APC inactivation.

Molecular Characterization of Undifferentiated-Type Gastric Carcinoma

As the great majority of gastric cancers develop histologically differentiated, and a significant proportion of differentiated-type carcinomas progress to become undifferentiated, both histological types are likely to share several common genetic abnormalities, such as p53 mutations at advanced stages. However, a subset of gastric cancers develop as undifferentiated carcinomas, including signet-ring cell carcinoma and poorly differentiated adenocarcinoma, and the molecular pathogenesis of this tumor type remains largely unknown. To characterize the molecular features of undifferentiated-type gastric carcinomas that developed as undifferentiated-type, we examined for p53, APC, and epithelial (E)-cadherin gene mutations, microsatellite alterations including loss of heterozygosity (LOH) and microsatellite instability (MSI), and hypermethylation of the E-cadherin gene promoter in 26 early undifferentiated gastric carcinomas, consisting of 14 signet-ring cell carcinomas and 12 poorly differentiated adenocarcinomas. E-cadherin expression was evaluated immunohistochemically. p53 mutations were detected in only one poorly differentiated adenocarcinoma sample (3.8%; 1/26), whereas no APC or E-cadherin mutations were found. LOH was present only at D8S261 on the short arm of chromosome 8 in 2 of 14 (14%) informative tumors, both of which were poorly differentiated adenocarcinomas, and MSI was not observed in any of the tumors. No signet-ring cell carcinomas have been found to carry gene mutations or microsatellite alterations. In contrast, hypermethylation of the E-cadherin promoter occurred in 69% (18/26) of the tumors; 57% (8/14) of signet-ring cell carcinomas, and 83% (10/12) of poorly differentiated adenocarcinomas, and was significantly associated with loss or reduced expression of E-cadherin. Thus, whereas tumor suppressor gene mutation, LOH, and MSI were less common in undifferentiated-type early gastric carcinomas, epigenetic inactivation of E-cadherin via promoter hypermethylation may be an early critical event in the development of undifferentiated tumors.

Frequent loss of expression without sequence mutations of the DCC gene in primary gastric cancer

Loss of heterozygosity (LOH) on chromosome 18q21 is frequently found in various human cancers, suggesting the presence of tumour suppressor gene(s) in this chromosomal region. DCC is the most likely target of LOH because loss or reduction of DCC expression has been found in many types of cancers. However, few reports have focused on sequence mutations of this gene. We investigated sequence mutations and expression of DCC in primary gastric cancers. We studied mutations in 25 of the 29 DCC exons by PCR-SSCP in 17 primary gastric cancers exhibiting LOH on 18q21. No mutations of DCC were found in any of the tumours, although 78% (47/60) of the primary tumours showed apparent loss or reduction of DCC expression by immunohistochemistry. Analysis of methylation status of DCC revealed that methylation frequently occurred in both primary tumours (75%; 45/60) and corresponding non-cancerous gastric mucosae (72%; 43/60). Methylated status of DCC was significantly correlated with the loss of DCC expression in primary tumours (P < 0.01). These results indicate that DCC is frequently silenced, probably by epigenetic mechanisms instead of sequence mutations in gastric cancer. http://www.bjcancer.com

Analysis of genetic and epigenetic alterations of the PTEN gene in gastric cancer

Abstract. The PTEN tumor suppressor gene on 10q23.3, responsible for the Cowden and Bannayan-Zonana syndromes, encodes a dual-specificity phosphatase able to dephosphorylate both tyrosine phosphate and serine/threonine phosphate residues. Mutational inactivation of PTEN has been reported in various malignancies, including endometrial cancers, ovarian cancers, and glioblastomas. In this study, we investigated PTEN gene mutations in 10 gastric cancer cell lines and 58 primary gastric cancers by polymerase chain reaction single strand conformation polymorphism (PCR-SSCP). Hypermethylation of promoter region CpG islands, an alternative mechanism of gene inactivation to coding region mutations, was also evaluated by methylation specific PCR (MSP). Only one (1.7%) of the 58 primary tumors carried a somatic 5-bp deletion in intron 7 of PTEN, which did not alter the mRNA sequence, and no mutations were detected in any of the cell lines. Similar levels of PTEN mRNA expression were observed in all cell lines and primary tumors studied by RT-PCR, and PTEN promoter CpG islands remained unmethylated. Therefore, we conclude that PTEN does not participate in gastric carcinogenesis as a tumor suppressor gene.

Establishment of a Human Monoclonal Antibody to Hanganutziu-Deicher Antigen as a Tumor-associated Carbohydrate Antigen

We have established a human‐human hybridoma producing a monoclonal antibody against a tumor‐associated carbohydrate‐specific antigen, Hanganutziu‐Deicher (HD) antigen. Human spleen lymphocytes from a patient with esophageal varices complicated with liver cirrhosis were cultured in serum‐free medium and co‐stimulated with both anti‐human μ‐chain antibodies and supernatants of concanavalin A‐stimulated human spleen cell culture (ConA sup). The activated lymphocytes were subsequently primed in vitro with particulate HD3 antigen and fused with a parent hybrid myeloma cell line, KR‐12. A hybridoma, 1F43E31G7 produced anti‐HD human monoclonal antibody (IgM λ). This monoclonal antibody reacted strongly with N‐glycolyl‐neuraminyl α2‐3 lactosylceramide (HD3) and slightly with N‐glycolylneuraminyl α2‐3 lactoneotetraosylceramide (HD3), but did not react with N‐glycolylneuraminyl α2‐3 lactoneohexaosylceramide (HD7), N‐acetylneuraminyl α2‐3 lactosylceramide (GM3) and other derivatives of HD3 prepared by chemical modification of the sialic acid residue of HD3, which indicates that the monoclonal antibody is directed precisely toward the terminal sialic acid and whole structure of HD3.

Expression of acetylated and dimethylated histone H3 in colorectal cancer.

Aim: The expression of acetylated and dimethylated histone H3 in colorectal cancer was examined by immunohistochemistry and chromatin immunoprecipitation (ChIP)/Western blot (WB) assay. The correlation between the expression of histone H3 and clinicopathological findings was analyzed. Methods: Formalin-fixed and paraffin-embedded sections obtained from 80 operated cases of colorectal cancer were immunostained with anti-acetylated histone H3 (H3Ac) antibody and anti-dimethylated histone H3 lysine 4 (H3K4) antibody. Positive immunoreactivity was evaluated using the Allred scoring system. Furthermore, the expression was confirmed by ChIP/WB assay using formalin-fixed and paraffin-embedded sections. Results: There was good correlation between immunostaining and expression on ChIP/WB assay (p = 0.0005). There was a significant difference between the Allred score of H3K4 and the depth of tumor invasion (p = 0.0003) and the pathological stages (p = 0.0065). In overall survival classified by Allred scores of H3Ac (p = 0.0072) and H3K4 (p = 0.0187), the highest scores represented significantly worse prognoses than the other scores. Specifically, in stages II and III, the highest scores represented significantly worse prognoses than the other scores (p < 0.0001 and p = 0.0173, respectively). Conclusion: The expression of H3Ac and H3K4 may estimate patient prognosis.

Evaluation of salvage surgery for type 4 gastric cancer

Patients with type 4 gastric cancer and peritoneal metastasis respond better to chemotherapy than surgery. In particular, patients without gastric stenosis who can consume a meal usually experience better quality of life (QOL). However, some patients with unsuccessful chemotherapy are unable to consume a meal because of gastric stenosis and obstruction. These patients ultimately require salvage surgery to enable them to consume food normally. We evaluated the outcomes of salvage total gastrectomy after chemotherapy in four patients with gastric stenosis. We determined clinical outcomes of four patients who underwent total gastrectomy as salvage surgery. Outcomes were time from chemotherapy to death and QOL, which was assessed using the Support Team Assessment Schedule-Japanese version (STAS-J). Three of the patients received combination chemotherapy [tegafur, gimestat and otastat potassium (TS-1); cisplatin]. Two of these patients underwent salvage chemotherapy after 12 and 4 mo of chemotherapy. Following surgery, they could consume food adequately and their STAS-J scores improved, so their treatments were continued. The third patient underwent salvage surgery after 7 mo of chemotherapy. This patient was unable to consume food adequately after surgery and developed surgical complications. His clinical outcomes at 3 mo were very poor. The fourth patient received combination chemotherapy (TS-1 and irinotecan hydrochloride) for 6 mo and then underwent received salvage surgery. After surgery, he could consume food adequately and his STAS-J score improved, so his treatment was continued. After the surgery, he enjoyed his life for 16 mo. Of four patients who received salvage total gastrectomy after unsuccessful chemotherapy, the QOL improved in three patients, but not in the other patient. Salvage surgery improves QOL in most patients, but some patients develop surgical complications that prevent improvements in QOL. If salvage surgery is indicated, the surgeon and/or oncologist must provide the patient with a clear explanation of the purpose of surgery, as well as the possible risks and benefits to allow the patient to reach an informed decision on whether to consent to the procedure.

Successful resection of a liver metastasis from gastric leiomyoblastoma: report of a case.

A 20-year-old woman was referred to our hospital for detailed investigation of a gastric submucosal tumor. A leiomyoma was preoperatively diagnosed and laparoscopic-assisted enucleation was performed. The resected tumor was 4 × 3 × 1.5 cm in size and postoperative histological examination identified it as a gastric leiomyoblastoma. Therefore, a secondary resection in the form of a distal gastrectomy was carried out. No tumor cells were found in the gastric specimen or in the lymph nodes; however, 5 months after the operation, an abdominal computed tomography scan revealed a recurrence in the liver, and she was readmitted for further examinations. The lesion was diagnosed as a single liver metastasis from the gastric leiomyoblastoma and successfully resected. The histopathological findings of the liver tumor resembled those of the primary gastric tumor. Her postoperative course was uneventful and she has been well, without any evidence of recurrence, to date. Only 12 other cases of leiomyoblastoma of the stomach with liver metastasis have been reported in Japan, all of which were associated with a very poor prognosis. Therefore, patients with this unusual disease entity should be carefully followed up after resection of the primary tumor.

Tu1575 Conservative Treatment and Interval Appendectomy for Acute Appendicitis

BACKGROUND Conservative treatment for acute appendicitis (AA) is gradually being adopted as valuable therapeutic choice. Interval appendectomy (IA) after conservative treatment is controversial. OBJECTIVE To clarify the success and recurrence rate of conservative treatment for acute appendicitis, and necessity of IA Patients and Methods We reviewed 503 patients with AA between 2006 and 2013. RESULTS In 503 patients, of which 122 patient undergone emergency appendectomy within 3 days after admission, remaining 381 patients underwent conservative treatment. In 381 patients taken conservative treatment, the success rate of conservative treatment was 98%. Nine patients (2%) were required appendectomy because of progressive disease despite of conservative treatment. After conservative treatment 95 patients (27%) were diagnosed as a recurrent AA at median follow-up 18 months. The predictor for recurrence after conservative treatment was the only following multiple episodes of AA (p=0.002, HR 2.1, 95%CI [1.56-41.1]). The abscess formation and appendicolith did not predict the disease recurrence. At the recurrent AA, 37 of 95 (39%) patients underwent appendectomy including IA. The morbidity of appendectomy for recurrent disease was 5%. Especially the morbidity of IA for recurrent disease was 0%. CONCLUSIONS The success rate of conservative treatment was very high and the risk of recurrence after conservative treatment was 27%. The morbidity of appendectomy for recurrent disease was low (5%). The conservative treatment may be an effective alternative to emergency appendectomy. The routine IA is probably not warranted following successful management of AA, but IA should be required to patients with multiple episodes of AA. Morbidity rate of appendectomy by timing of operation