Kanji Kobayashi

Mutations of the β-Catenin Gene in Endometrial Carcinomas

To investigate the contribution of β‐catenin to the development of endometrial carcinoma, we searched for genetic alterations of the β‐catenin gene in primary endometrial carcinomas. Mutational analysis of exon 3 of the β‐catenin gene, encoding the serine/threonine residues for GSK‐3β phosphorylation, was performed for 35 tumors. Nucleotide sequencing analysis revealed that 5 tumors (5/35, 14%) contained mutations (S33C, S37C, S37F, T41A) that altered potential GSK‐3β phosphorylation sites. Each of the mutations resulted in the substitution of serine/threonine residues that have been implicated in the down‐regulation of β‐catenin through phosphorylation by GSK‐3β kinase. Furthermore, the incidence of β‐catenin mutations was significantly higher in early‐onset (3 of 5) than that in late‐onset tumors (2 of 30) (P=0.014, Fishers exact test). Replication error (RER)‐positive phenotype was not detected in tumors with the β‐catenin gene mutation, although 10 of 35 tumors revealed RER. We performed immunohistochemistry of β‐catenin in 17 cases for which tissue samples were available. We confirmed accumulation of β‐catenin protein in both the nucleus and cytoplasm in 3 tumors, including two in which amino acid alterations had occurred at codon 33 and 37. The other case had no mutation in exon 3. Our results suggested that mutations at serine/threonine residues involved in phosphorylation by GSK‐3β affected the stability of β‐catenin. Accumulation of mutant β‐catenin could contribute to the development of a subset of endometrial carcinomas, particularly those of the early‐onset type.

A Single Nucleotide Polymorphism in the Matrix Metalloproteinase-1 Promoter in Endometrial Carcinomas

Recent studies demonstrated that a single guanine insertion polymorphism in a matrix metalloprotease‐1 promoter created an Ets binding site and affected the elevation of the transcriptional level of matrix metalloproteinase‐1 (MMP‐1). Furthermore, in tumor cell lines derived from melanoma and breast cancer, the incidence of the 2G/2G genotype was significantly higher than that in the normal population. To evaluate the contribution of this polymorphism in endometrial carcinomas, we genotyped 100 endometrial carcinomas and then analyzed immunoexpression of MMP‐1 in these carcinomas. We found that endometrial carcinoma patients showed a significantly higher rate of 1G/2G or 2G/2G genotype than control individuals, and that tumors containing the 2G allele(a) expressed MMP‐1 protein more frequently than those with 1G/1G genotype. Therefore, the single nucleotide polymorphism at the MMP‐1 promoter affected the expression level of the MMP‐1 protein, which may result in the association with more aggressive character in endometrial carcinoma. Our result suggests that the presence of 2G polymorphism at the MMP‐1 promoter may be one of the risk factors for the development and/or progression of endometrial carcinoma.

Mutational analysis of mismatch repair genes, hMLH1 and hMSH2, in sporadic endometrial carcinomas with microsatellite instability.

Microsatellite instability, monitored by replication error (RER), bas been observed in both sporadic and hereditary types of endometrial carcinoma. In the hereditary tumors, this instability is considered to be caused by a germline defect in the DNA mismatch‐repair system. We previously reported that nearly one‐quarter of sporadic endometrial carcinomas examined revealed an RER‐positive phenotype at multiple microsatellite loci. To investigate the role of genetic alterations of DNA mismatch‐repair genes in sporadic endometrial carcinomas, we screened 18 RER(+) endometrial carcinomas for mutations of hMLH1 and hMSH2. Although we found no germline mutations, we detected two somatic mutations of hMLH1 in a single endometrial cancer; these two mutations had occurred on different alleles, suggesting that two separate mutational events had affected both copies of hMLH1 in this particular tumor. These data implied that mutations of hMLH1 or hMSH2 play limited roles in the development of sporadic endometrial carcinomas, and that the tumors with genetic instability might have alterations of other mismatch‐repair genes, such as hPMS1 and hPMS2, or of unknown genes related to the mismatch‐repair system.

Mutational Analysis of β-Catenin Gene in Japanese Ovarian Carcinomas: Frequent Mutations in Endometrioid Carcinomas

To investigate the contribution of the β‐catenin gene to the development of ovarian carcinomas, mutational analysis of exon 3 of the β‐catenin gene was conducted. We analyzed 61 primary ovarian carcinomas, consisting of 49 non‐endometrioid‐type and 12 endometrioid‐type tumors, for genetic alteration of the β‐catenin gene. Five carcinomas showed β‐catenin mutations (S37C, T41I, T41A), including 4 (33%) of 12 endometrioid‐type tumors and 1 (14%) of 7 mucinous‐type tumors. All of these mutations altered at the serine/threonine residues that are potential sites of GSK3‐β phosphorylation. We detected no carcinomas with interstitial deletion involving exon 3 of β‐catenin. Furthermore, we immunohistochemically studied 27 of the 61 ovarian carcinomas. Both nuclear and cytoplasmic β‐catenin expressions were demonstrated in 4 of the 27 ovarian carcinomas for which tissue samples were available for examination. All 4 cases exhibited mutations in exon 3 of β‐catenin, including a mucinous carcinoma. Our results suggested that β‐catenin gene mutation at potential GSK3‐β phosphorylation sites results in accumulation of β‐catenin protein within the cells and its translocation to nuclei. Accumulated β‐catenin protein may be involved in the development of endometrioid‐type ovarian carcinomas, and some mucinous‐type ovarian carcinomas.

Mutations in the BRCA1 gene in Japanese breast cancer patients

Predisposing germline mutations in the BRCA1 gene were identified recently in families with 17q‐linked breast and ovarian cancers. Using single‐strand conformation polymorphism (SSCP) analysis, we examined primary breast cancers for mutations in coding exons of BRCA1 in a panel of 103 patients, of whom all either represented early‐onset cases (< 35 of age), were members of multiply‐affected families, and/or had developed bilateral breast cancers. Mutations were detected in tumors from four patients, all of whom had developed breast cancers bilaterally: a frame‐shift due to a 2‐bp deletion at codon 797; a nonsense mutation at codon 1214; and two missense mutations, one at codon 271 leading to Val → Met substitution, and the other at codon 1150 leading to Pro → Ser substitution. In each case the same mutation was present in constitutional DNA. The mean age of onset was 49 years among the Japanese carriers of BRCA1 mutations identified in this study, in contrast to the mean age of 35 observed among carriers of BRCA1 mutations in a similar U.S. study (Futreal et al., 1994). The evidence reported here supports a rather limited role of BRCA1 in breast carcinogenesis.

Mutation of the SRC gene in endometrial carcinoma.

Recently, an activating mutation of the SRC Gene has been implicated in about one‐tenth of advanced colon cancers. The SRC 531 mutation results in truncation of SRC directly C‐terminal to the regulatory Tyr 530 and appears to activate the Tyr 530. To investigate whether mutation of SRC plays an important role in the development and progression of gynecological tumors, we performed mutational analysis of the entire coding region of SRC in 70 ovarian carcinomas, 68 endometrial carcinomas and 3 endometrial stromal sarcomas by means of polymerase chain reaction‐single strand conformation polymorphism (PCR‐SSCP) followed by nucleotide sequencing and restriction fragment length polymorphism (RFLP) analysis. We found one truncated mutation at codon 531 (Gln to Stop) in an endometrial carcinoma. However, we found no mutation of this gene in ovarian carcinoma or endometrial stromal sarcoma. Our results suggest that mutation of SRC may be implicated in a small proportion of endometrial carcinomas.

Mutational Analysis of STK11 Gene in Ovarian Carcinomas

Recently STK11,the causative gene of Peutz‐Jeghers syndrome (PJS) was identified on chromosome 19p13.3. PJS is often accompanied by several malignancies, including breast tumor, adenoma malignum of the uterine cervix, and ovarian tumor. To investigate the involvement of STK11 gene in the development of ovarian carcinomas, we analyzed 30 ovarian carcinomas for loss of hetero‐zygosity (LOH) and STK11 gene mutations. We found one missense mutation (codon 281, Pro to Leu) with heterozygous and somatic status. This mutation occurred at codon 281, which lies within the mutational hot spot (codon 279‐281) of STK11 gene previously reported in PJS. We also detected LOH in 2 (11%) of 19 informative ovarian carcinomas. Our results suggest that mutations of the STK11 gene may play a limited role in the development of ovarian carcinomas.

Telomerase activity in malignant ovarian tumors with deregulation of cell cycle regulatory proteins.

Using a semiquantitative telomeric repeat amplification protocol assay, telomerase-positive frequencies and enzyme levels were measured. Out of 95% of 49 human ovarian tumors, the highest level of telomerase activity was observed in malignant tumors. Furthermore, by immunohistochemical staining of cell cycle regulatory proteins (pRB, p16, cyclin D1, cyclin E and p53) at the G1 checkpoint, we evaluated the relation between each protein alterations and the levels of telomerase activity. We could not demonstrate a clear relation with each molecule except for cyclin E, but suggesting that aberrant accumulation of these proteins was considered as a reason for telomerase deregulation, which may play an essential role in the pathway of telomerase regulation.

Combination Therapy with Granisetron, Methylprednisolone and Droperidol as an Antiemetic Prophylaxis in CDDP-Induced Delayed Emesis for Gynecologic Cancer

Objective: To better control both acute and delayed emesis resulting from cisplatin(CDDP)-based chemotherapy for gynecological malignancies, we designed a ‘cocktail therapy’ (CCT) using granisetron (GRN) in combination with methylprednisolone (MPD) plus droperidol (DRP). Methods: Two crossover clinical trials were carried out to compare the efficacy and safety of (a) GRN alone (3 mg/patient) with that of GRN, MPD (250 mg/patient) and DRP (0.5 ml/patient) in 42 patients (CCT group) and (b) GRN and MPD (CMB group) with that of the CCT group in 27 patients during the first 7 days of chemotherapy, independent of the weight/body surface of the patients. One of these regimens was administered intravenously for the first 3 days of chemotherapy, in case of failure for a maximum of 5 days. Results: For acute emesis, complete protection from nausea and vomiting by the end of the 1st day was achieved in 64.3% receiving GRN and in 92.9% receiving CCT (p < 0.01). For delayed emesis, complete protection was best achieved in CCT on days 2–3, showing statistical significance compared to GRN treatment (p < 0.01). Comparing the three kinds of treatment during 7 days, the lowest protection was 38.1% in the GRN group, 51.9% in the CMB group and 72.5% in the CCT group, especially on days 2 or 3. Conclusions: The CCT combination is useful for the control of delayed and/or anticipatory emesis resulting from CDDP-based chemotherapy for women with gynecological malignancies.

Clinical Factors and Biomarkers Which Affect a New Universal Grading System for Ovarian Epithelial Carcinoma

Objective: To detect clinical factors and biomarkers which affect a new grading system for ovarian epithelial cancer that was proposed by Shimizu et al. and to analyze the impact of those factors on malignant behaviors of the tumor.