Osamu Nunobiki

Murine double‐minute 2 homolog single nucleotide polymorphism 309 and the risk of gynecologic cancer

A functional T to G germline polymorphism in the promoter region of murine double-minute 2 homolog single nucleotide polymorphism 309 (MDM2-SNP309) has been reported to profoundly accelerate tumor formation, suggesting that it may also represent a powerful cancer predisposing allele. In this study, MDM2-SNP309 was examined in a total of 400 blood samples from 108 normal, 88 cervical, 119 endometrial and 85 ovarian cancer cases using two independent polymerase chain reaction assays for each allele. When the MDM2-SNP309 genotype was classified into two subgroups of TT+TG and GG, the GG genotype was associated with an increased risk for the development of endometrial cancer (odds ratio [OR] = 1.91, 95% confidence interval [CI] = 1.05 to 3.47) compared with the TT+TG genotype (P = 0.0353). The G allele also increased the risk of endometrial cancer (OR = 1.20, 95% CI = 0.83 to 1.74) compared with the T allele, but no statistical difference was found (P = 0.3333). The homozygous GG genotype was also associated with postmenopausal status and type I endometrial cancer (P = 0.0306 and 0.0326, respectively). There was no significant difference in the genotype or allele prevalence between control subjects and cervical or ovarian cancer patients. These results suggest that homozygous GG genotype of MDM2-SNP309 may be a risk factor for postmenopausal and type I endometrial cancer in a Japanese population.

Polymorphisms of p53 codon 72 and MDM2 promoter 309 and the risk of endometrial cancer

Genetic polymorphisms of p53 and its negative regulator murine double minute 2 homolog (MDM2) have been shown to be closely associated with tumorigenesis in a variety of human cancers. In the present study, single nucleotide polymorphism (SNP) at p53 codon 72 and MDM2 promoter 309 was examined for germline DNA samples from 102 endometrial cancer cases and 95 controls using polymerase chain reaction-based fragment analysis. There were no significant differences in the genotype and allele prevalence between control subjects and endometrial cancer patients for p53 codon 72. The GG genotype frequency of MDM2-SNP309 was statistically higher in endometrial cancer patients than that in normal healthy women when compared with the TG genotype (P = 0.0088). However, no statistically significant differences were found between the TT and TG or GG genotype frequencies and allele prevalence. Interestingly, the combination of the homozygous Arg/Arg genotype of p53 codon 72 and homozygous GG genotype of MDM2 SNP309 polymorphisms was significantly associated with the risk of endometrial cancer (odds ratio = 3.28, 95% confidence interval = 1.13 to 9.53, P = 0.0212). The homozygous variants of wild p53 codon 72 and mutant MDM2 promoter 309 may cooperatively increase the risk of endometrial cancer in a Japanese population.

Genetic Polymorphism of Cancer Susceptibility Genes and HPV Infection in Cervical Carcinogenesis

It is widely accepted that specific human papillomavirus (HPV) types are the central etiologic agent of cervical carcinogenesis. However, a number of infected women do not develop invasive lesions, suggesting that other environmental and host factors may play decisive roles in the persistence of HPV infection and further malignant conversion of cervical epithelium. Although many previous reports have focused on HPV and environmental factors, the role of host susceptibility to cervical carcinogenesis is largely unknown. Here, we review the findings of genetic association studies in cervical carcinogenesis with special reference to polymorphisms of glutathione-S-transferase (GST) isoforms, p53 codon 72, murine double-minute 2 homolog (MDM2) gene promoter 309, and FAS gene promoter -670 together with HPV types including our recent research results.

MDM2 SNP 309 human papillomavirus infection in cervical carcinogenesis.

OBJECTIVE To investigate the biological significance of single nucleotide polymorphism (SNP) at murine double-minute 2 homolog (MDM2) promoter 309 in cervical carcinogenesis. METHODS SNP at MDM2 promoter 309 (T/G) together with human papillomavirus (HPV) types was examined in a total of 195 cervical smear samples and 8 human cervical squamous carcinoma cell lines using two independent PCR assays and PCR-RFLP techniques. RESULTS Forty-one patients with high-grade squamous intraepithelial lesion (HSIL) had higher frequency of high-risk HPV than 102 with low-grade SIL (LSIL) and 52 controls. There was an increased OR (8.88; CI=2.34-33.63; P=0.003) for TG+GG genotype in HSIL cases compared to controls among 68 patients with high-risk HPV. Twenty-one cases with HPV types 16 and/or 18 had significantly higher frequency of the TG+GG genotype and G allele than 47 with other types of high-risk HPV. Seven of 8 cervical carcinoma cell lines also showed TG or GG genotype. CONCLUSION MDM2-SNP309 (T/G) and high-risk HPV infection may be closely associated with cervical carcinogenesis in a Japanese population.

Germline polymorphism of cancer susceptibility genes in gynecologic cancer.

The multifactorial process of carcinogenesis involves mutations in oncogenes, or tumor suppressor genes, as well as the influence of environmental etiological factors. Common DNA polymorphisms in low penetrance genes have emerged as genetic factors that seem to modulate an individual’s susceptibility to malignancy. Genetic studies, which lead to a true association, are expected to increase understanding of the pathogenesis of each malignancy and to be a powerful tool for prevention and prognosis in the future. Here, we review the findings of genetic association studies of gene polymorphisms in gynecologic cancer with special reference to glutathione-S-transferase, FAS/CD95 and p53 genes including our recent research results.

Mutational analysis of the BRAF gene in human tumor cells

Genes of the RAF family, which mediate cellular responses to growth signals, encode kinases that are regulated by RAS and participate in the RAS, RAF, mitogen/extracellular signal-regulated kinase, extracellular signal-regulated kinase and mitogen-activated protein kinase pathway. As BRAF is a serine/threonine kinase that is commonly activated by somatic point mutation, it may provide possible diagnostic and therapeutic targets in human malignant tumors. We analyzed exon 15 of the BRAF gene for mutations in 58 lung, 12 breast, six kidney, 14 cervical, four endometrial and 10 ovarian carcinoma cell lines by PCR-SSCP and direct sequencing. The T1796A transversion was found in one (2.9%) of 34 small cell lung carcinoma and one (8.3%) of 12 breast carcinoma cell lines, resulting in a valine-to-glutamate substitution at residue 599 (V599E). One (4.2%) of 24 non-small cell lung carcinoma cell line showed the C1786G transversion, leading to a leucine-to-valine substitution at residue 596 (L596V). No BRAF point mutations were found in any of the other cell lines examined. Our present results suggest that BRAF may not be a frequent target of mutations involved in the pathogenesis of human lung, breast, kidney, cervical, endometrial and ovarian carcinomas.

Adrenomedullin, Bcl-2 and microvessel density in normal, hyperplastic and neoplastic endometrium

Adrenomedullin (ADM) is a multifunctional 52‐amino acid peptide involved in numerous physiological and pathological processes, including angiogenesis, growth regulation, differentiation, and vasodilation. ADM is thought to act through the G protein‐coupled receptor calcitonin receptor‐like receptor, with specificity being conferred by receptor‐associated modifying protein 2. The aim of the present study was to clarify the roles of ADM status, and tumor vessels in endometrium. Specimens were examined for ADM, microvessel density (MVD), area of venules (AV) and Bcl‐2 oncoprotein using an immunoperoxidase method. The difference of ADM between normal proliferative phase and hyperplasia without atypia was significant (P < 0.05). The level of Bcl‐2 was significantly different between hyperplasia without atypia and hyperplasia with atypia (P < 0.05). ADM, MVD and AV in the endometrium increased in a stepwise manner from normal, simple or complex hyperplasia with or without atypia to grade 1 adenocarcinoma. In contrast, expression of Bcl‐2 oncoprotein was decreased. These parameters identify the role of ADM expression and Bcl‐2 protein in relation to cell growth and vasodilating in the neoplastic changes.

Germline polymorphisms of glutathione-S-transferase GSTM1, GSTT1 and p53 codon 72 in cervical carcinogenesis

The clinical significance of glutathione-S-transferase GSTM1, GSTT1 and p53 codon 72 polymorphisms in cervical carcinogenesis was investigated. Germline polymorphisms of GSTM1, GSTT1 and p53 codon 72 together with human papillomavirus (HPV) types were examined in a total of 457 blood and cervical smear samples from normal healthy women and the patients with premalignant and malignant cervical lesions. The 167 patients with low-grade squamous intraepithelial lesion (LSIL), 49 with high-grade SIL (HSIL) and 83 with squamous cell carcinoma (SCC) had significantly higher frequency of high-risk HPV than 1 58 controls. The 49 patients with HSIL and 83 with SCC had statistically higher frequency of null GSTT1 genotype than 158 controls. There was an increased odds ratio for null GSTT1 genotype in HSIL and SCC cases compared with controls among 191 patients with high-risk HPV. The 67 cases with HPV types 16 and/or 18 had higher frequency of the GSTT1 null genotype than 186 with other types of HPV. There was no statistical difference in the polymorphic frequency of GSTM1 and p53 codon 72 genotypes between SILs and controls with or without high-risk HPV. These results suggest that GSTT1 null genotype may increase the risk of cervical cancer particularly in the cases with high-risk HPV types in a Japanese population.

A case of pituitary adenocarcinoma in which tumor cells were detected in cerebrospinal fluid: Immunocytochemical and immunoelectron microscopic studies.

今回, 脳脊髄液中に腫瘍細胞を認めた下垂体腺癌の1例を経験したので報告する.患者は48歳女性で, CT上鞍上槽に加え左前頭葉に伸展する腫瘍を確認し, 最終的には左後頭葉, 脳幹部, 両側小脳橋角部および脊髄にも転移がみられた.細胞像: 脳脊髄液 (CSF) 中の腫瘍細胞は大小不同著明な類円形細胞が, 散在性およびシート状に認められた.少数ではあるが広い細胞質に多くの空胞がみられる大型の多核巨細胞も散見された.免疫細胞化学所見: CSF中の腫瘍細胞を用いてGH, ACTHおよびProlactin反応を行った結果, 腫瘍細胞の細胞質には著明なGH陽性の褐色陽性顆粒を認めた.ACTHおよびPRL反応は陰性であった.免疫電子顕微鏡所見: CSF中の腫瘍細胞を用いてProtein A-gold法を用いたGH反応を行った結果, 腫瘍細胞の細胞質には多数のミトコンドリアと電子密度の高い直径約200nmの分泌穎粒が認められた.GI {反応陽性のgold顆粒は円形分泌顆粒のみに一致して陽性所見を認めたことより, この腫瘍細胞は周囲のGHを取り込んだものではなく, 腫瘍細胞自身がGHを産生していることが証明された.

A new technique for preparation of endometrial cell samples.

子宮内膜細胞診の判定は, 細胞個々の示す異型だけでなく, 病理学的な「組織構築」の異常を観察することも大切である. 著者らは細胞採取から標本作製に至る手技について改良を加え, 子宮腔内での組織構築が可能な限り推定しうる標本作製法を開発した.すなわち従来の直接塗抹標本作製法に対して組織構築が保たれた状態で観察できるように, 以下の3項目について工夫および改良を行った.1) 細胞採取後, ただちに塗抹することなく器具に付着する細胞および細胞集塊を生理的食塩水中に洗い落とし液状検体とする.2) 上記液状検体にメンブレンフィルターを用い判定の障害となる赤血球を除去する.3) 赤血球除去後のメンブレンフィルター上の細胞集塊の上にさらにほかのメンブレンフィルターを重ねて圧挫し, 固定後クロロホルムでフィルターを溶解. 残された細胞集塊を染色し2枚のカバーガラスで挟むように封入することにより顕微鏡下, 集塊の裏表の観察を可能とした.この結果, 3次元的な細胞診標本の作製が可能となった. 本法を多くの症例に応用することにより, 子宮内膜細胞診の診断精度向上をはかりたい.