Germline p53 Mutation in a Case of Li-Fraumeni Syndrome Presenting Gastric Cancer

Li-Fraumeni syndrome is a relatively rare disease entity characterized by sarcomas of the soft tissues, bone and miscellaneous rumors of juvenile onset and frequent occurrence of metachronous tumors (1). Germline mutation of the p53 tumor suppressor gene has been reported in approximately 70% of the Li-Fraumeni kindreds (2). Here we report a case of Li-Fraumeni syndrome presenting a gastric cancer in the proband and a hepatosarcoma in his son. The proband was a 38-year-old Japanese male referred to a clinic with a diagnosis of gastric carcinoma with multiple liver metastases. There was no previous history of malignant disorders, but his 11-year-old son suffered from a liver tumor diagnosed as a hepatosarcoma and underwent surgical resection followed by systemic chemotherapy (Fig. 1). The proband received systemic chemotherapy comprising seven courses of 5FU and MTX. However, the disease was progressive during the course. The proband died 3 months after admission and was autopsied. The family history of pediatric cancer and the relatively young onset of the gastric carcinoma prompted genetic analysis of the p53 gene with suspicion of Li-Fraumeni syndrome. PCR/direct sequencing analysis revealed a germline missense mutation in exon 8 at codon 273 of the p53 gene, causing a substitution of CAT (His) for CGT (Arg) (Fig. 2). Analyses of the primary tumor and liver metastasis showed overexpression of the p53 protein in immunohistochemistry (Fig. 3) and loss of heterozygosity (LOH) in the opposing allele carrying the wild-type p53 gene (Fig. 4). In segregation analysis of the kindred, the same mutation was detected in his two children. However, his mother and sister were negative for the germline p53 mutation (Fig. 1). His father died at 63 years of age due to a hepatocellular carcinoma (HCC) which was caused by viral hepatitis. Hence it is likely that the mutation was a new germline mutation arising at the proband. At autopsy, the tumor was limited to the stomach and the liver and there was no other tumor originating from the other organs. So far, four cases of Li-Fraumeni syndrome have been reported showing the mutation at codon 273 of the p53 gene, i.e. two TGT (Cys), one GGT (Gly) and one CAT (His) mutation (5–8). To our knowledge, this is the second report of the missense mutation to CAT (His) at codon 273 in exon 8 of the p53 gene. Gene testing revealed the proband and two sons were heterozygous [E+ (mut/wt)] for the p53 germline mutation and his mother and sister were homozygous for the wild-type p53 gene (E−). ‘E’ stands for Evaluation and is used to represent clinical and/or test information on the pedigree. An arrow indicates the proband. HCC, hepatocellular carcinoma. An asterisk indicates the kindreds examined for the p53 gene mutation at the National Cancer Center Hospital. The pedigree was constructed according to the recommendations of the Pedigree Standardization Task Force of the National Society of Genetic Counselors (3). Direct sequencing of the PCR product using the forward primer revealed a missense mutation at codon 273 of the p53 gene, substituting CAT (His)forCGT(Arg). Germline mutation of the p53 gene and allelic status of the p53 locus, (a) PCR/SSCP analysis of the p53 gene exon 8. Arrowheads indicate mutated alleles. Signals from wild-type p53 allele were faint in tissues of gastric cancer and liver metastasis (lanes 2 and 3). indicating LOH in the allele carrying wild-type p53 gene, (b) Allelic status of the p53 locus was analyzed by blunt-end single-strand DNA conformation polymorphism analysis, using three polymorphic markers (Ex4, Int7 and Int1) in the p53 gene (4). In analysis of peripheral blood lymphocyte (PBL), the proband was heterozygous for all three markers (panel 1), while the analyses of gastric cancer tissue and liver metastasis indicate the allelic loss of the p53 locus (panels 2 and 3). An asterisk indicates the decreased signal derived from the lost allele. H-E stain and immunostaining of the primary gastric cancer (a, b) and liver metastasis (c, d). (a) Histological diagnosis was a moderately differentiated adenocarcinoma of the stomach; (b) immunohistochemistry of the gastric cancer using an anti-p53 antibody (RSP53) showed intense nuclear staining of rumor cells; (c) histological diagnosis was a moderately differentiated adenocarcinoma that was compatible with the metastatic gastric carcinoma (a); (d) immunohistochemistry using the anti-p53 antibody (RSP53) showed overexpression of the p53 protein at cell nuclei. PCR/direct sequencing was performed with the following conditions and parameters: PCR primer, forward: 5′CCT ATC CTG AGT AGT GGT AA3′ PCR primer, reverse: 5′CCA AGA CTT AGT ACC TGA AG3′ Size of PCR product: 331 bp Thermal cycle profile: Initial denaturation: 94°C, 5 min 30 cycles of 94°C, 30 s/58°C, 30 s/72°C, 60 s Final extension: 72°C, 7 min Sequencing primer: the same as the PCR primers. Cancer Genetics Report is a new section for JJCO and presents a brief, 1–2 page report focusing on genetic analysis of a case which merits publication. Specifically, Typically, the section reports a previously undescribed germline mutation or polymorphism of a gene which is associated with a cancer. A case report with known mutation or polymorphism may also be considered, if the report can be expected to contribute substantially to the advancement and/or accumulation of our knowledge in the field of clinical cancer genetics. The nucleotide sequence of the mutation or polymorphism must be defined on the genomic DNA. The method of the mutation/polymorphism detection should be described explicitly, such as PCR conditions and primer sequences. Whenever appropriate, a pedigree (family tree) must be presented. The pedigree should be drawn according to the ‘Recommendations for Standardized Human Pedigree Nomenclature’, Am J Hum Genet 1995;56:745–52. Some basic keys of the Nomenclature are summarized in: http://wwwinfo.ncc.go.jp/jjco/pdf_files/pedigree.pdf The privacy of a patient and any other relevant family member(s) should be strictly protected in the manuscript and it is the responsibility of author(s) to obtain appropriate informed consent for publication. A manuscript for Cancer Generics Report should be prepared according to the Instructions to Authors for regular papers, except: (1) Running Head, Abstract and Mini-abstract are not required, (2) a genetic Summary should be provided describing Disorder, Ethnicity, Gene and its GenBank, EMBL or DDBJ accession number and Chromosomal assignment, Type of DNA variant. Mutation, Allelic frequency and Method of mutation detection, etc.. (3) the text may have a minimum number of sections, but a Methods section is mandatory and (4) two copies of the manuscript with original figures should be sent to the Editorial Office with a covering letter signed by all co-authors. This work was supported in part by Grants-in-Aid for Cancer Research and for the 2nd Term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health and Welfare, Japan.