Anne‐Lise Børresen

BACKGROUND: Acquired mutations in the p53 tumor-suppressor gene have been detected in several human cancers, including colon, breast, and lung cancer. Inherited mutations (transmitted through the germline) of this gene can underlie the Li-Fraumeni syndrome, a rare familial association of breast cancer in young women, childhood sarcomas, and other malignant neoplasms. We investigated the possibility that p53 mutations in the germline are associated with second primary cancers that arise in children and young adults who would not be considered as belonging to Li-Fraumeni families. METHODS: Genomic DNA was extracted from the blood leukocytes of 59 children and young adults with a second primary cancer. The polymerase chain reaction, in combination with denaturant-gel electrophoresis and sequencing, was used to identify p53 gene mutations. RESULTS: Mutations of p53 that changed the predicted amino acid sequence were identified in leukocyte DNA from 4 of the 59 patients (6.8 percent). In three cases, the mutations were identical to ones previously found in the p53 gene. The fourth mutation was the first germline mutation to be identified in exon 9, at codon 325. Analysis of leukocyte DNA from close relatives of three of the patients indicated that the mutations were inherited, but cancer had developed in only one parent at the start of the study. CONCLUSIONS: These findings identify an important subgroup of young patients with cancer who carry germline mutations in the p53 tumor-suppressor gene but whose family histories are not indicative of the Li-Fraumeni syndrome. The early detection of such mutations would be useful not only in treating these patients, but also in identifying family members who may be at high risk for the development of tumors.

The cytochrome P450 enzyme debrisoquine 4-hydroxylase metabolizes many different classes of commonly used drugs, such as antidepressants and neuroleptics. Deficient hydroxylation of debrisoquine, known as the poor metabolizer (PM) phenotype, affects 5-10% of Caucasians and may lead to adverse reactions upon administration of drugs in standard doses. This autosomal recessive metabolic deficiency is caused by the possession of two PM-associated mutations in the human CYP2D6 gene locus coding for the enzyme. These mutations include at least four different single base mutations and two different large gene deletion alleles. The single base mutations can be rapidly detected by PCR methods. In contrast, the large gene deletions have so far only been directly identified by RFLP analysis. By the use of sequence data previously published by others, we report here an alignment of different CYP2D alleles to focus on the presence of almost completely identical sequences immediately downstream of both CYP2D7 and CYP2D6 which may seriously complicate and interfere with PCR-based detection of the gene deletion. Based on this analysis, we have developed a rapid assay which, for the first time, detects the 13kb (also called 11.5 kb) Xba I gene deletion allele by the use of long-PCR technology. The primers were designed to amplify a 3.5 kb PCR product in the presence of this D6(D) allele. We have evaluated the method on 23 different DNA samples heterozygous (n = 22) or homozygous (n = 1) for the 13 kb gene deletion allele (previously typed by RFLP analyses). All samples were correctly identified by the assay. The PCR method did not detect the rare 11 kb Xba I gene deletion allele (n = 5), and there was no false positive amplification from deletion negative DNA samples (n = 47). This sensitive and specific PCR-based assay for detection of the D6(D) allele will improve the scientific and clinical use of CYP2D6 genotyping.

Acquired mutations in TP53 as well as immunohistochemically detectable protein expression have been implicated as prognostic factors for breast cancer. We have evaluated the relationship between mutations detected in 119 breast tumours and various clinicohistopathological indices, stratifying the mutations according to the functional domains as defined by the recent elucidation of the crystal structure of the protein. Patients with missense mutations located in regions encoding parts of the protein involved in zinc-binding had significantly decreased disease-free and overall survival relative to patients whose tumours had mutations in other domains. These results indicate that these biochemically defined domains also have biological relevance in terms of breast cancer disease course, and suggest that some mutations in TP53, more than others, can contribute to the development of clinically more aggressive and perhaps treatment resistant breast tumours. When confirmed, this will be of potential importance in predicting the clinical behaviour of breast cancer and its responsiveness to therapy.