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Previous investigations of a Li ± Fraumeni like family (Barnes et al., 1992) demonstrated that both the proband and her mother had elevated p53 protein levels in both tumour tissue and normal tissue at sites distant from the tumour, although no mutation was found in the p53 gene. In the present study two recently described functional assays for p53, an apoptotic assay and the functional assay for the separation of alleles in yeast (FASAY), have been employed to study the functional activity of p53 from this patient. The results of the apoptotic assay demonstrated that this patient had a p53 functional defect and the FASAY result suggested that this defect was in fact a germline mutation of the p53 gene. A point mutation of codon 337, which results in an amino acid substitution of a cysteine for an arginine, was demonstrated initially in cDNA and was con®rmed by sequencing of genomic DNA. This is an unusual mutation as it is in the oligomerisation domain of p53, in contrast to the majority of p53 mutations which are in the core DNA binding domain. This mutation results in a protein which still retains partial transactivational activity in the FASAY. The mutation of codon 337 is only the second reported case of a germline missense mutation occurring in the oligomerisation domain of p53.Keywords: p53; Li ± Fraumeni syndrome; oligomerisation domain; functional assaysThe tumour suppressor gene, p53, was ®rst discovered in 1979 (Lane and Crawford, 1979;Linzer and Levine, 1979). p53 is a 393 amino acid nuclear phosphoprotein which plays a central role in the cellular processes involved in the response to DNA damage (reviewed Kastan et al., 1995). It is made up of at least four domains, a transactivation domain, a central DNA binding domain, an oligomerisation domain and a regulatory domain, the latter two residing at the carboxy end of the protein.Following initial studies by Malkin et al. (1990) it is now established that germline p53 mutations are associated with Li ± Fraumeni syndrome (LFS), a dominantly inherited syndrome ®rst proposed by Fraumeni in 1969 (Li andFraumeni, 1969). However mutations in the coding region of p53 have only been con®rmed in about 50% of families with LFS (Birch et al., 1994). Families with this syndrome exhibit multiple primary neoplasms in childhood and early adulthood and the spectrum of cancers which commonly occurs in LFS families includes brain tumours, breast tumours, sarcomas, leukaemia and adrenal cortical tumours (Li et al., 1988). Families have now been recognised in which there is a similar spectrum of malignancies to LFS but the families do not ®t exactly into the criteria of LFS. These families have been named Li ± Fraumeni like (LFL) (Birch et al., 1994) and p53 germline mutations have been found in 7 ± 20% of these families (Eeles, 1995; Varley et al. manuscript in preparation). The patient who is the subject of this study is a member of a LFL family and meets the criteria for LFL families as set out by Birch et al. (1994).In 1992 Barnes et al. described a member of a LFL ...

Adjuvant systemic therapy for women with node-negative breast cancer is most easily justified for those patients at highest risk of relapse. We have examined the impact of tumor size, histologic grade, estrogen receptor (ER) status, tumor ploidy, and S-phase fraction (SPF) on relapse-free survival (RFS) for 169 patients with node-negative breast cancer in order to identify groups of patients at high and low risk of relapse. Patients with small tumors (less than or equal to 1.0 cm) had a significantly better RFS than those with larger tumors (P = .005), with 96% remaining relapse-free at 5 years. Patients with tumors less than or equal to 1.0 cm were thus excluded from analysis when attempting to define a group with a poor prognosis. Within the group of patients with tumors greater than 1.0 cm, tumor ploidy (P = .63), ER status (P = .3), or progesterone receptor (PgR) status (P = .24) did not predict for RFS. Patients with grade 1 or 2 infiltrating ductal tumors had a significantly better prognosis than those with grade 3 tumors (P = .04). The prognostic factor that gave the widest separation between subgroups, however, was SPF. Patients whose tumors were greater than 1.0 cm with an SPF less than or equal to 10% had a 5-year RFS of 78% compared with a 5-year RFS of 52% for those with an SPF greater than 10% (P = .006). We have combined tumor size and SPF to identify three prognostic groups: (1) tumor less than or equal to 1.0 cm, 5-year RFS 96%; (2) tumor greater than 1.0 cm plus SPF less than or equal to 10%, 5-year RFS 78%; 3) tumor greater than 1.0 cm plus SPF greater than 10%, 5-year RFS 52%. These prognostic groupings may help identify patients most suitable for adjuvant therapy.

SummaryThe relationship between c-erbB-2 gene expression (assessed immunohistochemically), S-phase fraction (SPF) and prognosis has been analysed in 172 women with primary breast cancer. c-erbB-2 staining was independent of age, tumour size, number of nodes involved, tumour grade and DNA ploidy, but was more common in oestrogen receptor (ER) negative tumours (P = 0.02) and progesterone receptor (PgR) negative tumours (P = 0.03). A weak correlation between c-erbB-2 staining and SPF was observed (r = 0.18). Amongst women with node negative disease, SPF was significantly related to relapse free survival (RFS, P = 0.04) while c-erbB-2 staining was not (P = 0.2). In contrast, both SPF (P = 0.002) and c-erbB-2 staining (P = 0.016) provided significant prognostic information on RFS for women with node positive disease. Multivariate analysis showed that c-erbB-2 staining and SPF gave independent information on RFS for women with node positive disease.

Summary The reactivity of 95 breast carcinomas with the antibody Ki-67, which recognises a nuclear antigen in proliferating cells, has been assessed and compared to their histological grade and, for 47 tumours, DNA index and S-phase content. The effects of freezing and section handling on the stability of the nuclear antigen have been assessed.Evidence of nuclear staining was seen in 56% of carcinomas, with a range of positive cells from <1% to 60%. Cytoplasmic rather than nuclear staining was observed in 26% and 18% of carcinomas were negative. A significant correlation was observed between the presence of nuclear staining and poorer histological grade and higher S-phase content, and between the percentage of positive nuclei and S-phase content, but not grade. Three groups of carcinomas were identified: those in which Ki-67 reactivity, grade and S-phase content were similar; ones in which there was prominent nuclear reactivity with Ki-67 but low grade and S-phase content; and a group showing the converse. These patients will be followed to assess which of these three markers of proliferation is of greatest prognostic value.