The role of the tumour suppressor gene, TP53, in determining breast tumour subtype

Background and hypothesis A germline TP53 mutation predisposes to young breast cancer and other tumours recognized clinically as Li-Fraumeni Syndrome. There is a growing recognition that HER2 amplified breast cancer is more frequent in TP53 germline mutation carriers than amongst sporadic cases. Frequently HER2 amplification in pure ductal carcinoma in situ (DCIS) presenting in sporadic breast disease does not typically progress to HER2 amplified invasive breast cancer. The hypothesis for this project is that an inherited TP53 gene mutation is important in driving the HER2 amplified breast tumour subtype. I have explored the timing of loss of TP53 function and the type of both inherited and acquired TP53 mutation, (missense or nonsense mutation) in order to evaluate the importance of each in determining breast tumour subtype. Materials and methods The Cohort study Of p53 related Early onset breast cancer (COPE) cohort comprises 136 breast tumour formalin fixed paraffin embedded (FFPE) blocks from 45 patients with a germline TP53 mutation. Full-face H&E sections were made for morphological review and cores were selected from the invasive tumour and DCIS for tissue microarrays (TMA) with immunohistochemistry staining. A HaloPlex® targeted enrichment kit was used to characterise the genetic landscape in the COPE tumours and in a HER2 amplified control group of 9 young breast cancer cases drawn from the Prospective study of Outcomes in Sporadic versus Hereditary breast cancer (POSH) cohort. The early biochemical mechanisms involved in the development of these breast tumours were investigated using the MCF10A cell line for three-dimensional cell culture in order to study the breast glandular architecture and to mimic ductal carcinoma in situ (DCIS). Cells were grown as spheroids on a reconstituted basement membrane in a growth arrest polarized fashion. The tumour suppressor genes TP53 and BRCA1 were transiently knocked down and stable over amplification of the oncogene ERBB2 was performed on the MCF10A cell. This was to understand the influence these key genes have on the morphogenetic processes including luminal clearing and proliferation during the early stages of tumourgenesis. Primary fibroblasts derived from associated HER2+ and triple receptor negative breast cancers (TNBC) were grown in culture and the expression of stromal markers investigated. Results Pathological analyses: I confirmed a high prevalence of HER2 amplified, high grade, ductal no special type tumours in TP53 associated invasive breast tumours. HER2 overexpression was confirmed in 19/36 (52.8%) of TP53 associated cases compared with 717/2956 (24%) from the large young onset POSH cohort (aged 40 or younger at diagnosis) (p=<0.001). I also noted that 13/36 (31.1%) were ER+/HER2+ tumours which were significantly higher than the POSH cohort (p=0.002). Frequent widespread high grade ductal carcinoma in situ (DCIS), a high frequency of sclerotic tumour stroma (80.6% of cases) and confirmed upregulation of TGFβ signalling was reported. All tumour cases showed abnormal p53 immunohistochemistry and patchy staining in the DCIS was suggestive of p53 signalling deregulation and stabilisation of mutant p53. Our work supports the hypothesis that a germline TP53 mutation strongly predisposes to HER2 amplified, high grade, ER+ve tumour subtypes in contrast to triple negative breast cancers typically reported in BRCA1 carriers. Genomic analysis: Next generation sequencing (NGS) data from invasive tumour and DCIS samples revealed a loss of heterozygosity (LOH) of the wild type allele in 14/16 (87.5%) tumour samples and 4/4 (100.0%) DCIS samples. Clonality data suggests that cases typically acquire few somatic mutations and are clonally distinct with consistent widespread LOH of TP53. NGS data from HER2+ cases from POSH revealed a high variant allele frequency (VAF) of mutant TP53 reads consistent with widespread clonal TP53 loss and indicative of an early event. Cellular biology: ErbB2 overexpression and a loss of TP53 complement one another and drive a proliferative cell type losing cellular contact inhibition. This was further confirmed in threedimension with ErbB2 overexpression and loss of TP53 driving an aggressive invasive phenotype. Cells devoid of BRCA1 and p53 were shown to not cooperate suggesting why TP53 carriers develop HER2+ and BRCA1 carriers develop triple receptor negative tumours. Conclusions: An early loss of TP53 seems to be fundamental in driving a HER2 breast tumour phenotype. Early loss of TP53 and HER2 overexpression cooperate and give DCIS lesions an invasive and selective advantage driving the evolution of Li-Fraumeni Syndrome associated breast tumours. Patients with a missense TP53 mutation seem to be predisposed to develop a sclerotic tumour stroma