A study of the relationship of the internal endocrine environment to the p53 network, cyclin D1 and replication licensing proteins in breast cancer
Aim To assess the change in expression of various tumour markers (p53, mcm2 and cyclin D1) with change in internal endocrine environment in patients with primary breast cancer and to study the expression and prognostic association of cyclin D1 and replication licensing proteins (mcm2 and geminin) in invasive breast cancer. Hypothesis The endocrine environment may be critical in the development and efficacy of treatment in breast cancer. Oestrogen has the potential to modulate the function of p53 network and cell cycle proteins (cyclin D1) in breast cancer, the consequences of which could profoundly affect treatment efficacy. It is proposed that the serum oestrogen level may effect the measurement of protein markers in primary breast cancer. It is hypothesised that the replication licensing protein family, mcm, may be a more consistent marker of proliferation than the conventional immunohistochemical marker, Ki-67, in primary breast cancer. Material and Methods Women with a diagnosis of primary, operable, symptomatic breast cancer were eligible and a total of 99 patients (47 premenopausal and 52 postmenopausal) were included in the study. Of these, 30 pre-menopausal and 33 post-menopausal women had paired samples of breast cancer biopsy and venous blood taken at diagnosis and later at the time of resectional surgery. The samples were collected at two time points to study the change in expression of tumour proteins, if any, with the change in serum oestradiol levels in pre-menopausal women, the postmenopausal women served as the control. The rest of the patients included had tissue samples and blood taken at one time point only i.e. at resectional surgey. Samples comprised formalin fixed breast cancer tissues from both time points for immunohistochemistry and FISH; serum was drawn for 17-? oestradiol, progesterone, FSH, LH. Tumour micro-arrays were prepared from the surgical specimens (corresponding to the second time point) and whole tissue sections were used to study the diagnostic specimen (corresponding to the first time point).Results Overexpression of cyclin D1 was seen in 63% of cancers studied whilst CCND1 amplification was identified in 30%. CCND1 amplification was significantly associated with cyclin D1 protein overexpression (p<0.001; Fisher’s exact test) and both CCND1 amplification and cyclin D1 protein expression with oestrogen receptor (ER) expression (p=0.003 and p<0.001; Fishers exact test). Neither CCND1 amplification nor cyclinD1 expression was associated with tumour size, pathological node status or HER2 amplification. High CCND1 amplification (CNG > 8) was significantly associated with high tumour grade and higher Nottingham Prognostic Index (p=0.001). Increased expression of both Mcm2 and geminin was associated with high tumour grade (p<0.05 ANOVA). Mcm2: Ki-67 and geminin: Ki-67 ratios were similar across different tumour grades suggesting similar G1 transit time in different grades of tumour, with higher grade tumours containing a larger proportion of in-cycle cells with proliferative potential. Expression of Mcm2 and geminin was associated with a high Nottingham Prognostic Index NPI (p<0.001, Spearman correlation) and inversely with ER-positive tumours (p<0.0001, t-test), hence correlated with poor prognosis. We explored a potential change in p53 expression with change in serum oestradiol levels in premenopausal women, following the generation of hypothesis from an animal study done in MCF-7 mouse xenograft model. The observation from MCF7 xenograft model suggesting an inverse relationship between p53 expression and serum oestradiol did not hold good in humans, although the numbers tested were small. Conclusion High level CCND1 amplification could be used to identify a subset of ER-positive breast cancers that are associated with poor prognosis. Such patients could be offered adjuvant chemotherapy, in keeping with evidence that CCND1 amplified cancers respond poorly to endocrine agents. This study demonstrates that replication licensing factors; Mcm2 and geminin correlate with poor prognosis (ER-negative and high NPI) breast cancers, and therefore merit consideration as poor prognostic markers in breast cancer. We did not observe any significant change in expression of p53 protein with a change in serum oestradiol levels in premenopausal patients with primary invasive breast cancer.
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