P–446 Controlled ovarian stimulation protocols for oocyte vitrification induce differential gene expression profiles in primary tumours of breast cancer
Authors: Soriano M J , De. Castillo L M , Martínez J , Herraiz S , Díaz-García C 
Published: August 2021

Abstract

Study question
Could controlled ovarian stimulation (COS) protocols used in fertility preservation (FP) impact on malignant cell proliferation and tumour molecular profiling of breast cancer (BC) patients?

Summary answer
Letrozole supplementation during ovarian stimulation for oocyte vitrification could be considered as a safe procedure in estrogen-dependent BC patients undergoing FP.

What is known already
High estradiol levels associated to COS could promote changes in gene expression in estrogen-positive BC tumors. Estradiol levels reached during the ovarian stimulation could aggressively promote malignant cell proliferation and cell migration to adjacent organs. Aromatase inhibitors such as letrozole, are added to standard stimulation protocols to avoid this undesirable potential side effect. Despite the reassuring clinical results achieved by using letrozole for FP in BC patients, there is still a lack of evidence regarding its impact on malignant cell behaviour. For this reason, specific molecular studies to properly evaluate safety of letrozole in this specific population are still required.

Study design, size, duration
Experimental in vivo study. Thirty 5-week-old Nude-nu female mice were divided into three different groups: BC (n = 10), BC and FSH stimulation (BC-FSH, n = 10), or BC and letrozole stimulation (BC-LTZ, n = 10). BC was considered the control group, whereas BC-FSH and BC-LTZ represented distinct COS protocols. Hormone-dependent BC was induced in all mice. Animals were followed-up for 5 months and then euthanized to collect kidney, ovary, spleen, and liver tissues for gene expression and immunohistochemistry (IHC) analysis.

Participants/materials, setting, methods
One million of human MCF–7 BC cells were injected into the mouse left kidney capsule. Two days after xenograft, COS was induced by 10IU FSH or 1mg/ml letrozole + 10IU FSH, followed by ovarian triggering with 10IU hCG at 48h. Human BC RT2 Profiler PCR Arrays were performed to evaluate the impact of COS on tumour behaviour. BC biomarkers (Ki67, Erα, PR and HER–2) were also analyzed by IHC to validate gene expression results.

Main results and the role of chance
The differential gene expression was firstly assessed in kidney samples, as they represent the xenograft site, and different expression profiles were obtained depending on the COS protocol used. The BC-FSH group showed a global over-expression pattern of all genes of the array when compared to BC and BC-LTZ. Further gene ontology analysis revealed that cellular process, biological regulation, metabolic process, and proteases were the most over-represented biological terms, with a 20.5-fold over-expression for MMP2 compared to the other groups. On the other hand, BC-LTZ mice presented gene expression profiles similar to that of controls. When other tissues were analysed to detect malignant cell presence, our results revealed a significant up-regulation of matrix-proteases, cell cycle and proliferation related-genes, in liver samples from the BC-FSH group, but no amplification of any of the studied genes was detected in ovarian tissue or spleen. IHC findings confirmed the presence of human BC cells in 100% of samples from kidney tissue and in 30% of samples from liver tissue in the BC-FSH group. No human cells were detected by IHC in the BC and BC-LTZ groups.

Limitations, reasons for caution
Since this is an animal model of estrogen-dependent BC induced through a cell line, further validation with human tumour breast cancer samples would be required.
Wider implications of the findings: Adjuvant letrozole in COS protocols prevents BC cell migration. The present study suggests that this protective effect could be mediated by interfering ER-pathway downstream genes involved in cell proliferation and matrix digestion. Altogether, letrozole could safely be used as a supplement during COS procedures for oocyte vitrification in BC women.

Trial registration number
Not applicable