Abstract P1-10-12: Menstrual cycling critically affects the Oncotype DX 21-gene signature: Implications for predictive biomarker assays in premenopausal women
Authors: Bernhardt Sarah M , Dasari Pallave , Glynn Danielle J , Woolford Lucy , Raymond Wendy , Moldenhauer Lachlan M , Walsh David , Townsend Amanda R , Price Timothy J , Ingman Wendy V 
Published: March 2020

Abstract
Introduction: The 21-gene Oncotype DX Recurrence Score is predictive of adjuvant chemotherapy benefit for women with early stage, hormone receptor (HR)-positive, HER2-negative breast cancer. In premenopausal women, fluctuations in estrogen and progesterone during the menstrual cycle can impact gene expression in these hormone-responsive cancers. However, the extent to which hormonal fluctuations affect Oncotype DX Recurrence Scores remains unclear.
Aim: To determine the extent to which ovarian cycling affects the Oncotype DX 21-gene signature using paired premenopausal breast cancer samples and mouse models.
Methods: To investigate menstrual variation in the Oncotype DX 21-gene signature within the same tumour, paired formalin-fixed paraffin-embedded, HR-positive invasive breast cancer samples were collected on different days of the menstrual cycle from women &lt;50 years old (n=18), and compared to women &gt;50 years old (n=11). Samples were collected an average of 18 days apart and were neoadjuvant therapy naive. To determine the effect of progesterone on gene expression, HR-positive T47D breast cancer cells were xenografted into the mammary fat pad of BALB/c nude mice, treated with exogenous estrogen±progesterone (n=12, 11 respectively). Additionally, HR-positive mammary tumours were collected from naturally cycling Mmtv-Pymt mice at the estrus or diestrus phase of the ovarian cycle (n=25, 28 respectively). The 21-gene Oncotype DX signature was assessed through quantitative RT-PCR and an experimental recurrence score (RS) was calculated using the Oncotype DX Recurrence Score algorithm.
Results: Increased discordance in RS was observed between paired samples collected from younger women (3.2±2.5; mean±stdev), compared to older women (2.0±1.7; p=0.04). In young women, discordance was primarily driven by variable expression of proliferative genes, compared to older women, where discordances were a result of variable expression of invasive genes. Variable concentrations of progesterone at the time of tissue collection may influence proliferative gene expression and contribute to discordant RS. In support of this, in HR-positive xenograft tumours, expression of proliferative genes Ki67 (p=0.03) and STK15 (p=0.04), and Ki67 protein expression (p=0.02), were reduced following progesterone treatment. Furthermore, in naturally cycling Mmtv-Pymt mice, mammary tumours collected at diestrus, analogous to the luteal phase of the menstrual cycle in humans, show significant differences in expression of 6 Oncotype DX signature genes (Ki67, Ccnb1, Esr1, Her2, Grb7, Bag1; p≤0.05) and a significant increase in RS (21.1±2.4; mean±SEM) compared to tumours dissected at estrus (15.5±1.9; p=0.03). Clustering analysis revealed a subgroup of Mmtv-Pymt mammary tumours collected at diestrus characterised by increased expression of proliferative (p&lt;0.001) and invasive (p=0.01) genes and a significant increase in RS (p=0.01). These tumours also exhibited higher expression of estrogen regulated genes (p=0.005) suggesting increased sensitivity to hormonal fluctuations during the ovarian cycle, and possibly greater variability in RS.
Conclusion: Our results suggest that menstrual cycling affects the expression of genes included in the Oncotype DX 21-gene signature and influences experimental recurrence scores. Oncotype DX may be less effective for guiding chemotherapy treatment decisions for cycling premenopausal women compared to older postmenopausal women.
Citation Format: Sarah M Bernhardt, Pallave Dasari, Danielle J Glynn, Lucy Woolford, Wendy Raymond, Lachlan M Moldenhauer, David Walsh, Amanda R Townsend, Timothy J Price, Wendy V Ingman. Menstrual cycling critically affects the Oncotype DX 21-gene signature: Implications for predictive biomarker assays in premenopausal women [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-10-12.