Abstract 495: The impact of preoperative hydroxyprogesterone on serial levels of circulating tumor cells [CTCs] in patients undergoing surgery for operable breast cancer
Authors: Warawdekar Ujjwala M. , Parmar Vani , Singh Akshita , Aher Swapnil , Kulkarni Abhay , Chaudhari Meenal , Gupta Sudeep , Badwe Rajendra A. 
Published: July 2016

Abstract
Introduction: Assessment of Circulating Tumour Cells [CTCs] from the peripheral blood of patients diagnosed with cancer is emerging as a valuable biomarker for prognostication and tailoring therapy. A previous study demonstrated that a single depot injection of hydroxyprogesterone prior to surgery improved the DFS of patients with large tumours or positive nodes, forming the basis for this investigation, designed to assess effects of this drug on serial levels of CTCs on patients with operable breast cancer.
Methods: Seventy patients were enrolled, randomized, and as previously described a single depot injection of hydroxyprogesterone was administered prior to surgery in patients in the experimental arm. Peripheral blood samples were obtained at three time points during surgery; prior to induction
of anaesthesia, intra operative and at completion. Peripheral Blood Mononuclear Cell [PBMC] fractions separated on Ficoll hypaque were enriched for tumour cells with double positive immunomagnetic selection for EpCAM [Epithelial Cell Adhesion Molecule]. Half the enriched fraction was routed for multistep sequential labelling of cell-surface markers; EpCAM and CD45, cytoplasmic Cytokeratin and staining with DAPI for the nucleus, facilitating acquisition with multi parametric flow cytometry and imaging using confocal microscopy. With pre-amplification of other half of the enriched fraction, a quantitative expression analysis for a panel of epithelial genes was performed.
Results:With multi parametric flow analysis, events that were CD45-/ EpCAM+/ CK+ with DAPI positivity were
defined as CTCs, reported as events/ml blood and in all samples were in the range of 0 to 155 /ml of blood with no significant difference observed between the two arms. Detailed analysis with pooled data of all time points, in premenopausal patients, using the Mann Whitney test, showed that the mean number of CTCs is significantly lower[p = 0.005] in the experimental arm[n = 30] as compared to the control arm[n = 36] .Analysis within the hydroxyprogesterone group using the Wilcoxon signed rank test showed that the mean number of CTCs is lower in the post-surgery time point when compared to the pre surgery time point, in patients with positive nodes[n = 16][p = 0.044], large tumours [n = 18] [NS; p = 0.085] and post-menopausal node positive group [n = 7] [NS; p = 0.063] The expression analysis for the selected markers and confocal images validated the identity of tumour cells.
Conclusions: Patient follow-up will ascertain the benefits of reduced numbers observed and establish CTCs as a surrogate marker for prognostication.
Citation Format: Ujjwala M. Warawdekar, Vani Parmar, Akshita Singh, Swapnil Aher, Abhay Kulkarni, Meenal Chaudhari, Sudeep Gupta, Rajendra A. Badwe. The impact of preoperative hydroxyprogesterone on serial levels of circulating tumor cells [CTCs] in patients undergoing surgery for operable breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 495.