Abstract P3-05-01: Molecular analysis of cancer tissue, circulating tumor cells (CTC) and cell-free plasma tumor DNA (ptDNA) suggests variable mechanisms of resistance to endocrine therapy (ET) in estrogen receptor (ER) positive metastatic breast cancer (MBC)
Authors: Paoletti C , Aung K , Cannell EM , Darga EP , Chu D , Kidwell KM , Thomas DG , Tokudome N , Brown ME , McNutt LM , Gersch C , Schott AF , Park BH , Robinson DR , Chinnaiyan AM , Rae JM , Hayes DF 
Published: May 2016

Introduction: Fifty percent of ER positive MBC patients do not benefit from ET. Potential mechanisms of resistance to ET in this patient population include absence of ER expression by deletion or suppression, alteration in ER signaling pathway genes, or upregulation of multiple growth factor receptor pathways. We hypothesized that genotyping and phenotyping of CTC combined with genomic analysis of ptDNA will provide important insights into the multiple mechanisms of ET resistance and that a set of blood tests might serve as a "liquid biopsy" abrogating the need for tissue specimens.

Methods: Twenty-four patients providing informed consent were enrolled into the Mi CTC-ONCOSEQ study, a companion trial to Mi-ONCOSEQ (the Michigan Oncology Sequencing Program). Seven of these patients (5 with ER immunohistochemistry (IHC) positive and 2 with ER negative cancers) who had available archived primary and metastatic cancer tissue, a research metastatic biopsy for genomic analysis, and who had ≥5CTC/7.5 ml whole blood (WB) characterized for ER protein (CTC-ER) are the focus of this report. All the patients were ET refractory. None of them was progressing on fulvestrant at the time of study entry. CTC enumeration and phenotyping was performed with CellSearch©. Circulating ptDNA was analyzed by droplet digital polymerase chain reaction (ddPCR). ER status from archived tissue was obtained from chart review. ER mRNA expression was determined in the research biopsy of metastatic tissue by using quantitative RNA sequencing. Mutational status of ER gene, ESR1, was determined by Next-gen Sequencing using the Illumina HiSeq 2500 platform.

Results: The 2 control patients with triple negative breast cancer had negative CTC-ER. Discordance between CTC-ER and tissue ER by IHC was observed (Table). Two of the 5 ER positive patients retained CTC-ER positivity (39% and 19% of the CTC). One of them (#7) harbored an ESR1 mutation in the research biopsy tissue and in ptDNA, whereas the other (#14) had wild type (WT) ESR1. CTC-ER protein levels in patients #12, 17 and 24 were negative. All had WT ESR1 in the research biopsy tissue. Of note, patient #12 had WT ESR1 in the research biopsy, but an ESR1 mutation was detected in her ptDNA.

Pt#CTC-ER Tissue-ER  ESR1 status in research biopsyESR1 status in ptDNA N[deg]CTC/7.5ml WB% CTC-ER +Primary by IHCMet by IHCMet research biopsy by mRNA  71839%+++Y537SY537S141619%+NA+WTWT12130%+++WTD538G17160%++weakly+WTWT242750%+weakly+weakly+WTWT

Conclusions: These exploratory data suggest heterogeneous mechanisms of resistance to ET in patients with previously determined ER-positive MBC, including ESR1 mutations in ER positive cases (seen in 2 patients) and loss of ER expression (seen in CTC of 3 patients). In contrast, other cancers continue to express WT ESR1, and therefore must have developed alternative mechanisms of resistance. At least 2 of these mechanisms can be detected and monitored with complementary circulating assays: CTC and ptDNA. Further investigations are needed to understand the heterogeneous mechanisms of resistance to ET.

Citation Format: Paoletti C, Aung K, Cannell EM, Darga EP, Chu D, Kidwell KM, Thomas DG, Tokudome N, Brown ME, McNutt LM, Gersch C, Schott AF, Park BH, Robinson DR, Chinnaiyan AM, Rae JM, Hayes DF. Molecular analysis of cancer tissue, circulating tumor cells (CTC) and cell-free plasma tumor DNA (ptDNA) suggests variable mechanisms of resistance to endocrine therapy (ET) in estrogen receptor (ER) positive metastatic breast cancer (MBC). [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-05-01.