Abstract B018: Restoration of p16INK4A expression in circulating tumor cells in patients with advanced solid tumors treated with deoxycytidine analogs and associated dynamic EMT phenotypic changes

Abstract

Background. In vitro experiments from multiple laboratories have shown that the methylation-induced silencing of the p16INK4A gene, a tumor suppressor gene, can be reversed by DNA methyltransferase (DNMT1) inhibitor-based epigenetic modulation. However, it remains unclear whether DNMT1 inhibitors induce re-expression of p16INK4A in patients treated with demethylation promoting agents. Here we report p16INK4A expression status in human circulating tumor cells (CTCs) and its dynamic changes during DNMT1 inhibitor therapy in patients with advanced solid tumors, by using a novel CTC-based p16INK4A immunofluorescence assay. Methods. We demonstrated in vitro re-expression of p16 by Western blot and whole cell immunofluorescence in EJ-6 cells treated with decitabine, 4-thio-2’-deoxycytidine (TdCyd) and 5-fluoro-2’-deoxyctidine (FdC) but not in the p16-deleted line A549. We analyzed p16 re-expression in epithelial /mesenchymal CTC subpopulations as a clinical pharmacodynamic (PD) marker to assess the effects of DNMT1 inhibitors (analyzed using CellSearch® and fluorescence microscopy). Results. Over 300 specimens from 70 patients were analyzed from patients enrolled in both Phase 1 and Phase 2 clinical trials of the DNMT1 inhibitors TdCyd or FdC in combination with tetrahydrouridine (THU). Circulating tumor cells were characterized as DAPI+/CD45-/CEA or MUC1 positive, then classified as epithelial or mesenchymal based on expression of (pan-)cytokeratin (CK) or vimentin (VIM), respectively. The p16INK4A-positive CTC baseline level ranged from 0 to 15% in 57 of 58 patients with assessable CK+CTC numbers and in 46 of 53 patients with assessable VIM+CTC numbers. There were no patient specimens with CTCs that were positive for only VIM, but most specimens had higher numbers of VIM+ CTC compared to CK+ CTC, and not all patients had both CTC phenotypes present at baseline. During treatment with DNMT1 inhibitors, p16INK4A-positive CTCs increased in 46 of 58 patients with assessable CK+ CTCs (epithelial phenotype), but only 6 patients with VIM+ CTCs (mesenchymal phenotype) had p16 re-expression, and these patients had both CTC phenotypes present. The restoration of p16INK4A expression in CTC, not the CTC number, was correlated with evidence of drug clinical activity in patients. Drug treatment longer than 2 cycles was associated with both EMT phenotypic conversion and obvious morphological changes in CTCs that accompanied increased p16INK4A expression. Conclusions. Our studies indicate that monitoring dynamic changes of CTC-based p16INK4A expression is a sensitive PD biomarker for assessing DNMT1-targeted epigenetic anticancer therapeutics in clinical development. CTCs were predominantly mesenchymal phenotype. In addition, the CTC phenotype (epithelial vs mesenchymal) appeared to be quite dynamic in treated patients. Funded by NCI Contract No. HHSN261200800001E, and Cooperative Agreements U01CA062505 and UM1CA186717. Citation Format: Lihua Wang, Brandon Miller, Sonny Khin, Geraldine O’Sullivan-Coyne, Alice P. Chen, Shivaani Kummar, Naoko Takebe, Kate Ferry-Galow, Ralph E. Parchment, Larry Rubinstein, Richard Piekarz, Edward M. Newman, James H. Doroshow, Robert J. Kinders. Restoration of p16INK4A expression in circulating tumor cells in patients with advanced solid tumors treated with deoxycytidine analogs and associated dynamic EMT phenotypic changes [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B018. doi:10.1158/1535-7163.TARG-19-B018