Endocrine Abstracts

JOINT2949

Objectives: Distant metastases (DM) and/or biochemical persistent disease (BPD) in MTC, adversely affect disease prognosis. Calcitonin and CEA doubling-times (DTs) are the main prognostic indicators. Liquid-biopsy based on CTCs enrichment and molecular characterization is a non-invasive tool providing information about tumor biology and molecular identity. The aim of this study was molecular characterization of CTCs in spMTC with DM and/or BPD using epithelial, mesenchymal and MTC-specific markers while a droplet-digital polymerase chain reaction (ddPCR) was performed to detect in CTC-derived genomic (g)DNA the most common somatic mutation in spMTC (RET-M918T).

Methods: Twelve spMTC patients (DM:7, BPD:5) carrying somatic mutations in RET (n = 7, RET-M918T=4/7) and HRAS (n = 5) were included. Peripheral blood (10mL-EDTA) was obtained every 3-6 months depending on disease progression. In total of 53 samples, CTCs enrichment by EpCAM-based positive immunomagnetic selection (EpCam-IMS) was performed following our previous announcement where EpCam-IMS proved to be superior to the size-based Parsortix microfluidics system. CTCs gene expression analysis was based on RT-qPCR for epithelial (CK-8, CK-18, CK-19), mesenchymal (Vimentin-VIM), MTC-specific (Calcitonin-CALCA) and chemokine-receptor markers (CXCR4). A ddPCR assay was developed to detect the RET-M918T. Calcitonin, CEA DTs and disease status according to RECIST, were recorded.

Results: During a mean f-up of 24. 6 months (range 12-39) Calcitonin and CEA DTs were >24 months. Structural disease progression (SDP) was documented in four patients (2/4 harboring RET-M918T). Overexpression of CALCA was detected in 10 samples related to 8 patients, including those with SDP, at time-points 1-6 months before calcitonin levels showed a mean percentage increase from the preceding time point of 28. 65±24. 23% vs 14. 09±8. 59%, P = 0. 058, which was observed in 11 samples without CALCA overexpression. CXCR4 was overexpressed in 5 samples related to 3 patients (2/3 with SDP). In 2/17 samples related to RET-M918T patients the mutation was detected by ddPCR at time-points 6 months before SDP was documented, albeit calcitonin and CEA were slightly increased; the samples related at the time-point of SDP documentation were found negative. Epithelial markers were expressed in 8/53 (15. 1%) samples, while VIM in 22/53 (41. 5%).

Conclusions: Molecular characterization of CTCs in progressive spMTC, may add valuable information in disease monitoring. CALCA and CXCR4 expression in CTCs as well as gDNA detection by ddPCR may be used as ancillary biomarkers towards efficient therapeutic management and precision medicine implementation. Increased expression of VIM advocates towards an epithelial to mesenchymal transition-EMT process. Larger patients’ series and for longer follow-up periods should be studied to validate these Results