Endocrine Abstracts

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 for disease progression. Liquid biopsy based on CTCs enrichment and characterization seems to be an intriguing non-invasive tool providing information about tumor biology and molecular identity. The aim of this study was the molecular characterization of CTCs in spMTC patients with DM and/or BPD using epithelial, mesenchymal as well as MTC-specific markers.

Methods: Nine spMTC patients (DM:3, BPD:6) carrying somatic mutations in RET (n = 7) and HRAS (n = 2) were included. Peripheral blood (20mL-EDTA) was obtained every six months. Using identical blood draws for 31 PB-samples, CTCs enrichment was directly compared by EpCAM-based positive immunomagnetic selection (EpCam-IMS) and the size-based Parsortix microfluidics system (Angle PLC, UK). The EpCam-IMS was superior in terms of sensitivity since a significantly higher percentage of identical PB-samples was found positive at the gene expression level (P < 0.05) while specificity was not affected. 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). Calcitonin and CEA DTs were calculated, and disease status was determined according to the RECIST criteria.

Results: Calcitonin and CEA DTs were > 2 years in all but one patient (mean: 11.28 and 9.23 years, respectively). No structural disease progression (SDP) was documented except for one patient with HRAS mutation (pt-X). Interestingly, Calcitonin and CEA DTs of pt-X were 5.08 and 3.00 years, respectively, although there was an upward trend in CEA while serum Calcitonin levels were significantly elevated one month after SDP. Overexpression of CALCA was detected in one sample, related to pt-X, at a time-point set 60-days before marked serum calcitonin increase and 30-days before SDP was documented. CXCR4 was strongly expressed in 3 samples related to pt-X; CXCR4 expression was absent only at the final time-point of pt-X, when disease stabilization (biochemical and structural) was documented, after changing systemic treatment. Epithelial markers were not expressed in any of our samples while VIM was overexpressed in most of them (n = 20/31 samples).

Conclusions: EpCAM-IMS seems to be a better method for CTCs isolation in MTC patients with DM and/or BPD. Expression of VIM in most of our patients advocates towards an epithelial to mesenchymal transition (EMT) process possibly occurring in progressive MTC. CALCA and CXCR4 expression in CTCs, along with other epithelial and mesenchymal markers, should be studied in larger patients’ series and for longer follow-up periods.