Abstract
BACKGROUND
~5% of melanoma-associated brain tumor cases also develop LMDz, which is universally fatal. The aim of this study was to determine whether diagnosis and personalized treatment for melanoma-LMDz could be improved using the Veridex CellSearch® System adapted to enumerate CTCs from CSF. Cell-free DNA (cfDNA) and cell-associated DNA (caDNA) were analyzed. Culturing conditions were optimized for CSF-CTCs expansion ex vivo.
METHODS
Peripheral blood (PB)/CSF-CTCs were detected by Veridex CellSearch® System and the circulating melanoma cell kit. Melanoma cell enrichment and detection was based on anti-CD146 and anti-high molecular weight melanoma associated antigen (HMW-MAA-PE (MEL-PE)). Anti-CD34-APC and anti-CD45-APC were used for ruling out endothelial cells and leukocytes respectively, and DAPI was used for nuclear staining. CfDNA and caDNA were extracted and sequenced with TruSight Tumor 26 panel, Illumina Inc and then profiled in Mela Carta MassArray System.
RESULTS
PB-CTCs & CSF-CTCs: 12 patients with definite LMDz and 8 melanoma patients without LMDz were studied. Of the 12 LMDz patients, all but 1 patient (92%) had CSF-CTCs (range, 23 CTCs/ml CSF to 3055 CTCs/ml CSF). 3/12 patients (25%) had PB-CTCs (range, 0.4 CTCS/ml PB to 6.5 CTCs/ml PB). In contrast, only 3/8 (37%) melanoma patients without LMDz had CSF-CTCs detected, with significantly lower CTC counts per ml CSF (range, 0.13 CTCs/ml CSF to 0.6 CTCs/ml CSF). In their PB, 2/8 patients (25%) had PB-CTCs (range, 0.5 CTCs/ml PB to 1.6 CTCs/ml PB). CSF-CTCs Profile: LMDz patients showed GNAQ Q209P mutation (uveal melanoma), NRAS Q61R mutation (nasal melanoma) and also BRAF V600E mutation. Ex vivo culture of CSF-CTCs: We successfully demonstrated ex vivo expansion of isolated CSF-CTCs (possible for ~25% of samples) in the presence of FBS, FGF and EGF. CONCLUSIONS Taken together, the results indicate the potential value of CSF as a biopsy surrogate for assessing genetic makeup of CSF malignancies.