Clinical Application of Circulating Tumor Cells and Circulating Tumor DNA in Uveal Melanoma

Abstract

Purpose: To evaluate the feasibility of using circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) for the management of uveal melanoma (UM).

Patients and methods: Low-coverage whole-genome sequencing was used to determine somatic chromosomal copy number alterations (SCNAs) in primary UM tumors, ctDNA, and whole-genome amplified CTCs. CTCs were immunocaptured using an antimelanoma-associated chondroitin sulfate antibody conjugated to magnetic beads and immunostained for melanoma antigen recognised by T cells 1 (MART1)/glycoprotein 100 (gp100)/S100 calcium-binding protein β (S100β). ctDNA was quantified using droplet digital polymerase chain reaction assay for mutations in the GNAQ, GNA11, PLCβ4, and CYSLTR2 genes.

Results: SCNA analysis of CTCs and ctDNA isolated from a patient with metastatic UM showed good concordance with the enucleated primary tumor. In a cohort of 30 patients with primary UM, CTCs were detected in 58% of patients (one to 37 CTCs per 8 mL of blood), whereas only 26% of patients had detectable ctDNA (1.6 to 29 copies/mL). The presence of CTCs or ctDNA was not associated with tumor size or other prognostic markers. However, the frequent detection of CTCs in patients with early-stage UM supports a model in which CTCs can be used to derive tumor-specific SCNA relevant for prognosis. Monitoring of ctDNA after treatment of the primary tumor allowed detection of metastatic disease earlier than ¹⁸F-labeled fluorodeoxyglucose positron emission tomography in two patients.

Conclusion: The presence of CTCs in localized UM can be used to ascertain prognostic SCNA, whereas ctDNA can be used to monitor patients for early signs of metastatic disease. This study paves the way for the analysis of CTCs and ctDNA as a liquid biopsy that will assist with treatment decisions in patients with UM.

Fig 1.

Comparison between the genetic profile of the primary tumor, cell-free DNA (cfDNA), two circulating tumor cells (CTCs), and a single peripheral blood mononuclear cell (PBMC) in a patient with metastatic uveal melanoma. (A) Whole-body fluorodeoxyglucose–positron emission tomography scan of a patient with uveal melanoma at the time of blood collection. (B) Brightfield and florescent images of the two CTCs used for somatic chromosomal copy number alteration analysis. Cells were stained with a combination of antibodies against the melanoma markers melanoma antigen recognised by T cells 1 (MART1)/glycoprotein 100(gp100)/S100 calcium-binding protein β (S100β; green), CD45 (red), and 4',6-diamidino-2-phenylindole (DAPI; blue), taken at ×200 magnification. (C) Whole-genome sequencing somatic chromosomal copy number alteration profile of primary formalin-fixed paraffin-embedded tumor, cfDNA, two CTCs, and a single PBMC. The obtained sequence depth is indicated for each plot. Red and blue bars represent chromosomal losses or gains, respectively.

Fig 2.

Circulating tumor cell (CTC) and circulating tumor DNA (ctDNA) quantification in a primary uveal melanoma (UM) cohort. (A) Example of immunocytochemical staining of a UM CTC and peripheral blood mononuclear cell (PBMC). Green fluorescence (AF488, Mel) indicates staining with a combination of antibodies against the melanoma markers MART1/gp100/S100β; red fluorescence (phycoerythrin [PE]) indicates CD45 positivity; and blue fluorescence (4',6-diamidino-2-phenylindole, DAPI) indicates the presence of a nucleus. CTCs were identified as Mel-positive and DAPI-positive and CD45-negative cells. (B-E) Graphs illustrate (B) CTC count versus basal median diameter (n = 26; P = .874; r = −0.034), (C) tumor size as apical height (n = 26; P = .237; r = −0.250), or (D) tumor volume (n = 26; P = .338; r = −0.244). Spearman rank r and P values are indicated. (E) Comparison of CTC counts in patients with UM with and without chromosome 3 monosomy (n = 10). Mann-Whitney U test P value is indicated. (F-I) Graphs illustrate (F) ctDNA copies/mL versus CTC count in 8 mL of blood (n = 26), (G) ctDNA copies/mL versus basal median diameter (n = 30; P = .787; r = 0.053), (H) tumor size as apical height (n = 30; P = .384; r = −0.170), or (I) tumor volume (n = 30; P = .982; r = −0.004). Spearman rank r and P values are indicated. No correlation was found between CTC, ctDNA, and tumor size.

Fig 3.

Analysis of circulating tumor DNA (ctDNA) in patients with liver metastasis. (A) Comparison of ctDNA levels in patients with primary (n = 27) and metastatic (n = 8) uveal melanoma (UM) showed statistically significant differences (Student t test, P < .001). (B) Plasma ctDNA levels in longitudinally collected samples from patient 656 with UM. (C) Plasma ctDNA levels in longitudinally collected samples from patient 433 with UM, before and after the development of overt metastatic disease as shown by fluorodeoxyglucose–positron emission tomography imaging of the liver. NAD, no active disease; PD, progressive disease. (*) Resection of a solitary liver metastasis.