Circulating tumor DNA tracking through driver mutations as a liquid biopsy-based biomarker for uveal melanoma

Prisca Bustamante,Julia V Burnier,Emma Youhnovska,Miguel N Burnier,Claude Laure Dugé,Mohamed Abdouh,Thupten Tsering,Jacqueline Coblentz,Rita P Proença,Cristina Fonseca,Sonia A Callejo,Christina Mastromonaco,Rafaella Atherino Schmidt Andujar,Nadya Blanchard

doi:10.1186/s13046-021-01984-w

Abstract

BackgroundUveal melanoma (UM) is the most common intraocular tumor in adults. Despite good primary tumor control, up to 50% of patients develop metastasis, which is lethal. UM often presents asymptomatically and is usually diagnosed by clinical examination and imaging, making it one of the few cancer types diagnosed without a biopsy. Hence, alternative diagnostic tools are needed. Circulating tumor DNA (ctDNA) has shown potential as a liquid biopsy target for cancer screening and monitoring. The aim of this study was to evaluate the feasibility and clinical utility of ctDNA detection in UM using specific UM gene mutations.MethodsWe used the highly sensitive digital droplet PCR (ddPCR) assay to quantify UM driver mutations (GNAQ, GNA11, PLCβ4 and CYSTLR2) in cell-free DNA (cfDNA). cfDNA was analyzed in six well established human UM cell lines with known mutational status. cfDNA was analyzed in the blood and aqueous humor of an UM rabbit model and in the blood of patients. Rabbits were inoculated with human UM cells into the suprachoroidal space, and mutated ctDNA was quantified from longitudinal peripheral blood and aqueous humor draws. Blood clinical specimens were obtained from primary UM patients (n = 14), patients presenting with choroidal nevi (n = 16) and healthy individuals (n = 15).ResultsThe in vitro model validated the specificity and accuracy of ddPCR to detect mutated cfDNA from UM cell supernatant. In the rabbit model, plasma and aqueous humor levels of ctDNA correlated with tumor growth. Notably, the detection of ctDNA preceded clinical detection of the intraocular tumor. In human specimens, while we did not detect any trace of ctDNA in healthy controls, we detected ctDNA in all UM patients. We observed that UM patients had significantly higher levels of ctDNA than patients with nevi, with a strong correlation between ctDNA levels and malignancy. Noteworthy, in patients with nevi, the levels of ctDNA highly correlated with the presence of clinical risk factors.ConclusionsWe report, for the first time, compelling evidence from in vitro assays, and in vivo animal model and clinical specimens for the potential of mutated ctDNA as a biomarker of UM progression. These findings pave the way towards the implementation of a liquid biopsy to detect and monitor UM tumors.

Highlights

Uveal melanoma (UM) is the most common intraocular tumor in adults
These findings pave the way towards the implementation of a liquid biopsy to detect and monitor UM tumors
By targeting well-known driver mutations that characterize UM, we performed digital droplet PCR (ddPCR) analyses to determine its feasibility and value for UM patient staging

Summary

Introduction

Despite good primary tumor control, up to 50% of patients develop metastasis, which is lethal. UM is characterized by constitutive activation of G proteincoupled receptor signaling, with a hotspot mutation in either G protein subunit alpha Q (GNAQ) or alpha 11 (GNA11). Choroidal nevi are the most common pigmented intraocular lesions, with a prevalence of 4.6 to 7.9% in the USA [16]. Because these lesions are generally asymptomatic and found on ophthalmic exams performed for other reasons, it is believed that the true incidence may be much higher. Risk factors for malignant transformation include tumor thickness greater than 2 mm, subretinal fluid, visual symptoms, orange lipofuscin pigment, tumor margin within 3 mm of the optic disc (i.e. peripapillary), ultrasonographic hollowness, and halo absence [18]

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