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1. Introduction

This review discusses the most recent data on liquid biopsy in patients with melanoma, the most common skin cancer with high prevalence in US and European populations [1], or Merkel Cell Carcinoma (MCC), a skin tumor with a disease-associated mortality rate even higher than that of melanoma [2]. Due to their high mortality rate, new technologies are needed to improve the patient outcome. Particularly, specific biomarkers are required to facilitate their diagnosis and management. For a long time, cancer study was based on the analysis of specimens from the primary tumor or its metastases and on imaging data. The current limitations of tumor tissue biopsies and clinical imaging for cancer diagnosis and molecular profiling have led to the development of liquid biopsy. Indeed, tumor biopsy is an invasive procedure, and tumor tissue (especially in patients with cutaneous melanoma) is not always available. Therefore, liquid biopsy, a blood-based analysis of tumor-specific biomarkers, has been introduced as a new diagnostic approach that relies on circulating tumor cells (CTCs) and on circulating tumor-derived factors, such as cell-free tumor DNA (ctDNA), microRNAs (miRNA) and exosomes. The ultimate goal of liquid biopsy is to use the information gathered from such cells and factors to predict early cancer progression and to longitudinally monitor the treatment response, for a personalized medicine of patients with cancer. In melanoma, liquid biopsy has been already used to study many biomarkers (e.g., CTCs, ctDNA and exosomes), and its clinical pertinence is currently investigated in various clinical trials. For MCC, studies are in the early days and very few articles have been published. Moreover, no clinical trial is assessing liquid biopsy in MCC. In this review, we describe the circulating biomarkers and discuss the technical challenges and the clinical relevance of liquid biopsy for these two skin malignancies.

2. Merkel Cell Carcinoma

MCC is a rare skin cancer that usually appears as a pink/red, rapidly growing skin nodule on UV-exposed areas, such as head, neck and upper limbs. Usually, it is characterized by aggressive behavior and high metastasis rate without specific location ^[3][120]. Despite the currently low (but rapidly increasing) incidence (0.7 per 100,000) ^[4][5][6][121–123], MCC is associated with shorter disease-free and OS and higher cancer-related death rates than melanoma. To date, two main different oncogenic pathways have been identified ^[7][8][124,125]. The first is related to UV exposure with high tumor mutational burden, while the second one is related to a ubiquitous DNA virus, Merkel Cell Polyomavirus (MCPyV). Although the involvement of this virus in MCC development has been clearly established, the underlying molecular mechanisms have not been fully characterized. MCPyV was first described in 2008 by Feng et al. ^[9][126], and is the first human polyomavirus clearly linked to a human cancer ^[10][127]. Moreover, its epidemiological link with immunosuppressive conditions, including chronic lymphocytic leukemia and solid organ transplantation, is well established ^[11][12][128,129].

The hypothesis that MCC originates from epidermal Merkel cells ^[13][130] is supported by some common features between Merkel and MCC cells, such as the presence of a cytokeratin network as a dot, and the expression of cytokeratin-20 and neuron-specific enolase ^[14][15][131,132]. However, alternative theories have been proposed, involving, for example, a common cell ancestor with B lymphocytes ^[16][133].

Besides these debates on MCC cellular origin ^[17][131,134], recent studies tried to better understand the mechanisms underlying this malignancy, notably the involvement of the MCPyV virus that is detected in 80% of cases ^[18][19][20][124,127,135–137]. Currently, it is known that MCPyV is first present in cells in an episomal conformation ^[21][138] and is subsequently integrated in the cell DNA.

As this tumor remains poorly understood, liquid biopsy might help to decipher its nature, the underlying mechanisms and might ensure a real-time follow-up of the disease and of its response to different treatments. Research on this topic is in its early days, but some studies have already investigated different circulating biomarkers in MCC (Table 1).

Table 1. Studies on liquid biopsy in Merkel cell carcinoma.

CTC: circulating tumor cell, OS: Overall Survival, MCPyV: Merkel Cell Polyomavirus, ctDNA: circulating tumor DNA, miRNA: microRNA, ELISA: Enzyme-linked immunosorbent assay, RT-qPCR: Reverse-transcriptase Polymerase Chain Reaction.

2.1. CTCs and Circulating miRNAs

The few studies on this topic show how little is known about MCC. Moreover, the existing data concern small patient cohorts, due to MCC rarity. Nevertheless, some circulating biomarkers might help to better understand MCC. CTCs have been associated with patient survival and MCC aggressiveness ^[28][139,140,143,145], and high miRNA-375 concentration in plasma with tumor burden ^[29][142,146]. Therefore, they are candidate biomarkers for MCC follow-up. In patients with MCC, CTCs are usually detected using the CellSearch^® system ^[30][31][32][147–149], based on the positive enrichment of EpCAM-expressing cells. A new CTC detection method based on negative enrichment was investigated by Boyer et al. [144]. They evaluated CTC number to follow the disease course, and also characterized MCC CTCs (e.g., PD-L1 status). They found that CTC detection was associated with the cancer stage. The few studies on circulating miRNA in MCC used RT-qPCR as detection technology ^[33][142,150], like for other cancers ^[34][35][151,152]. The MCC miRNome has been investigated mostly in formalin-fixed paraffin-embedded tumor samples ^[36][37][153,154], and the miRNAs identified as MCC-specific could now be evaluated in liquid biopsies.

2.2. Exosomes

Exosomes are small vesicles secreted by different types of cells under the influence of cellular conditions and environment ^[38][155]. They might be used as an MCC biomarker. However, they have been investigated only in MCC cell lines and more data are needed. These preliminary studies suggest that exosomes might be a good candidate biomarker. Indeed, they showed that MCC-derived exosomes transport proteins linked to cancer, such as LDH and factors implicated in the p38 MAPK and Wnt signaling pathways. Importantly, these proteins were detected independently of the cell line MCPyV status ^[38][155], thus they could be used to monitor all patients with MCC.

2.3. Anti-MCPyV Antibodies

The presence of MCPyV in most MCC specimens and the higher incidence in immune-deficient patients indicate the implication of the immune system in MCC [123,124]. The large T (LT) and small T (sT) antigens of MCPyV are involved in oncogenesis, for example their presence has been linked to cell cycle disturbance or viral replication. Moreover, LT is required for the survival of cancer cell lines ^[39][156]. Titration of the VP1 capsid protein of MCPyV has been used as a circulating biomarker of the viral load in patients. In these studies, high levels of anti-VP1 and anti-sT antibodies in blood was correlated with better outcome ^[40][157], and anti-LT antibodies are a prognostic factor of recurrence if they are detected more than one year after diagnosis [141].

2.4. Immunotherapy

An immunohistochemical analysis of MCC specimens found that many cancer cells express PD-L1, particularly when they are in close proximity to infiltrating immune cells ^[41][158]. This mechanism is used by MCC cells to escape immunity. Specifically, cancer cells express PD-L1 at their surface, and its interaction with PD-1 at the surface of immune cells will block their identification as cancer cells. This phenomenon has been highlighted in many different cancers and is one of the immune checkpoints targeted by immunotherapy. Recently, immunotherapy based on PD-1/PD-L1 inhibition ^[42][43][44][159–161] has been approved by the US FDA for patients with metastatic MCC. This therapy has already been used in other cancers for some years ^[45][162] and is also efficient in patients with metastatic MCC, although the response rate remains unsatisfactory ^[46][159,163]. Detection of PD-L1 at the surface of CTCs could help for MCC patient management. This emphasizes the need of better understanding this disease to develop more appropriate treatments.

A query of the ClinicalTrials.gov database with the keyword “Merkel cell carcinoma” in November 2019 did not retrieve any ongoing study on circulating biomarkers in MCC. Most of the listed studies were testing new treatments.

2.5. Conclusion

Liquid biopsy in MCC could be of clinical interest for patient management, as suggested by the correlation of CTCs and circulating miRNAs with disease outcomes and tumor burden. However, more research must be done in larger cohorts and on different potential candidate biomarkers.