Evaluation of HLA-G immune checkpoint molecule in melanocytic lesions

The human leukocyte antigen-G (HLA-G) is a non-classical molecule of the major histocompatibility complex. HLA-G has been associated with the process of tumorigenesis and tumor escape. In this study, we aim to evaluate the HLA-G expression in melanocytic lesions and in melanoma for determining when melanocytic lesions start its expression. Twenty-two skin biopsies samples were submitted to immunohistochemistry; HLA-G expression was detected in 63.6% of the samples. This expression in melanocytic cells was significantly higher in melanoma than in benign melanocytic lesions ( p < 0.002). Our results suggest that HLA-G expression starts late in the process of tumorigenesis.

Evaluation of HLA-G immune checkpoint molecule in melanocytic lesions e1632020 introduction Melanoma is an aggressive tumor, accounting for most of the deaths caused by skin cancer. Although surgical excision is effective for the treatment of primary cutaneous melanoma, metastatic melanoma presents a high mortality rate. Standard chemotherapy has a limited effect in these cases (1) .
However, in the last decades there has been a revolution in the treatment of melanoma. Studies on the molecular mechanisms of this disease and cancer immunology have been performed. Currently, two new classes of systemic treatments are available: immunotherapy and targeted therapy (2)(3)(4) .
Immunotherapeutics target immune molecule checkpoints. However, the response to treatment is not uniform and is directly influenced by the tumor microenvironment. It is fundamental to know the infiltrate of immunocompetent cells in the tumor, as well as the factors and molecules produced by them (5) .
Most cancers produce several immunosuppressive factors that allow the immune response to be avoided. Among the mechanisms of immune escape of tumor cells, we can mention the expression of the human leukocyte antigen-G (HLA-G) molecule. HLA-G is distinguished from other HLA class I molecules, mainly because it: has a limited gene polymorphism; undergoes systematic alternative recombination of the primary transcript; presents tissue protein expression restricted to the placenta (invasive cytotrophoblast and amniotic epithelial cells) and certain adult tissues (thymus, pancreas, proximal nail matrix, and cornea) and erythroblasts; presents immune tolerance functions (6) .
Notably, the HLA-G molecule expression can be observed ectopically under pathological conditions. HLA-G expression in tumors is capable to protect cancer cells from natural killer (NK) cells and cytotoxic T lymphocytes. Thus, HLA-G expression becomes an important mechanism in which cancer cells are used to escape host immune surveillance. It is now widely accepted that HLA-G is a critical marker of immunotolerance in cancer cell immune evasion and is strongly associated with disease progress and prognosis for cancer patients (7) . HLA-G is considered an immune checkpoint and its heterogeneity in cancers have been related to disease stage and outcomes, metastatic status and response to different therapies (8) .
In this context, our aim was to evaluate the HLA-G molecule expression in melanocytic lesions and in melanoma for determining when melanocytic nevus evolution begins its expression.
methodology Samples This study was approved by the Institutional Ethics Committee for Human Subjects (protocol 419.570). Medical records were reviewed to determine age and gender. These studies were performed at the Preventing Health System, Goiânia, Goiás, Brazil.
The samples were obtained after surgical resection. For this study, 22 melanocytic lesions of different histological subtypes (acral melanoma, extensive superficial melanoma, lentigo maligna melanoma, nodular melanoma, and melanocytic nevus) from 16 patients were evaluated.

Immunohistochemistry
Sections from paraffin-embedded specimens were immunostained with MEM-G/02 monoclonal antibody (1/100) that recognizes the free heavy chain of all HLA-G isoforms (Exbio, Praha, Czech Republic). Invasive cytotrophoblast from the third-trimester human placenta served as a positive HLA-G protein control. The reaction product was visualized using the Ultratech HRP Streptavidin-Biotin Universal Detection System (Immunotech-Coulter, Villepinte, France) according to the manufacturer's recommendations. Ten skin biopsies of healthy women who had undergone breast reduction surgery were used as normal controls.

Evaluation of stained sections
Immunoreactivity was scored by evaluating the percentage of positive cells, using a semi-quantitative scoring method. The scoring system was performed as previously described by Gonçalves et al. (2015) (9) . Two independent pathologists (E. L. Z. and B. W. K.) interpreted the HLA-G staining results.

Statistical analysis
The immunostaining scores were compared to the Mann-Whitney U test. Significance was defined as a p < 0.05 at a confidence interval of 95%. All statistical analyses were performed using the RStudio front-end 0.98.507 software.

results
The immunohistochemical staining revealed that HLA-G+ cells revealed a cytoplasmic brown-stained pattern. This staining was also observed in immune-inflammatory cells. From the 22 samples subjected to immunohistochemistry, 14 expressed the HLA-G, whereas no control sample expressed its. The expression in the melanocytic lesions and melanoma is shown in detail in Table. HLA-G antigen expression in melanocytic cells was significantly higher in melanoma than in nevi (p < 0.002). Female samples presented higher staining intensity than males (p < 0.001).  differentiated progression rates of melanocytic lesions, which corroborates with previous studies demonstrating the expression of HLA-G in melanoma.
Positivity for HLA-G in benign lesions suggests vigilance over melanocytic nevi. About 30% of melanomas are derived from benign melanocytic lesions. In a retrospective study involving 850 cutaneous patients, Lin et al. (2015) (10) , demonstrated that 235 patients presented melanoma associated with a melanocytic nevus.
Histological types of superficial extensive melanoma and acral lentiginous melanoma expressed HLA-G, suggesting that HLA-G increasing is associated with malignant transformation in this type of cell, since HLA-G is often expressed in tumor cells, and its expression is associated with unfavorable prognosis in various malignancies (11) .

conclusion
Our results suggest that the HLA-G expression begins at latestage of the tumorigenesis process. HLA-G could allow prediction of malignancy potential and affords new insights on how to improve the targeting of immunotherapies.

acknowledgments
The authors acknowledge the financial support given by Fundação de Amparo à Pesquisa do Estado de Goiás.

discussion
In this study, lesions characterizing the evolution of melanocytic lesions changes during melanoma development were evaluated. We observed the expression of HLA-G in more