# NEW THERAPIES AND IMMUNOLOGICAL FINDINGS IN MELANOMA AND OTHER SKIN CANCERS

EDITED BY : Atsushi Otsuka and Reinhard Georg Dummer PUBLISHED IN : Frontiers in Oncology and Frontiers in Immunology

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# NEW THERAPIES AND IMMUNOLOGICAL FINDINGS IN MELANOMA AND OTHER SKIN CANCERS

Topic Editors: Atsushi Otsuka, Kyoto University, Japan Reinhard Georg Dummer, UniversitätsSpital Zürich, Switzerland

Citation: Otsuka, A., Dummer, R. G., eds. (2019). New Therapies and Immunological Findings in Melanoma and Other Skin Cancers. Lausanne: Frontiers Media. doi: 10.3389/978-2-88945-712-0

# Table of Contents

Kazuyasu Fujii

Judith A. Seidel, Atsushi Otsuka and Kenji Kabashima

Keitaro Fukuda and Takeru Funakoshi

Hassan Sadozai, Thomas Gruber, Robert Emil Hunger and Mirjam Schenk

*113 Strategies to Improve the Efficacy of Dendritic Cell-Based Immunotherapy for Melanoma*

Kristian M. Hargadon

# Biomarkers for immune Checkpoint inhibitors in Melanoma

*Shigehisa Kitano1,2\*, Takayuki Nakayama1 and Makiko Yamashita1*

*1Department of Experimental Therapeutics, National Cancer Center Hospital, Tokyo, Japan, 2Division of Cancer Immunotherapy, Exploratory Oncology Research and Clinical Trial Center, National Cancer Center, Tokyo, Japan*

Immune checkpoint inhibitors have now become a standard therapy for malignant melanoma. However, as immunotherapies are effective in only a limited number of patients, biomarker development remains one of the most important clinical challenges. Biomarkers predicting clinical benefit facilitate appropriate selection of individualized treatments for patients and maximize clinical benefits. Many biomarkers derived from tumors and peripheral blood components have recently been reported, mainly in retrospective settings. This review summarizes the recent findings of biomarker studies for predicting the clinical benefits of immunotherapies in melanoma patients. Taking into account the complex interactions between the immune system and various cancers, it would be difficult for only one biomarker to predict clinical benefits in all patients. Many efforts to discover candidate biomarkers are currently ongoing. In the future, verification, by means of a prospective study, may allow some of these candidates to be combined into a scoring system based on bioinformatics technology.

#### *Edited by:*

*Atsushi Otsuka, Kyoto University, Japan*

#### *Reviewed by:*

*Satoshi Fukushima, Kumamoto University, Japan Rodabe N. Amaria, University of Texas MD Anderson Cancer Center, United States*

> *\*Correspondence: Shigehisa Kitano<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Cancer Immunity and Immunotherapy, a section of the journal Frontiers in Oncology*

*Received: 31 January 2018 Accepted: 29 June 2018 Published: 18 July 2018*

#### *Citation:*

*Kitano S, Nakayama T and Yamashita M (2018) Biomarkers for Immune Checkpoint Inhibitors in Melanoma. Front. Oncol. 8:270. doi: 10.3389/fonc.2018.00270*

Keywords: biomarker, immune checkpoint inhibitor, malignant melanoma, cytotoxic T-lymphocyte-associated antigen 4, programmed death-1

# INTRODUCTION

In recent years, immune checkpoint inhibitors have increasingly been applied to the clinical development of cancer immunotherapy. For malignant melanoma, ipilimumab, a humanized monoclonal antibody (mAb) that blocks cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and nivolumab, as well as pembrolizumab, a humanized mAb that blocks programmed death-1 (PD-1) on primed T cells, have been approved and are now used as standard therapies. Several clinical trials have investigated new agents, alone and in combination, for use in the treatment of advanced malignant melanoma. However, immunotherapies are effective in only a limited number of patients and severe immune-related adverse events (irAEs) develop in some patients. Biomarkers predicting clinical benefit support appropriate the selection of individualized treatments for patients and maximize clinical benefits. Thus, one of the most important tasks for advancing this form of therapy is to identify "baseline (pretreatment)" biomarkers predicting responses or toxicities. In general, biomarkers are mainly divided into two functional categories, "prognostic" and "predictive." A prognostic biomarker can be defined based on the effects of patient or tumor biology on the patient's clinical outcome. This includes patients at high risk for disease relapse who may thus derive benefit from earlier treatments. On the other hand, a predictive biomarker is defined by the effects of treatment, including tumor response and improvements in overall survival (OS), disease-free survival (DFS), and progression-free survival (PFS). Many biomarker candidates have been identified, to date, in retrospective settings. This review summarizes recent findings of biomarker studies designed to identify means of predicting the clinical benefits of immunotherapies in melanoma patients, focusing on three categories: tumor tissue, peripheral blood, and others (**Table 1**).

Table 1 | Biomarkers for metastatic melanoma patients treated with immune checkpoint inhibitor therapy.

Peripheral blood Number of lymphocytes (42, 43) Number of Tregs (38, 44, 45) Number of MDSCs (46–50) Number of proliferating CD8+ T cells (52–57) Number of memory CD4+ T cells (58–60) Concentrations of cytokines (e.g., IL-6, IL-8, IL-10, and TGF-β) (62–64) Concentration of VEGF (66) PD-L1 expression on circulating tumor cells (8) Soluble PD-L1 (67) Others Microbiome (68–70) Fatty acids (71) Vitiligo and rash (72–75)

# BIOMARKERS IN TUMOR TISSUE

### PD-L1 Expression on Tumor Cells

Programmed death-ligand 1 (PD-L1) expression has been investigated as a potential biomarker for PD-1 or the PD-L1 inhibitor. In phase I trials, PD-L1 expression on tumor cells correlated with the response to anti-PD-1 antibody (1). Given these promising results, several companies developed PD-L1 companion diagnostic tests for anti-PD-1/PD-L1 antibody and patients with PD-L1-positive tumors were considered to be good candidates for anti-PD-1/PD-L1 antibody treatment. In fact, the U.S. Food and Drug Administration has approved pembrolizumab, an anti-PD-1 antibody, for the treatment of PD-L1-positive non-small cell lung cancer (NSCLC) and gastric cancer. However, there are several problems while using PD-L1 expression as a biomarker for immunotherapy. First, PD-L1 expression levels show heterogeneity within tumors (2). Second, PD-L1 is a dynamic marker that can be affected by treatment and local inflammation (3). Third, the optimal threshold level of PD-L1 expression remains uncertain (4). In fact, some PD-L1 negative patients also derive benefit from treatment with an anti-PD-1/PD-L1 inhibitor.

Interestingly, PD-L1 expression on tumor infiltrating immune cells may be more predictive of responsiveness to anti-PD-1 antibody than the level of PD-L1 expression by the tumor (5). Furthermore, while PD-L1 expression on tumor cells did not tend to be related to the response rate in melanoma patients treated with anti-PD-1 antibody (nivolumab) and anti-CTLA-4-antibody (ipilimumab), there was a correlation with a good response in non-small lung cancer patients treated with these drugs (6, 7). On the other hand, Schott et al. reported that PD-L1 expression on "circulating" tumor cells might also be a potential biomarker (8). They suggested circulating tumor cells to possibly be precursors of metastatic disease, with PD-L1 expression allowing stratification according to the anticipated response to therapy. Further study is needed to determine the clinical significance of PD-LI expression.

# Genes: Mutation-Burden and Gene-Expression

Melanoma is characterized by having one of the highest mutation burdens of any cancer (9, 10). These somatic mutations generate immunogenic-neoantigens recognized as tumor-antigens, possibly triggering effective anti-tumor immune responses (11–13). Genomic analysis revealed that a high mutational load at baseline may predict better survival but not treatment responses (13), and the mutation burden after PD-1 therapy was reportedly decreased in melanoma patients who responded to treatment (14).

Genes harboring significant mutations included *BRAF*, *CDKN2A*, *NRAS*, *PTEN*, and *TP53* in cutaneous melanoma, *BRAF*, *NRAS*, *NF1*, and *KIT* in acral melanoma (hands and feet), and *SF3B1* in mucosal melanoma (internal body surfaces) (15–17). The *BRAF* mutation was the most common, being detected in approximately half of metastatic melanoma patients. In the current treatment of melanoma, only *BRAF* V600 mutations are regarded as being molecular markers applicable to treatment decision-making strategies (10, 18). Several studies of CTLA-4 and PD-1 therapy have revealed that *BRAF* V600E mutations do not correlate with either the response to CTLA-4 therapy or the resulting OS, whereas the correlation with the response of melanomas to PD-1 therapy was significant (11, 19). On the other hand, inactivation of *CDKN2A* and/or *PTEN* is regarded as an important mechanism underlying resistance and/or durable responses to BRAF-inhibitor-based therapy, but is not currently taken into consideration in the clinical decision-making process (10).

Previous sequence studies, such as The Cancer Genome Atlas study, used exome and low-pass whole-genome sequencing (WGS). In 2017, Hayward et al. reported the first large, high-coverage WGS study of melanomas (cutaneous, acral, and mucosal subtypes), including analysis of the non-coding region. Their report showed that the number of mutations in the non-coding region was detected as a number equivalent to that in the coding region, and that the most common mutations in the non-coding region were in the *TERT* promoter upstream from the initiation codon (69% of all melanomas and 86% of cutaneous melanomas) (17). Moreover, Ishida et al. preliminarily reported a correlation between HLA-A\*26 alleles and the response to anti-PD-1 (nivolumab) therapy in Japanese patients with metastatic melanoma (20). HLA accounts for some of the individual differences in antigenspecific immune responses, and might provide useful information for devising individualized immunotherapeutic regimens. The associations of these new findings with clinical responses to immunotherapies merit further investigation.

On the other hand, there have been several investigations of the gene expressions on tumor tissues, for their value in predicting responses to immune checkpoint inhibitors. Immunohistochemistry and gene profiling assays have suggested the presence of a "T-cell-inflamed tumor microenvironment," with an abundance of chemokines and an IFN-γ signature, to correlate with the clinical efficacy of immune checkpoint inhibitors in melanoma patients (21, 22). Numerous studies have revealed the molecular mechanisms underlying lack of T-cell infiltration and resistance of melanomas to immune checkpoint therapy, such as the melanomaintrinsic active WNT/β-catenin-signaling pathway (23) and enrichment for mutations in *PTEN* (24), loss-of-function mutations in Janus kinase (JAK1)/JAK2 (which are involved in IFNγ signaling), and β2 microglobulin (an MHC class I subunit) (25, 26).

# Tumor Infiltrating Lymphocytes (TILs)

Tumor infiltrating lymphocytes, such as T cells, macrophages, and various types of immune suppressive cells, are considered to be the most important players in the regulation of antitumor immune responses. Several studies have demonstrated an increase in the TIL number to correlate with good clinical responses and a higher survival rate of patients with melanoma and various other cancers (27, 28).

In melanoma patients, immune suppressive cells, such as regulatory T cells (Tregs) (29, 30), monocytic myeloid-derived suppressor cells (m-MDSCs) (31–35), and tumor-associated (activated) macrophages (TAM; M2) (36), were reportedly increased in number and thereby inhibited effector T cells, resulting in an increase in tumor growth.

In contrast, a number of investigators have reported the quantity of infiltrating CD8+CD45RO+ effector memory T cells to be clearly associated with longer DFS and OS, for many cancer types including melanoma (37–39). Recently, Wei et al. comprehensively profiled the effects of CTLA-4/PD-1-targeted immunotherapy on tumor infiltrating immune cells. Their study revealed that PD-1 blockade and CTLA-4 blockade both led to a subset of exhaustedlike CD8+ T cells (CD45RO+PD-1+T-bet+EOMES+). They also showed that CTLA-4 blockade induced the expansion of an ICOS+ Th1-like CD4 effector population (CD45RO+PD-1loTBET+ and CD69+) in melanoma patients. These observations suggested that these two immunotherapies target specific subsets of exhaustedlike CD8+ T cells, but drive different cellular mechanisms to induce tumor rejection (40). Moreover, Canale et al. described high expression of CD39 on CD8+ infiltrating T cells as being increased in melanoma lesions. CD39 is the immunosuppressive enzyme termed ATP ectonucleotidase, and CD39highCD8+ T cells reportedly exhibit features of cellular exhaustion, such as reduced production of tumor necrosis factor and interleukin (IL)-2, as well as expressions of co-inhibitory receptors (41).

# BIOMARKERS IN PERIFERAL BLOOD

# Peripheral Blood Mononuclear Cells (PBMCs)

Blood biomarkers have most frequently been analyzed for correlations with clinical responses to immunotherapies. Baseline and/or post-treatment changes in absolute counts of white blood cells, lymphocytes, eosinophils, neutrophils, and monocytes, as well as ratios of neutrophils or monocytes to lymphocytes may both be promising and routinely available blood markers that have shown associations with responses to immune checkpoint inhibitors (11, 42, 43).

Recently, several studies have raised the possibility of circulating immune cells as predictive biomarkers for immune checkpoint inhibitors. The frequency of circulating Tregs is reportedly associated with disease progression and poor patient survival for many carcinomas treated with immunotherapy (38, 44, 45). Numerous studies have found that high levels of circulating m-MDSCs in various forms of cancer, including melanoma, correlate with poor survival (46–48). In patients treated with anti-PD-1 antibody, m-MDSCs were reported to be a blood cytology marker showing significant correlations with all outcome parameters (49, 50). However, human MDSCs have yet to be clearly characterized both biologically and phenotypically. A very recent study demonstrated that the frequency of CD14+CD16- HLA-DRhi monocytes predicts both PFS and OS of melanoma patients treated with anti-PD-1 antibody, based on analysis employing high-dimensional single-cell mass cytometry (51). This CD14+ population including MDSCs might be useful as a predictive and/or prognostic biomarker for cancer patients receiving immunotherapy, but further investigation is needed to clarify the phenotype and biological characteristics of this diverse population of cells.

On the other hand, several studies examining circulating T cells have shown the involvement of CD8+ T cells, such as the proliferating (Ki67+) CD8+ effector-like T cells, in NSCLC patients receiving PD-1-therapy (52), and neoantigen-specific circulating CD8+ T cells in melanoma (53, 54). The latter are CD8+ T cells expressing PD-1. In addition, two complementary reports showed that CD28, a member of the same family as PD-1 (including CTLA-4 and ICOS), expressed on CD8+ T cells is a key molecule in PD-1-targeted therapy (55). Hui et al. showed that "CD28 is the primary target of PD-1 signaling," using a cell-free membrane reconstitution system. Their report revealed that PD-1 was phosphorylated in response to PD-L1 ligation, thereby preferentially inducing dephosphorylation of CD28 (but not the T cell receptor), resulting in the inhibition of T cell proliferation (56). On the other hand, Kamphorst et al. found that, in lung cancer patients, proliferating Ki67+PD-1+CD8+ T cells were increased in peripheral blood, and subsequently activated (CD38+, HLA-DR+) and mostly expressed CD28 (57), implying that CD28 signaling is associated with rescue of the exhausted CD8+ T cells in PD-1 targeted therapies. These findings are reasonable and it is interesting that CD28, belonging to the same family as PD-1, is a key molecule in PD-1-targeted therapy, although its applicability as a predictive/ prognostic biomarker in melanoma patients is as yet unclear. Moreover, whether other family members, including CTLA-4 and ICOS, have similar features in immune checkpoint therapy, remains unknown. Elucidating these issues might reveal novel useful biomarkers for use alone and/or in combination with PD-1-targeted therapy. Another interesting, and potentially important, finding of these studies is that proliferating CD8+

effector-like T cells were reportedly increased following PD-1 targeted therapy.

Several recent studies, focusing on circulating CD4+ T cells, found that increases in central memory CD4+ T cells (CD27+, FAS−, CD45RA−, and CCR7+) (58), and IL-9-producing CD4+ T helper (Th9) cells (59), correlated with good clinical responses of melanoma patients to anti-PD-1 therapy. Moreover, in lung cancer patients treated with nivolumab, the frequencies of CD62LlowCD4+ T cells and Tregs (CD25+Foxp3+CD4+) in pretreatment PBMC were reported to correlate significantly with clinical responses (60). Their ASCO presentation outlined the major differences in pre-existing immunity, among patients showing a partial response, stable disease, or progressive disease, in response to anti-PD-1 Ab, as reflected by the status of CD4+ T cells, i.e., the balance between primed effector and Tregs. These recent reports raised the possibility that, in peripheral blood, not only T cell exhaustion but also activation of effector CD8+ T cells and increases in memory T cells appear to be highly important, and not only phenotyping markers but also functional molecules can serve important roles as prognostic and/or predictive factors for immune checkpoint inhibitors. Although peripheral blood analysis may provide valuable insights into the responses of cancer patients to immune checkpoint inhibitors, more investigation is needed before these biomarkers can be applied in clinical settings.

# OTHERS

# Soluble Factors (Serum/Circulating Factors)

Lactate dehydrogenase was frequently investigated in previous studies and showed significant correlations with OS and PFS, whereas there were no correlations with responses to treatments (61). Recently, several studies have revealed that serum cytokine levels to correlate with responses to immune checkpoint inhibitors. Sanmamed et al. showed serum IL-8 levels to be highly correlated with tumor burden changes in metastatic melanoma and NSCLC patients during treatment with anti-PD-1/anti-CTLA-4 therapy (62, 63), and Yamazaki et al. reported that pretreatment serum IFN-γ, IL-6, and IL-10 levels were significantly higher in those with tumor progression among patients with advanced melanoma given nivolumab (64). In addition, in patients with metastatic melanoma receiving nivolumab, the activity of soluble CD73, which is an enzyme that hydrolyzes extracellular AMP to adenosine, in blood was shown to be significantly associated with clinical outcomes (65). Moreover, Frankhauser et al., studying metastatic melanoma patients, reported gene expression of vascular endothelial growth factor-C (VEGF-C) to correlate markedly with both CCL21 and T cell inflammation, and that serum VEGF-C concentrations were associated with both T cell activation/ expansion and clinical responses to checkpoint blockade (66).

# Soluble PD-L1 (sPD-L1)

Pretreatment sPD-L1 levels reportedly correlate with progression of advanced melanoma treated with anti-CTLA-4 or anti-PD-1 antibody. Although changes in circulating sPD-L1 in the early phase after starting treatment did not distinguish responders from non-responders, patients who had increased circulating sPD-L1 after 5 months of treatment tended to show partial responses (67). The biology of sPD-L1 remains unclear and merits further research.

# Microbiome

A vast number of microbes colonize the human body. This colonization is associated with many diseases, including various malignancies. During the past decade, the advent of metagenomic sequencing that combines next-generation DNA sequencing technologies with computational analyses has allowed us to analyze the relationships between the microbiome and various cancers. Recent studies have suggested that the gut microbiome may affect the efficacy of immune checkpoint inhibitors and, consequently, that changing the gut microbiome of a mouse or even a human patient might make tumors more responsive to immune checkpoint inhibitors. This possibility was first evaluated using preclinical models. Vétizou et al. showed that the efficacy of anti-CTLA-4 therapy was diminished in a germ-free mouse model. In addition, the use of broad-spectrum antibiotics to eliminate gut microbiota altered the anti-tumor effect of anti-CTLA-4 therapy (68). Sivan et al. reported that *Bifidobacterium* counts decreased in parallel with the anti-tumor effects of anti-PD-L1 therapy in a mouse model (69). Furthermore, Gopalakrishnan et al. indicated that anti-PD-1 immunotherapy in melanoma patients may be modulated by the gut microbiome. These researchers reported significantly higher alpha diversity and a relative abundance of Ruminococcaceae bacteria in the gut microbiome of responders (70). These findings indicated that specific organisms comprising the gut microbiome enhanced anti-tumor responses in patients treated with immune checkpoint inhibitors. Although the gut microbiome is a potential predictive marker of immunotherapy, a larger prospective study is needed to confirm these results.

# Fatty Acids

Kim et al. investigated cellular metabolome and lipidome alterations related to melanoma metastasis. Their analysis showed a progressive increase in phosphatidylinositol species with saturated and monounsaturated fatty acyl chains, as the metastatic potential of the melanoma cells rose, highlighting these lipids as possible biomarkers (71).

# Vitiligo and Rash

Immune checkpoint inhibitors have a rather unique adverse event profile, generally described as irAEs, which are most commonly observed in the skin, the gastrointestinal tract, the lungs, the liver, endocrine system, and other organs. Cutaneous irAEs are much more common adverse events in patients with melanoma than in those with other solid tumors. Although vitiligo is attributed to an autoantibody to melanocytes, the etiology of vitiligo is not understood in detail. Vitiligo occurrence has long been speculated to be related to tumor shrinkage in melanoma patients (72). Vitiligo develops in 13–26% of patients treated with nivolumab (73, 74), though grade III/IV disease is rare.

Recent studies have shown vitiligo and rash to be associated with a significant OS improvement in metastatic melanoma patients treated with immune checkpoint inhibitors (73–75). Furthermore, Nakamura et al. suggested that the occurrence of vitiligo might not be regarded as an early marker of good clinical response because the mean time to vitiligo occurrence was approximately 5 months after starting nivolumab (73). The onset times of vitiligo vary considerably depending on the type of drug administered and patient features. Thus, when we use cutaneous irAEs as a biomarker for immune checkpoint inhibitors, we should take into consideration the characteristics of each drug.

# CONCLUSION

Numerous candidate biomarkers are currently the focus of research, based mainly on retrospective analyses. Most notably, tumor mutation burden, intratumoral or immune cell expressions of PD-L1, and CD8+ T cell infiltration into the tumor have been documented in several cohorts. For example, not only melanoma but also lung carcinoma, one of the carcinomas which also has a high mutation burden, shows good clinical responses to PD-1/PD-L1 therapy. In lung carcinoma, mutation burden, TIL accumulation, and/or PD-L1 expression on tumor cells correlated with good clinical responses. However, renal cell carcinoma is also reportedly responsive to PD-1 therapy, despite having a low mutation burden, while TIL accumulation and PD-L1 expression did not correlate with treatment effectiveness. These observations suggest that these factors are not always applicable to predicting clinical benefits. Taking into account the complex interactions between the immune system and malignancies *via* cell surface molecules, such as immune checkpoint molecules, humoral factors, including proteins, cytokines, and so on, it is not unreasonable to speculate that a single biomarker would not allow clinical benefits to be predicted in all patients. In the near future, by applying bioinformatics technology, several biomarkers might be combined to produce a useful scoring system, depending on the type of cancer, the stage, individual treatments, and the timing of intervention. Recent advancements in assay technology, such as mass cytometry (CyTOF), multicolor IHC, multiplex gene analyzer, and so on (**Figure 1**), have the potential to provide an abundance of biological and/ or phenotypical observations in a range of environments. Now is the time to discover the candidate biomarkers which might comprise such a future scoring system. Finally, needless to say, a prospective study on a large patient population is essential.

# AUTHOR CONTRIBUTIONS

SK: conception/design of the manuscript. SK, TN, and MY: writing of the manuscript.

# REFERENCES

**Conflict of Interest Statement:** The authors have no conflicts of interest to disclose. Outside of the submitted work, SK reports personal fees from Astra Zeneca, personal fees from Chugai, personal fees from Pfizer, personal fees from Sanofi, personal fees from Nippon Kayaku, personal fees from Boehringer Ingelheim, personal fees from Meiji Seika Pharma, personal fees from Taiho, personal fees from Novartis, personal fees from Daiichi-Sankyo, personal fees from MSD, personal fees from Kyowa Hakko Kirin, personal fees from Celgene, personal fees from Sumitomo Dainippon Pharma, grants and personal fees from Eisai, grants from REGENERON, grants from Astellas, grants from Gilead Sciences, grants from AMED (Japan Agency for Medical Research and Development), grants from JSPS (Japan Society for the Promotion of Science), personal fees from Ono Pharmaceutical Co., Ltd., and personal fees from Bristol-Myers Squibb.

*Copyright © 2018 Kitano, Nakayama and Yamashita. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# New Therapies and immunological Findings in Cutaneous T-Cell Lymphoma

#### *Kazuyasu Fujii\**

*Department of Dermatology, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan*

Primary cutaneous lymphomas comprise a group of lymphatic malignancies that occur primarily in the skin. They represent the second most common form of extranodal non-Hodgkin's lymphoma and are characterized by heterogeneous clinical, histological, immunological, and molecular features. The most common type is mycosis fungoides and its leukemic variant, Sézary syndrome. Both diseases are considered T-helper cell type 2 (Th2) diseases. Not only the tumor cells but also the tumor microenvironment can promote Th2 differentiation, which is beneficial for the tumor cells because a Th1 environment enhances antitumor immune responses. This Th2-dominant milieu also underlies the infectious susceptibility of the patients. Many components, such as tumor-associated macrophages, cancer-associated fibroblasts, and dendritic cells, as well as humoral factors, such as chemokines and cytokines, establish the tumor microenvironment and can modify tumor cell migration and proliferation. Multiagent chemotherapy often induces immunosuppression, resulting in an increased risk of serious infection and poor tolerance. Therefore, overtreatment should be avoided for these types of lymphomas. Interferons have been shown to increase the time to next treatment to a greater degree than has chemotherapy. The pathogenesis and prognosis of cutaneous T-cell lymphoma (CTCL) differ markedly among the subtypes. In some aggressive subtypes of CTCLs, such as primary cutaneous gamma/delta T-cell lymphoma and primary cutaneous CD8+ aggressive epidermotropic cytotoxic T-cell lymphoma, hematopoietic stem cell transplantation should be considered, whereas overtreatment should be avoided with other, favorable subtypes. Therefore, a solid understanding of the pathogenesis and immunological background of cutaneous lymphoma is required to better treat patients who are inflicted with this disease. This review summarizes the current knowledge in the field to attempt to achieve this objective.

Keywords: cutaneous T-cell lymphoma, mycosis fungoides, Sézary syndrome, primary cutaneous CD30+ T-cell lymphoproliferative disorders, adult T-cell leukemia/lymphoma

# OVERVIEW OF CUTANEOUS T-CELL LYMPHOMAS (CTCLs)

Non-Hodgkin lymphomas can occur at extranodal sites in approximately 27% of cases, with the gastrointestinal tract and skin being the first and second most common sites of extranodal involvement (1). Most nodal non-Hodgkin lymphomas are B-cell derived, which is in contrast to the approximately 75–85% of primary cutaneous lymphomas that are T-cell derived (2–6). The incidence of CTCLs has been increasing (7); consequently, 4–8 people per million currently suffer from these cancers (8, 9). CTCL represents a series of skin-based neoplasms of T-cell origin,

#### *Edited by:*

*Atsushi Otsuka, Kyoto University, Japan*

#### *Reviewed by:*

*Yu Sawada, University of Occupational and Environmental Health Japan, Japan Daniel Olive, Aix Marseille Université, France*

*\*Correspondence: Kazuyasu Fujii<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Cancer Immunity and Immunotherapy, a section of the journal Frontiers in Oncology*

*Received: 13 March 2018 Accepted: 17 May 2018 Published: 04 June 2018*

#### *Citation:*

*Fujii K (2018) New Therapies and Immunological Findings in Cutaneous T-Cell Lymphoma. Front. Oncol. 8:198. doi: 10.3389/fonc.2018.00198*

**12**

*NR, not reached; CTCL, cutaneous T-cell lymphoma.*

*a A portion of these diseases is considered as primary CTCL.*

*bProvisional entity in World Health Organization classification (2016).*

predominantly comprised of peripheral CD4+ T-cells. There are 12 distinct CTCL subtypes (**Table 1**), with mycosis fungoides (MF) being the most common (10). Primary cutaneous CD30+ T-cell lymphoproliferative disorders are the second most common, except in some countries in the Pacific where adult T-cell leukemia/lymphoma (ATL) ranks second (6, 11).

# MF/SÉZARY SYNDROME (SS)

Mycosis fungoides and SS constitute the most common types of primary CTCLs. MF is characterized by erythematous patches, plaques, or tumors on the skin (**Figure 1**), with the involvement of lymph nodes, blood, and viscera also possible. MF can mimic benign inflammatory skin disorders, such as atopic dermatitis or psoriasis; thus, it is not unusual for MF to remain undiagnosed for years. Although MF is typically an indolent disorder, the disease may progress toward or exhibit *de novo* more advanced forms including tumors and erythroderma (>80% of the body surface area showing patches/plaques without overt leukemia). This can lead to lymph node or organ involvement, accompanied by increased morbidity and mortality. Patients are classified as having either early-stage (patches/plaques) or advanced-stage (tumors, erythroderma, lymph node, and/or visceral involvement) (12, 13). SS is the leukemic form of the disease, in which erythroderma is accompanied by measurable levels of malignant lymphocytes with cerebriform nuclei [i.e., Sézary cells (SC)] in the blood. Typical SC counts would be ≥1,000/μL, with a CD4/ CD8 ratio of ≥10 and a loss of one or more T-cell antigens (CD4+CD7− > 30% or CD4+CD26− > 40%). Furthermore, CD30 expression is associated with a significantly reduced diseasespecific survival and is often associated with histologically detectable large cell transformation, hallmarking a more aggressive clinical course (14).

In the past, SS has been considered a leukemic and aggressive variant of MF. However, a recent study determined that MF and SS arose from distinct T-cell subsets: SS from central memory

Figure 1 | Clinical findings of mycosis fungoides/Sézary syndrome. (A) Patches, (B) plaques, (C) and nodules on the plaque. Written informed consent was obtained from each patient.

T-cells and MF from skin-resident effector memory T-cells (15). CD158k/killer cell immunoglobulin-like receptor 3DL2 represents a specific marker for the evaluation of SC (16); in particular, CD4+ CD158k+ lymphocytes in blood from patients with SS correspond to the malignant clonal cell population (17). In addition, immunohistological finding of CD158k in affected skin is reported to distinguish SS from MF (18). Clonal malignant T-cells from the blood of patients with SS coexpress the lymph node homing molecules C–C motif chemokine receptor 7 (CCR7)/CD197 and CD62L/l-selectin, as well as the CD27 differentiation marker, a characteristic of central memory T-cells. This is consistent with the clinical presentation of peripheral blood disease, lymphadenopathy, and diffuse erythroderma of the skin. In contrast, T-cells from MF skin lesions do not express CCR7, l-selectin, and CD27, but strongly express CCR4 and cutaneous lymphocyte antigen (CLA)/ CD162, characteristics of skin-resident effector memory T-cells. This difference in the putative origins between SS (central memory T-cell-derived) and MF (tissue-resident memory-derived) can explain their distinct clinical behaviors; central memory T-cells are long-lived, apoptosis-resistant cells that can be found in the peripheral blood, lymph nodes, and skin, whereas skin-resident memory T-cells remain in the skin and do not enter the general circulation. That MF and SS are derived from different T-cell precursors is also supported by comparative genomic hybridization and gene-expression profiling, demonstrating that the CTCL genotypes are distinct (19, 20). Overall, MF is characterized by gains on chromosomes 1 and 7 and losses on chromosome 9, whereas SS is characterized by gains on chromosomes 8 and 17 and losses on chromosome 10. A multiplatform genomic analysis of patients with SS detected (1) activating *CCR4* and caspase recruitment domain-containing protein 11 (*CARD11*) mutations in nearly one-third of patients; (2) deletion of zinc finger E-box binding homeobox 1 (*ZEB1*), encoding a transcriptional repressor essential for T-cell differentiation, in over one-half of patients; and (3) overexpression of interleukin 32 (IL-32) and interleukin-2 receptor subunit gamma in nearly all patients (21).

# ROLES OF CHEMOKINES IN DEVELOPMENT OF SKIN PATHOLOGY

Malignant T-cells are suggested exhibit phenotypes of mature CD4+ memory T-cells, along with type 2 or 17 (Th2 or Th17 (22)) T-helper cell phenotypes, or be comprised of FOXP3 regulatory T-cells (Treg) (23, 24). Many chemokines are also reportedly expressed in the affected skin of patients with CTCL, suggesting that chemokine–receptor interactions play important roles in disease progression (25). Chemokine receptor expression on tumor cells in MF varies with disease stage. In the patch and plaque stages of MF, most infiltrating cells express CXC chemokine receptor (CXCR) 3/CD183 in the affected skin (26). CXCR3 binds three distinct ligands, namely CXC chemokine ligand (CXCL) 9/monokine induced by gamma interferon (MIG), interferon-gamma-inducible protein-10 (CXCL10/ IP-10), and interferon-inducible T cell alpha chemoattractant (CXCL11/ITAC)/IP-9; all are expressed in the affected skin in the patch and plaque stages of MF (27–29). Various cell types express these chemokines including keratinocytes, dermal fibroblasts, and Langerhans cells. In the early stages of MF, expression of CXCL9, CXCL10, and CXCR3 is believed to be important for recruitment and accumulation of tumor cells in the skin (25, 28). However, later in the tumor stage, the tumor cells increase in size and tend to express CCR4 instead of CXCR3 (30). The expression levels of CXCL9 and CXCL10 also tend to be lower in the affected skin of patients with MF during the tumor stage than during the patch and plaque stages (31). Moreover, CCR6/CD196 and its ligand CCL20/macrophage inflammatory protein (MIP)- 3α are upregulated in advanced CTCL (32). Tumor MF cells exhibit high levels of CCR7 (33), which is considered to be associated with loss of epidermotropism and migration to peripheral lymph nodes, which constitutively synthesize the CCR7 ligands, CCL19 and 21 (34). CCR7 is also expressed at high levels in SC (35), as mentioned in Section "MF/Sézary syndrome (SS)".

Circulating SC and skin-infiltrating cells in SS also express CCR4 (30, 35, 36). CCR4-expressing T-cells were found in CTCL lesions along with high expression of two CCR4 ligands, namely CC chemokine ligand (CCL) 17/thymus and activation-regulated chemokine and CCL22/macrophage-derived chemokine (30). CCL17 is expressed by endothelial cells and keratinocytes in the affected skin of patients with MF and SS (30, 37). During the tumor stage of MF, serum CCL17 levels are much higher than those during the patch/plaque stages (37), suggesting the importance of CCL17–CCR4 interactions in tumor cell trafficking to the skin of these patients. CCL22 is expressed by dendritic-like cells and keratinocytes (30, 37). Serum CCL22 levels are significantly higher in patients with MF than in healthy controls or patients with psoriasis vulgaris (37).

CC chemokine ligand 27/cutaneous T cell attracting chemokine is a CCR10 ligand that is constitutively produced by activated keratinocytes in various diseases (38). CCR10 is expressed on the tumor cells of MF and SS (35, 39). Strong immunostaining of CCL27 has been observed in the affected skin of patients with MF compared to that of unaffected individuals (39, 40). Serum CCL27 levels and the number of circulating CCR10+ CD4+ cells are both increased in patients with MF compared to that of control patients (39). Therefore, CCR10–CCL27 interactions may also contribute to the migration of lymphoma cells to the affected skin in MF and SS. In addition to CCR4 and CCR10, expression of the receptor for CXCL12/stromal cell-derived factor 1, CXCR4, is observed in SC (36). CXCL12 is a chemoattractant for CXCR4-positive cells and is strongly expressed in the affected skin of patients with MF (41) and SS (36). Therefore, CXCL12–CXCR4 interactions may also facilitate the recruitment of lymphoma cells to the skin.

# Th2-DOMINANT MICROENVIRONMENT

As the microenvironment in early-stage MF consists of nonmalignant Th1 cells and CD8+ tumor-infiltrating T cells, MF and SS are considered Th2-type diseases, which are frequently accompanied by eosinophilia and high serum levels of IgE. In the early 1990s, peripheral blood mononuclear cells in patients with SS and non-leukemic CTCL were reported to be Th2 dominant (42, 43). In 1994, mRNA for Th2 cytokine was detected in the skin of patients with MF (44). T-cell clones from patients with SS were identified thereafter to have Th2-like properties (45). However, in the early stages of MF, affected skin and peripheral blood T-cells express a profile of Th1 cytokines (46, 47). The Th2 phenotype appears to be caused by leukemic T-cells, as culturing benign T-cells away from malignant clones reduces Th2 and enhances Th1 responses (48). The Th2-dominant microenvironment is advantageous for tumor cells, because interferon (IFN)-γproducing Th1 cells enhance immune responses against the tumor. Indeed, IFN-γ has been shown to be effective for CTCL treatment (49, 50). Adenoviral-mediated gene therapies that increase expression of IFN-γ have also been used successfully in CTCL (51–53). CTCL cells can inhibit T-cell proliferation and suppress dendritic cell (DC) maturation by secretion of Th2 cytokines (54). Skin and nasal colonization with *Staphylococcus aureus* is common in patients with MF/SS; in particular, a Th2-dominant microenvironment may underlie this susceptibility to infection (55). Infections of *S. aureus* and sepsis also frequently occur in patients with CTCL (56). Accordingly, the major cause of death in patients with erythrodermic MF and SS is intravenous line sepsis, with *S. aureus* often being the causative microorganism (57).

In early-stage MF, signal transducers and activators of transcription (STAT) 4, the activation of which is required for Th1 differentiation, are overexpressed by IL-12 signaling *via* JAK2/ TYK2 (58). In later stages, IL-2 and IL-15 signaling *via* JAK1 and JAK3 kinases activates STAT5, which increases the expression of oncogenic miR-155 (59) and subsequently inhibits STAT4 expression (60), resulting in a switch from Th1 to Th2 phenotype in malignant T cells. Downregulation of STAT4 is also induced by deficiencies in IL-12 expression (58, 61) and lack of the IL-12R β2 chain (58). During this switch, the expression of STAT6 is often upregulated in CTCL (60). STAT5 activation is seen in both early and late stages. Specifically, in the late stage, constitutive STAT5 activation is induced by cytokine-independent JAK1/ JAK3 signaling (59). In the advanced stage, such constitutive STAT3 activation, which increases survival and resistance to apoptosis and promotes Th2 and Th17 phenotypes, is induced by an IL-21 autocrine signaling loop (62), the presence of IL-7 and IL-15 in the microenvironment (63), and/or constitutive cytokine-independent activation of JAK1 and JAK3 signaling (64, 65). Moreover, GATA3, a transcriptional regulator of Th2 cells, is overexpressed in SC *via* proteasome dysregulation (66).

# CANCER-ASSOCIATED FIBROBLASTS

Fibroblasts are crucial components of the tumor microenvironment, promoting the growth and invasion of cancer cells through various mechanisms (67). The fibroblasts in the affected skin of patients with advanced CTCL promote a Th2-dominant microenvironment by augmenting Th2 and attenuating Th1 immune responses. Increased expression of CCL26/eotaxin-3 is observed in the dermal fibroblasts, keratinocytes, and endothelial cells of the affected skin of patients with advanced MF (68). In addition, serum CCL26 and CCL11/eotaxin-1 levels were shown to be higher in patients with CTCL than in healthy control patients, which correlated with serum soluble interleukin-2 receptor (sIL-2R) levels. However, CCR3/CD193, a receptor for CCL26 and other ligands, is not expressed on lymphoma cells in MF or SS (69). Because mRNA for CCR3 is detected in affected skin (68) and CCR3 is expressed on eosinophils and subpopulations of Th2 cells (70, 71), CCL26 and CCL11 are believed to support the Th2 dominant microenvironment in MF and SS disease lesions (25).

Periostin constitutes an extracellular matrix protein that is expressed in several cancers (72); it is prominent in the stromal area during the patch and plaque stages of MF, but decreases during the tumor stage (73). Fibroblasts are reportedly the source of periostin in MF (74). IL-4 and IL-13 can induce periostin secretion by dermal fibroblasts, periostin mediates thymic stromal protein (TSLP) production by keratinocytes, and TSLP subsequently activates immature myeloid DCs, which modulate Th2 immune responses *via* CCL17 production (75). Serum (76) and plasma (77) TSLP levels are increased in patients with CTCL, suggesting that TSLP contributes to the Th2-dominant microenvironment in MF lesions. TSLP also induces the growth of CTCL cells (74). Therefore, periostin can directly stimulate the growth of CTCL tumor cells in addition to inducing a Th2-dominant environment in CTCL tumors.

Expression of herpesvirus entry mediator (HVEM)/CD270, a member of the tumor necrosis factor-receptor superfamily, on dermal fibroblasts in the affected skin of patients with MF and SS is decreased as the disease progresses. In addition, low HVEM expression on dermal fibroblasts in the affected skin of patients with advanced CTCL attenuates the expression of Th1 chemokines, resulting in Th2-dominant microenvironments. This occurs because the interaction between HVEM and tumor necrosis factor superfamily member 14 (also termed LIGHT)/CD258 on dermal fibroblasts increases the secretion of CXCL9–11, which are chemokines that recruit CXCR3-positive Th1 cells (29).

# TUMOR-ASSOCIATED MACROPHAGES (TAMs)

Macrophages constitute a major component of the leukocyte infiltrate in the tumor microenvironment (78), in which they are termed TAMs. TAMs usually comprise polarized M2 macrophages that contribute to an immune-suppressive environment and promote tumor cell growth (79). CD163 is recognized as a marker for TAMs. As with many malignancies (80), the presence of M2 macrophages in the affected skin of patients with MF has been correlated with patient prognosis (81, 82), and the presence of M2 macrophages has been correlated with lymph node staging (83); this suggests that TAMs play a significant role in MF pathogenesis. Serum sCD163 levels in patients with CTCL are significantly higher than those in normal controls and they significantly correlate with serum sIL-2R levels. TAMs are believed to play a role in the formation of CTCL by secreting various chemokines (73, 82, 84). Periostin-stimulated macrophages produce CXCL5 and CXCL10 (73), which correlates with MF tumor formation in a xenograft CTCL mouse model (84). CCL18/alternative macrophage activation-associated CC chemokine 1/MIP-4 is secreted by M2 macrophages (85) and binds to its receptor (i.e., CCR8) on Th2 cells (86). The expression of CCR8 on MF or SS tumor cells has not been reported, although the mRNA expression of CCR8 is known to be upregulated in patients with SS (21). TAMs are known to express CCL18 in the skin of patients with CTCL (87, 88). Serum CCL18 levels were significantly higher in patients with CTCL than in healthy controls, and these levels significantly correlated with modified severity-weighted assessment scores, serum sIL-2R, lactate dehydrogenase, Th2 cytokines, and chemokines (88). Furthermore, high serum levels of CCL18 were associated with poor patient prognosis (88). In the affected skin of patients with MF/SS, TAMs highly express CD30, which is the target of the anti-CD30 antibody–drug conjugate, brentuximab vedotin (89).

# DENDRITIC CELLS

Dendritic cells are antigen-presenting cells with a unique capacity to induce primary immune responses (90). By secreting Th2 cytokines, CTCL cells can suppress the maturation of DCs (54). Notably, IL-10 downregulates DC functions and may promote tolerance by skin DCs, rather than immune defense (91). Immature DCs can induce tolerance by presenting antigens to T-cells without appropriate costimulation. A significant increase in various DC subsets is seen in the affected dermis of patients with MF/SS, with the majority being immature CD209/

DC-specific ICAM-3 grabbing non-integrin (DC-SIGN)-positive DCs. Increases in CD208/DC-lysosome-associated membrane glycoprotein-positive DCs (i.e., mature DCs) and CD303/blood dendritic cell antigen 2-positive DCs (i.e., plasmacytoid DCs) are also observed, but the numbers of cells expressing CD208 or CD303 are few, suggesting that many DCs in the dermis of CTCL lesions are immature. Increased number of immature DCs in CTCL lesions may be important for immunological tolerance against malignant T-cells (92). However, some CD208-positive, mature DCs may attempt to mount an immune response against the cancer cells, as mature CD208-positive DCs are elevated in the skin draining lymph nodes of patients with MF (83).

# OTHER KEY PLAYERS

The keratinocytes in the affected skin of patients with MF/SS release multiple chemokines including CCL17, CCL26, CCL27, CXCL9, and CXCL10, which help to attract T-cells to the epidermis, as mentioned above. Nerve growth factor (NGF) expression is also elevated in the affected skin of patients with SS, which stimulates the sprouting of nerve fibers. NGF is associated with the severity of pruritus in atopic dermatitis (93), and serum NGF levels are elevated in patients with SS (94). The enhanced expression of NGF is supposedly associated with pruritus in SS.

Mast cells also serve as critical stimulators of the tumor microenvironment (95). Patients with CTCL have increased number of mast cells in their affected skin and this correlates with disease progression (96). Moreover, in a model of cutaneous lymphoma, tumor growth in mast cell-deficient mice was significantly decreased. Therefore, mast cells represent key players in the development of CTCL.

Th22 cells, which produce IL-22 but not IFN-γ, IL-4, or IL-17, express CCR6, CCR4, and CCR10, thus enhancing skin infiltration. IL-22 mediates host defenses against bacterial infection (97). The affected skin of patients with MF/SS expresses high levels of IL-22 and low levels of IL-17 (32). A case of SS reportedly also had high IL-22 expression that was modulated by systemic bacterial infections (98). The serum levels of IL-22 and IL-22-induced CCL20 are increased in patients with MF/SS and are associated with disease severity (32); this suggests an important role of IL-22 in establishing the tumor microenvironment in MF and SS.

Myeloid-derived suppressor cells (MDSCs) are also recognized as key players in tumor immune escape mechanisms (99). The progression from early patch/plaque lesions to tumors in MF is related to an increase in MDSCs (100). Therefore, MDSCs play a role in MF progression by decreasing antitumor immune responses.

T-cell exhaustion *via* immune checkpoints also constitutes an important factor underlying tumor survival. The expression levels of PD-1 (101, 102), PD-L1 (102), CTLA-4 (103), and ICOS (104) have been described at different stages of the disease, suggesting a role for immune checkpoint inhibitor therapies.

# TREATMENT

There is currently no cure for CTCL, thus treatment is aimed primarily at improving symptoms and quality of life and maintaining remission. Therapies are tailored to the individual patient, based on age, performance status, extent of disease burden, rate of disease progression, and prior treatments (105). A typical MF progression starts at the patch and plaque stage and then advances to dermal-based tumors over many years. Effective immune control in the initial disease stages can slow disease progression. Hughes et al. reported that chemotherapy shortens the median time until the next treatment in patients with MF/ SS (106). Multiagent chemotherapy often induces immunosuppression, which leads to an increased risk of infection and poor tolerance (107). Therefore, chemotherapy should be limited until all other options are exhausted. In comparison, IFN and histone deacetylase inhibitors afford greater times to next treatment than those from chemotherapy.

Both IFN-α and IFN-γ represent effective clinical treatments for CTCLs, including MF, *via* their cytotoxic and immunological effects on tumor-associated T-cells (108–110). A meta-analysis suggested that the overall response rate (ORR) to IFN-α was 70% (109). In all stages of MF, IFN-α achieves a superior time to next treatment compared to that of chemotherapy (106). IFN-γ shifts the Th2-dominant tumor microenvironment to a Th1 environment, as mentioned above. IFN-α2a and IFN-γ have been shown to decrease the expression and production of CCL17 and CCL18 and increase those of CXCL10 and CXCL11. Furthermore, subcutaneous administration of IFN-α increased the number of CXCL11-producing cells in the affected skin of patients with advanced MF (111).

Toll-like receptor (TLR) agonists induce anticancer effects by stimulating the innate immune system. Imiquimod is a topical immunomodulator that stimulates Th1 responses by activating TLR7 on plasmacytoid DCs, which leads to the production of IFN-α, IL-12, and tumor necrosis factor-α (112). The effectiveness of topical imiquimod has been reported in early-stage (113–115), folliculotropic, and tumor-stage MF (116). Resiquimod, a TLR7/8 agonist, is also effective for early-stage MF (117). TLR8 is expressed by myeloid-derived DCs, which are the most abundant DCs in human skin. Resiquimod, but not imiquimod, potently activates these cells (118).

The acetylation of histones plays a critical role in gene expression regulation (119). Histone acetylation and deacetylation control gene transcription and are mediated by histone acetyltransferases and deacetylases, respectively. Histone deacetylase inhibitors enhance the acetylation of histones and non-histone proteins and can induce apoptosis (120). Histone deacetylase inhibitors are potential therapeutic agents for the treatment of lymphoid neoplasms (121–124). Pruritus relief has also been reported with these inhibitors (121, 122, 124–126), supposedly through the reduction in the levels of IL-31-expressing T-cells (127).

Brentuximab vedotin (mentioned above) is an antibody– drug conjugate, in which an anti-CD30 monoclonal antibody is linked with the anti-tubulin agent, monomethyl auristatin E (128). Brentuximab vedotin is effective in the treatment of CD30-positive relapsed/refractory Hodgkin's lymphoma (129) and anaplastic large cell lymphoma (130). In a phase II study for MF/SS with variable CD30 expression levels, an ORR of 70% was observed with brentuximab vedotin (127). In addition, a significant improvement in objective response was observed in a randomized, phase III clinical trial (131).

Mogamulizumab, a defucosylated humanized anti-CCR4 antibody that was first approved for relapsed ATL, as described in further detail in Section "Adult T-cell leukemia/lymphoma," is also effective for CTCL including MF/SS (132, 133), and approved for relapsed or refractory CCR4-positive CTCL. In addition, an anti-CD158k monoclonal antibody, IPH4102, has also recently been developed (134), for which clinical studies in CTCL are ongoing (135). Lenalidomide (136), bortezomib (137), and immune checkpoint blockade are also under investigation.

# PRIMARY CUTANEOUS CD30**+** T-CELL LYMPHOPROLIFERATIVE DISORDERS

Primary cutaneous CD30+ T-cell lymphoproliferative disorders (PC CD30+ T-LPD) constitute the second most common form of CTCL, representing approximately 30% of all cutaneous lymphomas (2). They comprise a spectrum of diseases from lymphomatoid papulosis (LyP) to primary cutaneous anaplastic large-cell lymphoma (PCALCL) (138). The expression of CD30, a cytokine receptor belonging to the tumor necrosis factor receptor superfamily, by atypical T-cells is the common immunophenotype of this disorder.

Primary cutaneous anaplastic large-cell lymphoma is characterized by large T-cells with prominent nuclear pleomorphisms along with CD30 expression by more than 75% of the tumor cells (2). A single tumor or a group of firm nodules is seen clinically (**Figure 2**). PCALCL was established as a distinct form of ALCL because its clinical course, phenotype, and genotype are significantly different from those of systemic ALCL, including ALK-positive and ALK-negative forms (139–141). Moreover, IFN regulatory factor-4 translocations are reported to be specific for PCALCL (142). In contrast to that of systemic ALCL, the prognosis of PCALCL is reportedly excellent (143), with the exception of cases in Japan that appear to have a less favorable prognosis (144). PCALCL arising on the legs tends to produce poorer outcomes (145). The typical histology of PCALCL is a

circumscribed nodular infiltrate of cohesively arranged large lymphoid cells that extends into the deep dermis or hypodermis. Neutrophil-rich and eosinophil-rich variants have been noted and appear to be associated with immunodeficiency (146). The abundant infiltration of neutrophils can be explained by the release of IL-8, a potent neutrophil chemoattractant, from the tumor cells (147).

The tumor cells in PCALCL possess an activated T-cell phenotype and express CD2, CD4, and CD45RO, with a loss of CD2 and CD5 occurring variably. CD3 may be lacking or expressed at lower levels owing to genetic alterations in the T-cell receptor (TCR) coding regions on chromosome 1 in the tumor cells (148). Additionally, CD25/IL-2R, CD71, human leukocyte antigen– antigen D related, and CLA/CD162, as well as cytotoxic proteins, such as T-cell intracellular antigen 1 (TIA-1), granzyme B, and perforin, are expressed in half of PCALCL cases. PCALCL is often negative for epithelial membrane antigen, which differentiates it from systemic ALCL. Numerous quantities of TAMs are also present.

As opposed to MF/SS, the tumor cells of PCALCL express CCR3. CCL11, a CCR3 ligand, is also expressed by PCALCL cells and is detected in the connective tissue cells in the tumor. The CCR3+ tumor cells abundantly express IL-4 but not IFN-γ (69). The expression of both CCL11 and CCR3 on the tumor cells can lead to homotypic aggregation, which can be observed as cohesive clusters of tumor cells, a characteristic finding in ALCL (149). As CCR3 is also expressed on eosinophils and subpopulations of Th2 cells (70, 71), CCR3+ cells secreting CCL11 and IL-4 may produce a Th2-dominant microenvironment, which is suitable for tumor growth.

Lymphomatoid papulosis was first described by the dermatologist, Warren L. Macaulay, as a chronic recurrent, selfregressing papulonodular skin eruption with histologic features of a malignant lymphoma (138). Five histological variants (types A to E) are recognized as original variants in the updated World Health Organization classification of 2016 (10). LyP type A is the most common subtype, accounting for 75% of LyP cases (150). Type A is characterized by wedge-shaped dermal infiltrates with scattered large CD30+ cells. Histiocytes, eosinophils, and neutrophils comprise the background inflammatory cells. Type B shows epidermotropic infiltrates of small to medium-sized lymphocytes with variable CD30 expression and atypical chromatin-dense nuclei. Type C shows nodular cohesive infiltrates of large CD30+ pleomorphic or anaplastic lymphocytes. Type D shows epidermotropic infiltrates of atypical, small to medium-sized pleomorphic CD8+ cytotoxic cells (151). Type E shows angioinvasive infiltrates of mainly medium-sized pleomorphic CD30+ cells (152). Vascular occlusion by atypical lymphocytes and/or thrombi, hemorrhage, ulceration, and extensive necrosis are observed. LyP can persist for years or decades, but is not life-threatening (143, 153). However, some patients with LyP can develop secondary lymphoid neoplasms, in particular MF, Hodgkin's lymphoma, and cutaneous or nodal CD30+ ALCL (140, 146, 154). Surgical excision or radiation therapy is the recommended therapy for solitary or grouped lesion(s) of PCALCL, whereas methotrexate is the most prescribed therapy for multifocal lesions (138). The brentuximab vedotin (128) has been granted breakthrough

obtained from the patient.

therapy designation; in addition, bexarotene, a retinoid X receptor-specific agonist, has also been shown to be effective for both PCALCL (ORR: 50%) and LyP (ORR: 60%) in clinical trials (155). HDAC inhibitors (156), crizotinib, an ALK inhibitor (157), and anti-PD-1 are under investigation.

## ADULT T-CELL LEUKEMIA/LYMPHOMA

Adult T-cell leukemia/lymphoma is a distinct T-cell malignancy caused by human T-lymphotropic virus type I (HTLV-1). HTLV-I infections are endemic in many parts of the world including southwest Japan, the Caribbean basin, and parts of central Africa and South America. Neoplastic T-cells are usually CD4+CD25+CCR4+ (158). The general characteristics of ATL are lymphadenopathy, hepatosplenomegaly, hypercalcemia, abnormal peripheral blood lymphocytes with multilobulated nuclei, and skin lesions (**Figure 3**).

There are four clinical subtypes of ATL (159): acute, lymphoma, chronic, and smoldering, based on peripheral blood involvement, organ complications, and laboratory examinations. Patients with ATL can be stratified into two groups: aggressive, which consists of the acute, lymphoma, and unfavorable chronic types, and indolent, which consists of the favorable chronic and smoldering types. The chronic type is separated into the favorable and unfavorable subgroups according to significant prognostic factors. This stratification is important for treatment selection, with most patients with aggressive ATL being given systemic chemotherapy, whereas those with indolent ATL are given topical therapy or are placed on observation.

Cutaneous involvement is frequently observed in patients with ATL at 30–70% (160, 161), regardless of ATL subtype. Cutaneous manifestation in the smoldering type of ATL has been suggested to reflect poor prognosis (162), and cutaneous ATL was recently proposed to include the lymphoma type as an extranodal variant (163). The majority of skin lesions are caused by the direct invasion of ATL tumor cells, forming various types of eruptions (164). In addition to these primary invasive lesions, patients with ATL may present with secondary inflammatory or infectious lesions (165). Compared to those of peripheral blood tumor cells, skin-infiltrating ATL tumor cells exhibit enhanced characteristics, such as increased expression of chemokine receptors. The interaction between chemokines and chemokine receptors drives T-cell migration and activation, which plays a critical role in the pathogenesis of various neoplastic and inflammatory disorders. ATL cells produce several chemokines including CCL3/MIP-1α, CCL4/MIP-1β (166), CCL2/monocyte chemoattractant protein-1 (MCP-1) (167), and CCL1/I-309 (168), as well as several chemokine receptors, including CCR4 (158, 169), CCR7 (170), and CCR8/CDw198 (168). Overexpression of chemokine CCL1 and its receptor, CCR8, contributes to autocrine anti-apoptotic effects ATL cells (168). Increased CCR7 expression is associated with lymphoid organ infiltration (170).

Adult T-cell leukemia/lymphoma cells not only express CCR4 but also its ligands, CCL17 and CCL22 (171). Neoplastic T-cells that highly express the Th2 chemokine receptor, CCR4, are found in the peripheral blood and affected skin of patients with ATL. In CTCL, extravasation of lymphoma cells into the skin is mediated by CCL17 and CCL22 released from epidermal cells (30). In contrast, one of the major sources of CCL17 in the affected skin of patients with ATL is the tumor cell itself (171). Moreover, CCL17 and CCL22 can also attract CCR4-expressing Treg cells, which may further suppress cytotoxic T-cells and prevent tumor immunosurveillance of the ATL cells (165). As ATL cells share the CD4+CD25+CCR4+ phenotype with Treg cells, ATL cells have been postulated as being Treg cells. In addition to CD25 and CCR4, ATL cells express CTLA-4 and FoxP3, both of which are expressed in Treg cells (172, 173). However, whether ATL cells can function as Treg cells is controversial because tumor cells possess very limited regulatory ability (174).

Th17 cells play an important role in cutaneous innate immunity. Th17-derived cytokines stimulate keratinocytes to produce antimicrobial peptides (175). ATL tumor cells can reduce the number and/or function of Th17 cells. Studies have shown that cellular immune responses are greatly impaired in patients with ATL, and ATL cells have been shown to secrete immunosuppressive cytokines such as IL-10 and transforming growth factor-β1 *in vitro*. In particular, ATL cells, as well as Treg or Th2 cells residing in the blood, produce IL-10, thereby suppressing Th17 activity (176). IL-17 enhances the synthesis of various antimicrobial peptides, such as human β-defensin 2, LL-37 (177), and S100A7, in keratinocytes. These peptides are active against fungi, such as those causing ringworm (178). More than 60% of patients with

Figure 3 | Clinical findings of adult T-cell lymphoma/leukemia. (A) Patch, (B) plaque, (C) multiple nodulotumoral lesion, and (D) tumor. Written informed consent was obtained from each patient.

ATL have tinea pedis/unguium/corporis, candidiasis, or other cutaneous fungal infections (165). Other skin infections may occur in these patients in addition to superficial fungal infections. It has been reported that scabies is sometimes superimposed on the skin lesions of patients with ATL (179).

Programmed cell death (PD)-1/CD279 constitutes a cell surface receptor that suppresses the immune system. PD-1 expression on HTLV-1-specific cytotoxic T-cells is dramatically upregulated in HTLV-1 carriers and patients with ATL (180). PD-1 is expressed at high levels on CD4+ neoplastic and non-neoplastic cells, but not on CD8+ cells (181). Because normal CD4+ T-cells can be infected with HTLV-1, they can sometimes express PD-1, leading to immunosuppression. Moreover, it is noteworthy that PD-L1 is expressed in ATL cells (181). Expression of both PD-1 and PD-L1 by the ATL cells suggests a self-destructive state of the tumor cells. However, it may be more important that the PD-L1 expressed by the tumor cells suppresses the function of PD-1-expressing normal CD4+ T-cells, resulting in immune evasion. Of note, 25% of patients with ATL have structural variations in the 3'-region of the gene for PD-L1, which leads to marked elevations of aberrant *PDL1* transcripts (182).

The fact that the tumor cells express CCR4 provides a therapeutic strategy for ATL. The anti-CCR4 monoclonal antibody mogamulizumab markedly enhances antibody-dependent cellular cytotoxicity and has been approved for the treatment of patients with CCR4-positive ATL, peripheral T-cell lymphoma, and CTCL. In a phase II trial of patients with relapsed CCR4 positive ATL, the ORR was 50%, with a complete response rate of 30% (183). Mogamulizumab is more effective against the peripheral blood tumor cells than those in the skin and lymph nodes. Cutaneous adverse reactions (CARs) are frequently observed during treatment (183, 184) and are supposedly indicative of favorable prognoses in ATL (185); a reduction in Treg by mogamulizumab is believed to induce CARs (186, 187). Recently, pretransplantation mogamulizumab has been reported to increase the risk of severe acute graft-versus-host disease (188, 189), and non-relapse mortality is significantly higher in patients with pretransplantation mogamulizumab. Therefore, mogamulizumab should be carefully considered and monitored for patients with ATL who are eligible for allogeneic hematopoietic stem-cell transplantation.

### PANNICULITIS-LIKE T-CELL LYMPHOMA

Subcutaneous panniculitis-like T-cell lymphoma (SPTCL) with α/β phenotype and SPTCL with γ/δ phenotype have been recognized as unique entities, considering their clinical, histological, and immunological characteristics (2, 190, 191). The term SPTCL is now used exclusively for cases with the α/β T-cell phenotype, whereas those of the γ/δ T-cell phenotype have been reclassified as primary cutaneous gamma/delta T-cell lymphoma (PCGD-TCL) (2). The differential diagnosis of these two diseases is important, as each has a different prognosis and therapeutic strategy. In addition, both entities should be differentiated from other types of malignant lymphoma with preferential subcutaneous involvement and from other forms of lobular panniculitis, especially lupus panniculitis (192, 193).

# SUBCUTANEOUS PANNICULITIS-LIKE T-CELL LYMPHOMA

Patients with SPTCL present clinically with multiple nodules or deeply seated plaques without ulceration. The skin lesions usually involve the legs, arms, and trunk. Systemic symptoms, such as pyrexia, fatigue, and weight loss, and laboratory abnormalities, including cytopenia and elevated liver function tests, are commonly observed. Hemophagocytic syndrome (HPS) is observed in <20% of patients (194). Dissemination to extracutaneous sites rarely occurs. As many as 20% of patients have associated autoimmune disease, which is commonly systemic lupus erythematosus (194).

The histopathological findings in SPTCL are dense, nodular, or diffuse subcutaneous infiltrates with a pattern similar to lobular panniculitis. The epidermis is not typically involved. The rimming of individual fat cells by neoplastic T-cells is a curious finding, although it is not diagnostic (193). The neoplastic T-cells are interspersed with small reactive lymphocytes and many histiocytes, whereas other inflammatory cells, including neutrophils and eosinophils, as well as the plasma cells and plasmacytoid DCs that are common in lupus panniculitis (195, 196), are usually lacking (193). High-throughput sequencing of the TCR genes can assist in the diagnosis of SPTCL (192). The neoplastic cells have a mature CD3+CD4−CD8+ T-cell phenotype and express cytotoxic proteins, such as granzyme B, TIA-1, and perforin (194). Although the exact mechanisms that neoplastic cells utilize to migrate into the hypodermis are still mostly unknown, CCR5 expression on neoplastic cells and its ligands, CCL3, CCL4, and CCL5, which can be secreted from immunologically activated adipocytes, may contribute to the pathogenesis of SPTCL (197, 198).

The differential diagnosis of SPTCL includes both PCGD-TCL and lupus panniculitis. Differentiation is critical because PCGD-TCL with panniculitis-like features generally has a poor prognosis and requires systemic chemotherapy. In contrast, SPTCL has an excellent prognosis, especially in the cases without HPS (194). Both SPTCL and PCGD-TCL have nodular skin lesions with panniculitis-like features and rimming of fat cells. In contrast to that of SPTCL, PCGD-TCL involves ulceration of the hypodermis, dermis, and/or epidermis (194). Expression of βF1, but not TCRγ/δ or CD56, is useful to differentiate between SPTCL and PCGD-TCL.

Multiagent chemotherapy is not recommended as a first-line treatment for SPTCL without HPS. Systemic corticosteroids or other immunosuppressive agents, such as cyclosporine or methotrexate, are preferred, which is also the case with relapsing disease (199–201). Oral bexarotene has also shown good response rates (202).

# PRIMARY CUTANEOUS GAMMA/DELTA T-CELL LYMPHOMA

Primary cutaneous gamma/delta T-cell lymphoma is a lymphoma composed of a clonal proliferation of mature, activated γ/δ T-cells with a cytotoxic phenotype. Most patients present with deep dermal or subcutaneous plaques or tumors, either with or without epidermal ulceration and necrosis (194, 203, 204). The skin lesions are often generalized and involve the extremities. Some patients may present with a single tumor, or scaly patches/plaques, clinically resembling early-stage MF (204). The involvement of mucosal and other extranodal sites is frequently noted, although lymph nodes, spleen, and bone marrow are rarely involved (204, 205). Most patients present with systemic symptoms including B symptoms. PCGD-TCL is frequently accompanied by HPS, particularly in patients with panniculitislike tumors (194, 203). Chronic antigenic stimulation has been hypothesized to be involved in the pathogenesis of PCGD-TCL (206). PCGD-TCL is also associated with opportunistic infections in patients with congenital or acquired immunosuppression and autoimmunity (207–209).

The lymphoid infiltrates have a variable histological pattern and may be epidermotropic, dermal, and/or subcutaneous (203, 204). In contrast to that of SPTCL, a pure panniculitic pattern is rarely observed (204), and variable patterns can be found in skin biopsies obtained from different sites or different parts of the same biopsy (190, 203, 204). Lichenoid or vascular interface dermatitis-like patterns of epidermal infiltration may occur, which may be associated with intraepidermal vesiculation and necrosis (204). Panniculitis-like lesions may show the rimming of fat cells observed in SPTCL. Angiocentricity, angiodestruction, and tissue necrosis may be seen. Hemophagocytosis may be present, especially in cases with HPS. The tumor cells have a characteristic phenotype of TCR γ/δ+, βF1−, CD3+, CD2+, CD5−, and CD56+, with a strong expression of cytotoxic proteins. PCGD-TCL with subcutaneous panniculitis-like infiltrate preferentially derives from the V2 subtype (205). PCGD-TCL is resistant to multiagent chemotherapy. The effectiveness of hematopoietic stem cell transplantation has been reported in some patients with PCGD-TCL (204, 210, 211).

# PRIMARY CUTANEOUS CD4**+** SMALL/ MEDIUM T-CELL LYMPHOPROLIFERATIVE DISORDER

Primary cutaneous small/medium-sized T-cell lymphoma (PCSM-TCL) has recently been reclassified as primary cutaneous small/medium-sized T-cell lymphoproliferative disorder (PCSM-TCLPD) because of its indolent behavior and uncertain malignancy (10). PCSM-TCL was originally associated with a favorable 5-year survival rate of 60–80% (2). However, fatal outcomes have not been documented in subsequent reports (212, 213).

Primary cutaneous small/medium-sized T-cell lymphoproliferative disorder characteristically presents with a single lesion on the head, neck, or upper arms, but rarely presents as multiple papules, plaques, or tumors (212, 214). Histopathologically, PCSM-TCLPD is characterized by many small- to medium-sized CD3+CD4+CD8− T-cells, with a small number of large CD4+ pleomorphic T-cells and variable admixtures of CD8+ T-cells, B-cells, histiocytes, plasma cells, and eosinophils (2).

The few, large pleomorphic CD4+ T-cells in PCSM-TCLPD express PD-1, BCL6, and CXCL13 (215), all of which are expressed on a particular germinal center T-cell subset, termed follicular helper T (TFH) cells. TFH cells are important in germinal center formation and plasma cell development. The expression of PD-1, BCL6, and CXCL13 by these large CD4+ T-cells suggests that PCSM-TCLPD originates from TFH cells (215). PD-1 is typically expressed by atypical cells in PCSM-TCL and pseudo-T-cell lymphomas (216). The clinical presentation, pathological features, and immunohistochemical findings of PCSM-TCLPD are very similar to those of pseudo-T-cell lymphomas (217, 218). The demonstration of a T-cell clone and loss of pan-T-cell antigens are useful diagnostic criteria for PCSM-TCL (218). The staining pattern for nuclear factor of activated T-cells, cytoplasmic 1 is also reported to be useful for the differential diagnosis between PCSM-TCLPD and pseudo-T-cell lymphomas (219), where NFAT1c nuclear staining indicates PCSM-TCLPD and cytoplasmic staining indicates pseudo-T-cell lymphoma. The cytoplasmic staining pattern is also seen in MF, ALCL, and LyP. The clinical behavior of PCSM-TCLPD is almost always indolent, with most patients showing localized disease. Treatment with local therapies, such as excision or radiation therapy, is often curative (214, 220, 221).

# HYDROA VACCINIFORME-LIKE LYMPHOPROLIFERATIVE DISORDER (HVLL)

Typical hydroa vacciniforme (HV) is characterized by lightinduced herpetiform vesiculopapules on the sun-exposed areas. The eruptions form crusts and then heal to leave varicelliform scars. Systemic symptoms are absent, and the disease usually improves spontaneously in adolescence and young adulthood (222). Routine laboratory tests are normal. Since the first report in 1986 (223), peculiar HV-like eruptions have been recognized in children mainly from Asia and Central and South America. HVLL was included for the first time in the 2008 World Health Organization classification of tumors of hematopoietic and lymphoid tissues (224). HVLL is defined as an Epstein–Barr virus (EBV)-positive CTCL that occurs in children and less often in young adults (225). Unlike typical HV, HVLL eruptions become more severe with age, presenting with marked facial edema and vesiclopapules followed by ulceration and crusting. Systemic symptoms, including high-grade fever and liver damage, are usually present. Hepatosplenomegaly and lymphadenopathy are frequently observed during the acute phase. The lesions are associated with EBV infection and frequently possess monoclonal rearrangements of the TCR genes (226, 227). Although the skin lesions are not limited to sun-exposed areas, there is an increased occurrence during the summer. Most cases have a CD8+ T-cell phenotype (228), whereas a small number of cases have been reported to have a natural killer-cell phenotype (229, 230). Regardless of cell-type derivation, the lymphoid cells are positive for cytotoxic markers, such as granzyme B and TIA-1 (231).

# SEVERE MOSQUITO BITE ALLERGY

An associated cutaneous disorder is a severe allergy/hypersensitivity to mosquito bites (232). It is defined as an EBV+ NK-cell lymphoproliferation that is characterized by high fever, ulcers, skin necrosis, and deep scarring, with the potential to progress into overt NK/T-cell lymphoma or aggressive NK-cell leukemia in the protracted clinical course (233). Severe mosquito bite allergy was included for the first time in the 2017 World Health Organization classification of tumors of hematopoietic and lymphoid tissues (234).

# PRIMARY CUTANEOUS ACRAL CD8**+** T-CELL LYMPHOMA

Primary cutaneous acral CD8+ T-cell lymphoma is characterized as a solitary, slow-growing nodule without prior patches or plaques (235), but with precedence of bilateral, symmetrical disease and recurrent disease (236). Most cases appear on the ear, although other peripheral locations, such as the nose, hands, and feet, have been noted (237).

Primary cutaneous acral CD8+ T-cell lymphoma and PCSM-TCLPD are often indistinguishable morphologically. Moreover, the overt clinical features of both diseases are similar, such as targeting adults, a preference for the face and neck, solitary tumors without ulceration, and an indolent behavior. However, T follicular markers, such as CD10, Bcl-6, PD-1, and CXCL13, which are expressed on neoplastic cells of PCSM-TCLPD, are negative in primary cutaneous acral CD8+ T-cell lymphoma (236). Granzyme B expression is also typically negative in the latter (238). The clinical course for primary cutaneous acral CD8+ T-cell lymphoma is invariably indolent; cutaneous relapse may occur, but there have been no reports of progression to extracutaneous sites, and overtreatment should be avoided (238). Localized therapy, such as topical steroids, radiotherapy, and surgical excision, or careful monitoring, is preferred. IFN,

#### REFERENCES

psoralen-ultraviolet A phototherapy, and methotrexate have been used for patients with multifocal cutaneous disease (238).

# PRIMARY CUTANEOUS CD8**+** AGGRESSIVE EPIDERMOTROPIC CYTOTOXIC T-CELL LYMPHOMA

Primary cutaneous CD8+ aggressive epidermotropic cytotoxic T-cell lymphoma (PCAETCL) is characterized by disseminated, rapidly developing papules, plaques, and nodules with central ulceration or necrosis. PCAETCL may spread to other visceral organs including the lungs, testes, central nervous system, and oral mucosa (239–241); it carries an overall poor prognosis. However, the lymph nodes are rarely involved. Histological findings demonstrate prominent epidermotropism, with necrotic keratinocytes and ulceration (240). Dermal infiltrates consist of atypical lymphocytes, often extending into the deep dermis and subcutaneous fat. Adnexal invasion is frequently observed (242). Blistering, angiocentricity, angioinvasion, riming of adipocytes, and destruction of adnexal structures may be seen (240). Cells invariably demonstrate CD8+CD4− phenotypes and usually express CD3, β-F1, and TIA-1. CD45RA is expressed in the majority of cases (239). T-cell clonality is usually demonstrated. Conventional therapies for CTCL are ineffective and multiagent chemotherapies have unsatisfactory outcomes (240). Hematopoietic stem cell transplantation is a reasonable treatment choice for PCAETCL (243).

# AUTHOR CONTRIBUTIONS

The author confirms being the sole contributor of this work and approved it for publication.

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**Conflict of Interest Statement:** The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2018 Fujii. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Melanoma immunotherapy: Next-Generation Biomarkers

#### *Sabrina A. Hogan1,2, Mitchell P. Levesque1,2 and Phil F. Cheng1,2\**

*1Department of Dermatology, UniversitätsSpital Zürich, Gloriastrasse, Zurich, Switzerland, 2 Faculty of Medicine, Universität Zürich, Zürich, Switzerland*

The recent emergence of cancer immunotherapies initiated a significant shift in the clinical management of metastatic melanoma. Prior to 2011, melanoma patients only had palliative treatment solutions which offered little to no survival benefit. In 2018, with immunotherapy, melanoma patients can now contemplate durable or even complete remission. Treatment with novel immune checkpoint inhibitors, anti-cytotoxic T-lymphocyte protein 4 and anti-programmed cell death protein 1, clearly result in superior median and longterm survivals compared to standard chemotherapy; however, more than half of the patients do not respond to immune checkpoint blockade. Currently, clinicians do not have any effective way to stratify melanoma patients for immunotherapies. Research is now focusing on identifying biomarkers which could predict a patient's response prior treatment initiation (or very early during treatment course), in order to maximize therapeutic efficacy, avoid unnecessary costs, and undesirable heavy side effects for the patient. Given the rapid developments in this field and the translational potential for some of the biomarkers, we will summarize the current state of biomarker research for immunotherapy in melanoma, with an emphasis on omics technologies such as next-generation sequencing and mass cytometry (CyTOF).

#### *Edited by:*

*Atsushi Otsuka, Kyoto University, Japan*

#### *Reviewed by:*

*Daniel Olive, Aix Marseille Université, France Yasuhiro Nakamura, Saitama Medical University International Medical Center, Japan*

#### *\*Correspondence:*

*Phil F. Cheng<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Cancer Immunity and Immunotherapy, a section of the journal Frontiers in Oncology*

*Received: 02 March 2018 Accepted: 08 May 2018 Published: 29 May 2018*

#### *Citation:*

*Hogan SA, Levesque MP and Cheng PF (2018) Melanoma Immunotherapy: Next-Generation Biomarkers. Front. Oncol. 8:178. doi: 10.3389/fonc.2018.00178*

Keywords: melanoma, immunotherapy, biomarkers, next-generation sequencing, review literature as topic

Immunotherapy has revolutionized the management of metastatic melanoma. Prior to 2011, the median survival for metastatic melanoma was 9 months, compared to greater than 18 months in 2017 (1). Patients now benefit from novel immune checkpoint inhibitors (ICIs), anti-cytotoxic T-lymphocyte protein 4 (CTLA-4) and anti-programmed cell death protein 1 (PD-1). From the latest survival data of the Checkmate 067 trial, progression-free survival (PFS) for ipilimumab is 2.9 months, for nivolumab 6.9 months, and for the combination of nivolumab and ipilimumab 11.5 months. Overall survival (OS) of the ipilimumab group was 19.9 and 37.6 months for the nivolumab group. Median OS was not reached in the combination nivolumab and ipilimumab group with a minimum follow-up time of 36 months (2–6). Although OS is extended, not all patients benefit from immunotherapy. Response rates for ipilimumab range from 11% to 19% (4, 5) and for pembrolizumab or nivolumab from 33% to 44% (2, 6, 7). These new ICIs clearly show superior median and long-term survivals compared to standard chemotherapy; however, more than half of the patients do not respond to immune checkpoint blockade. Currently, there are no clinically approved biomarkers to aid in patient selection in melanoma. In this review, we seek to delineate the current state of biomarker research for immunotherapy in melanoma, with an emphasis on omics technologies such as next-generation sequencing (NGS) and mass cytometry (CyTOF). Given the urgent clinical need for such biomarkers, we decided to focus on human studies only, which we think are more clinically relevant.

# IMMUNE CHECKPOINTS

CTLA-4 and PD-1 are two immune checkpoints regulating immune homeostasis. CTLA-4 is a negative regulator of T-cell priming that acts to control naïve T-cell activation by competing with the co-stimulatory molecule CD28 for binding to shared ligands CD80 and CD86 on antigen-presenting cells (APCs) in the lymph node (8). Ipilimumab, a monoclonal antibody against CTLA-4, was the first agent approved for the treatment of unresectable or metastatic melanoma that showed an OS benefit in a randomized phase III trial (4). PD-1 is a T-cell exhaustion marker which is upregulated by T-cells upon activation during priming or expansion and binds to one of two ligands: programmed cell death 1-ligand 1 (PD-L1) and -ligand 2 (PD-L2) (9–11). Pembrolizumab and nivolumab are monoclonal antibodies against PD-1 that have both shown OS benefit in randomized phase III trials and are approved for the treatment of metastatic melanoma (2, 7). Furthermore, nivolumab and pembrolizumab have both improved OS compared with ipilimumab in metastatic melanoma patients that are naïve to both agents. Combination therapy with ipilimumab and nivolumab has demonstrated additional clinical activity with objective response rates ranging from 50% to 60% and improved OS compared to ipilimumab alone. Although ipilimumab, nivolumab, and pembrolizumab have significantly improved the survival of melanoma patients, there are major toxicities associated with the use of these drugs [reviewed in Ref. (12)]. Grade 3 and higher adverse events are seen in about 20% of patients treated with ipilimumab, in 15% of patients treated with nivolumab, and in 50% of patients treated with the combination of both drugs (6). As these therapies result in objective responses for only a subset of patients, there is a crucial need to identify biomarkers that can potentially predict the efficacy of anti-CTLA-4 or anti-PD-1 treatment or identify a specific subset of patients who may benefit from immunotherapy. A summary of current potential biomarkers for immunotherapies in metastatic melanoma patients is listed in **Figure 1**.

# CLINICAL BIOMARKERS

Approved markers for melanoma monitoring have not substantially evolved over the past decade. Clinicians have mainly used the TNM staging system as a diagnostic and prognostic indicator. In 2009, lactate dehydrogenase (LDH) was shown to be an independent predictor of survival in melanoma and was therefore added to the AJCC guidelines (13). Accelerated metabolism in cancer cells requires increased glycolysis that creates a high amount of LDH as a byproduct, which is therefore a robust proxy to assess tumor burden (14). It is the only accepted serum biomarker with prognostic value for OS in melanoma (15). In the context of immunotherapies, elevated LDH is a negative prognostic marker for patients treated with ipilimumab (16) and with pembrolizumab (17, 18). However, subgroup analysis of anti-PD-1 treated cohorts recently pointed out that LDH level is not correlated with the duration of response (KEYNOTE-006). Indeed, once patients show response to the treatment, the LDH level is not associated with the duration of the remission period. As described by Diem et al. in a study specifically assessing the role of LDH as a marker for anti-PD-1 therapy, LDH is nevertheless a useful marker to monitor disease progression and help treatment decisions (19).

Another well-known marker to monitor melanoma is S100, which is a good indicator of advanced clinical disease stage (20). S100 was shown to be predictive of response to anti-CTLA-4 (16). However, similar to LDH, S100 seems to mainly be a proxy of disease stage, able to highlight very ill patients who are more unlikely to respond to the treatment due to the high tumor burden of the disease, but not actually able to predict response to immunotherapies. The same is true for the number of organs involved, which was another potential marker, proposed by Diem et al., to stratify patients prior to anti-CTLA-4 therapy (21).

C-reactive protein (CRP) was described as a negative prognostic factor for anti-CTLA-4 treatment (22). Unlike LDH and S100, CRP is directly related to immune response. However, it is a general marker of inflammation and is not specific to melanoma, ergo, an increase in CRP levels may also be the result of any other ongoing infection (23). For anti-PD-1 therapy, intra-tumoral PD-L1 expression, evaluated by immunohistochemistry, has been assessed as a predictive biomarker. The results have been inconclusive due to a lack of standards for PD-L1 "positivity". Different antibodies and different evaluation criteria have been used for PD-L1 expression in clinical trials. Some studies have used a >5% cutoff (Checkmate-066 and Checkmate-067), whereas others have used >1% cutoff (KEYNOTE-006). In the Checkmate-066 trial, both PD-L1 negative and positive patients had better outcomes than chemotherapy-treated patients, suggesting that PD-L1 status was not a relevant stratification marker (2). More research will be needed to standardize the assessment of PD-L1 expression for it to become a biomarker for anti-PD-1 therapy in melanoma. Blood markers which hold the most potential toward predicting response to immunotherapies are immune cell populations. Indeed, they are either themselves part of or directly influencing the immune response against the tumor. The different findings related to blood cytology as a biomarker for immunotherapy in melanoma are summarized in **Figure 1**. Briefly, for anti-CTLA-4 treatment, absolute neutrophil count, absolute lymphocyte count, neutrophil to lymphocyte ratio, absolute eosinophil count, relative lymphocyte count (RLC), absolute monocyte count, antibodies against NY-ESO1, T-regulatory cell count, and myeloid-derived suppressor cell (MDSC) count have been described as predictive biomarkers. In anti-PD-1-treated patients, RLC, relative eosinophil count (REC), and MDSC count seem to hold some predictive potential prior to treatment initiation. In addition, increased serum levels of TGFβ and increased frequency of Th9 cells in the peripheral blood were detected in responders to nivolumab prior to therapy initiation (24). Unfortunately, most of the studies were performed on small cohorts and the results have not been verified in larger prospective trials (25). Although guidelines have been published about how to best perform biomarker studies (26), most research groups have different evaluation criteria. In this review, we sought to document the most relevant biomarkers associated with immunotherapy outcome in melanoma patients. For a systematic review of clinical biomarkers, see Ref. (18).

Figure 1 | Summary of the biomarker studies performed on peripheral blood and tumor on anti-CTLA4 and anti-PD1 treatment.

# BIOMARKERS FROM NEXT-GENERATION SEQUENCING

Whole exome (WES) and RNA sequencing (RNAseq) are powerful tools for evaluating the genomic landscape of a tumor. WES only captures the exonic gene regions, so it enriches for mutations in coding regions, while RNAseq can provide the entire transcriptome of a sample and is useful for establishing gene signatures for specific cohorts within a patient group. In terms of immunotherapy, WES has been useful in determining mutational load and discovering neoantigens in melanoma tumors (27). Melanoma has one of the highest mutation rates of all cancers (28–30) and has a high probability for neoantigen generation. Neoantigens are the result of somatic mutations which translate into a mutated protein that is detected and presented by APCs. Neoantigens are an attractive target for immunotherapy as they are only expressed by the tumor and not by the normal tissue. Many studies have utilized WES and RNAseq to evaluate the mutation profile and gene expression changes in patients treated with anti-CTLA-4 and anti-PD-1, with the aim to find biomarkers to predict response.

Snyder et al. performed WES on 64 patients treated with anti-CTLA-4 (31). This study was the first to associate mutational burden to clinical benefit and they also defined a neoantigen signature associated with clinical benefit. They concluded that, although patients with high mutation burden are more likely to respond to anti-CTLA-4, the types of neoantigen the patient expresses will ultimately determine their response. Van Allen et al. performed WES on 110 patients and RNAseq on 40 patients treated with anti-CTLA-4 (32). They could confirm that mutational load is associated with clinical benefit to anti-CTLA-4 treatment. Neoantigen load was also measured and this parameter was also significantly associated with response; however, they could not detect the neoantigen signature seen in the study performed by Snyder and colleagues. They concluded that clinically beneficial neoantigens are most likely private events (specific to each individual) and recurrent neoantigens (consistent in the general population) are quite rare. Van Allen and colleagues also analyzed the transcriptome in a subset of these patients and found that expression of cytolytic markers, such as granzyme A and perforin, were beneficial for response. Expression of CTLA-4 and PD-L2 was also associated with clinical benefit. Riaz et al. performed WES on 174 patients treated with anti-CTLA-4 therapy (33). They discovered 48 patients with mutations in SERPINB3 or SERPIN4 and observed that those patients were more likely to be responders. Patients with SERPINB3 or SERPINB4 mutations also had higher mutational loads. Friedlander et al. performed a quantitative polymerase chain reaction (PCR) study on peripheral blood from 360 patients receiving anti-CTLA-4 therapy (34). From a panel of 169 genes, they established a 15 gene signature that was predictive and prognostic for response and 1-year OS to anti-CTLA-4 treatment. The 15 genes are ITGA4, LARGE, CDK2, TIMP1, DPP4, NRAS, ERBB2, NAB2, ADAM17, RHOC, TGFB1, CDKN2A, HLADRA, MYC, and ICOS.

In order to elucidate resistance mechanisms and biomarkers of response to treatment, Hugo et al. used WES (38 patients) and RNAseq (28 patients) on a set of melanoma patients treated with anti-PD-1 (35). Mutational load did not have a significant association to response to anti-PD-1 therapy and neoantigen load was not significantly correlated with response either. Nonetheless, mutational load was associated with OS suggesting that other factors influence response to anti-PD-1 and survival. BRCA2 mutations occurred in 30% of the responders to anti-PD-1. RNAseq analysis uncovered a co-enrichment of 26 gene signatures in 9 of the 13 non-responding patients, which the authors termed innate anti-PD-1 resistance (IPRES) signature. They validated the IPRES signature on three other datasets and found over-representation in anti-PD-1 non-responding samples.

In a small study of four patients treated with anti-PD-1, Zaretsky et al. used WES on patients that developed new lesions under anti-PD-1 therapy. They discovered that the progressive tumors acquired JAK1, JAK2, or B2M loss of function mutations. JAK1 and JAK2 mutations cause insensitivity to interferon gamma-induced arrest and B2M mutations led to a loss of MHC class 1 expression (36). In a follow-up study, Shin et al. performed WES on 23 patients before anti-PD-1 treatment (37). In their cohort, mutational load had no association to response and one of the non-responders had a loss of function mutation in JAK1. This study confirms the role of JAK1 as a marker for innate and adaptive resistance to anti-PD-1, although it might be a rare occurrence.

Roh et al. performed WES on sequential biopsies of patients treated with anti-CTLA-4 and then anti-PD-1. Thirty patients had WES at baseline, 3 on anti-CTLA-4 treatment, 25 at anti-CTLA-4 progression, 18 on anti-PD-1 treatment, and 12 at anti-PD-1 progression. Overall, they found that mutation burden was not associated with response, but high copy number loss was associated with poor response (38). In the regions with recurrent copy number loss, PTEN was one of the notable tumor suppressor genes suggesting that it could be a driver of resistance mechanisms to immunotherapy. Another study also observed that PTEN loss was associated with resistance to anti-CTLA-4 therapy (39).

Johnson et al. performed targeted panel sequencing on 65 patients treated with anti-PD-1 therapy. In their cohort, mutational load was associated with response to anti-PD-1 and patients with high mutational load (>23.1 mutations/MB) had longer PFS and OS compared to the intermediate mutational load group (3.3–23.1 mutations/MB) and the low mutation load (<3.3 mutations/MB) group. They observed more frequent BRCA2 mutations in responders than in non-responders (5/32 vs. 2/33). LRP1B mutations were significantly enriched in the responder group (11/32) compared to the non-responder group (1/33). LRP1B mutated patients also had a higher mutational load compared to LRP1B wild-type patients.

Riaz et al. performed WES on 68 patients treated with anti-PD-1 that had previously progressed on anti-CTLA-4 therapy (35 patients) or were naïve to anti-CTLA-4 (33 patients). Mutational load was associated with clinical benefit in the anti-CTLA-4 naïve group, but not the anti-CTLA-4-resistant group. No single gene mutations were significantly associated with response or resistance to therapy. Decreased mutational and neoantigen load during therapy was associated with response in both anti-CTLA-4-naïve and anti-CTLA-4-resistant groups. RNAseq analysis of the pretreatment samples showed an enrichment in T-cell activation and lymphocyte aggregation pathways. These signatures indicate an immunologically active tumor, or "hot tumor," and all patients with complete response or partial response in the anti-CTLA-4-resistant group had the "hot tumor" signature, although not all responders in the anti-CTLA-4-naïve group had the "hot tumor" signature. Riaz et al. also investigated the early effects of anti-PD-1 treatment (29 days after start) by RNAseq and uncovered a global increase in immune checkpoint genes such as PD-1, CTLA-4, CD274 (PD-L1), ICOS, and LAG3 in all samples. For responders, the significant pathways included inflammatory response and cytokine-mediate signaling pathways.

Finally, Davoli et al. investigated the role of aneuploidy in response to immunotherapy (40). They analyzed the copy number data from 5,255 tumor/normal samples, representing 12 cancer types from The Cancer Genome Atlas project, and found that for most tumors, there was a positive correlation between aneuploidy severity and mutational load. They also found that tumors with high levels of aneuploidy showed elevated expression of cell cycle and cell proliferation markers, as well as a reduced expression of markers for cytotoxic immune cell infiltrates. Aneuploidy levels were a stronger predictor of markers of cytotoxic immune cell infiltration than tumor mutational load. To correlate aneuploidy with response to immunotherapy, they used data from Snyder et al. and Van Allen et al. (31, 32) and found in both datasets that high levels of aneuploidy correlated with poorer survival.

# BIOMARKERS FROM T-CELL RECEPTOR (TCR) PROFILING

Antigen detection by T-cells is by definition dependent on tumorspecific T-cell generation and clonal amplification. In the context of immunotherapies, which aim at enhancing the recognition of the cancer cell by the immune system, there is an obvious rational basis for examining the T-cell repertoire in order to shed light on the specific mode of action of the drug and find potential biomarkers of response.

The main challenge when analyzing TCR repertoire is its immense diversity. The TCR is a heterodimer comprised of two chains αβ or δγ. The β and δ chains, are generated by the random rearrangement of a variable region (V), a diversity region (D), and a joining region (J) with a constant region (C). The α and γ chains, consists of segments from the V, J, and C regions. Additional complexity is introduced by random addition or deletion of nucleotides at the junction sites of V, D, and J. The theoretical limit of the TCR repertoire is in the range of 1015, which is several magnitudes higher than the total amount of T-cells in the body, approximately 4 × 1011 (41). The estimated number for the TCR repertoire is in the order of 106 to 108 (42). Most of the studies mainly assess the complementarity-determining region 3 (CDR3) from β chain, which is considered an acceptable proxy for estimating diversity since it is the most variable region of the receptor and that αβT-cells represent about 90% of all T-cells (43). Due to the advances in NGS, it is possible now to identify each individual TCR sequence in the CDR3 region (44). Multiplex PCR is one of the widely used methods to amplify the CDR3 region. Primers for the J alleles or the constant region of the TCR α and β chains are used together with a mix of primers for all known V alleles. A drawback of multiplex PCR is that it is limited to known V alleles. As a result, for TCR discovery experiments, other methods such as targeted enrichment—a technique where RNA baits capture the TCR receptor, usually the CDR3 region—are preferred. Since bait capture takes into account mismatches, it allows for discovery of new alleles and TCR receptors. In the context of immunotherapy, TCR repertoire analysis is useful for determining if the tumorreactive clones have undergone activation and clonal expansion. In an adequate immune response, the tumor-specific T-cells will represent a significant proportion of the whole repertoire and therefore be assessable at the level of the whole TCR population.

In 2014, Robert et al. compared pre- to post-treatment peripheral blood mononuclear cell (PBMC) samples from melanoma patients treated with anti-CTLA-4 (45). The results from deep sequencing of the multiplex PCR for the TCR Vβ CDR3 region showed that 19 out of 21 patients had an increased number of unique clonotype (richness). There was no significant difference in the V or J segment usage and no difference in the total of unique sequences between responders and non-responders. The number of unique productive sequences in the top 25% of clones showed a particularly high increase in diversity after treatment. Those changes were not associated with peripheral lymphocyte count; however, CD8+ tumor-infiltrating cells showed a positive correlation with the TCR repertoire diversity. Finally, they showed that patients experiencing more toxicities had more diverse sequences post treatment. Overall, this study reports that anti-CTLA-4 treatment increases TCR repertoire diversity in an unspecific manner. Subsequently, the same group performed a similar study on 9 anti-PD-1-treated patients and compared the results (46). Unlike the effect of anti-CTLA-4, anti-PD-1 therapy does not increase TCR repertoire complexity, on the contrary, 4/9 samples show a decrease >15% in the absolute number of unique sequences and only one had an increase >15%. Those results suggest that the mode of action of the two drugs is considerably different.

Cha et al. shed more light on the potential mechanism of anti-CTLA-4; their study assessed the changes in TCR repertoire between baseline and 4 weeks of treatment in PBMCs from 21 melanoma patients (47). They confirmed that anti-CTLA-4 treatment induces a significant change in the clonotypes frequency compared to healthy donors. They showed that the diversification is the result of a higher gain of new clonotypes and lower loss of existing ones. The number of therapy-induced expanding clones are not different between the responders and the non-responders, which is in line with what Robert et al. described. However, patients who survived longer exhibited less clonotypic changes overtime, they maintained the most abundant clones which were present at baseline and also had fewer clones significantly decreasing in frequency. Finally, they also demonstrated that the clones expanding in response to therapy are largely non-naïve T-cells, suggesting that patients who respond to the therapy already have pre-primed T-cells in circulation before the onset of anti-CTLA-4 treatment.

In light of these findings, Postow et al. hypothesized that the shape of the TCR repertoire prior to treatment initiation may influence the likelihood of a response to the treatment (48). Twelve baseline PBMC samples from 4 responders and 8 non-responders to anti-CTLA-4 were analyzed for richness and evenness of the TCR repertoire. In this small cohort, they first showed that patients who responded to the treatment have more similar VJ usage among each other than compared to the VJ usages in the non-responders. Furthermore, low richness or evenness of the TCR repertoire was significantly associated with a poor response to anti-CTLA-4. That is, a TCR repertoire composed of less unique clones (less diverse) or skewed toward a few specific clones (very clonal) is predictive of a non-response to the treatment.

The detection of clonally expanded tumor-specific T-cell in the blood indicates that the immune system mounted an immune response against a foreign entity, which could be the tumor. However, it is not certain that the activated T-cells would be able to home to the tumor efficiently and be able to kill the cancer cells. Therefore, it is also important to assess the immune status at the tumor site.

Tumeh et al. performed a very elegant study exploiting tumor samples from melanoma patients prior and during anti-PD-1 treatment (49). By qualitative and quantitative immunohistochemistry, they revealed that, at baseline, patients who eventually respond to the therapy, have more CD8+ T-cells at the invasive margin of the tumor compared to non-responders. This population increases and migrates toward the center of the tumor during treatment in responders. They showed that an efficient response to anti-PD-1 therapy requires pre-existing CD8+ T-cells, which are most likely tumor specific. To confirm this theory, they sequenced the TCR Vβ region of tumor baseline samples and found that responders indeed had a more clonal TCR repertoire. On treatment, samples of responders showed significantly more clonal expansion than non-responders.

Johnson et al. used NGS to assess differences between baseline samples of responders and non-responders to anti-PD-1 therapy (50). They assessed mutational load as well as specific mutations differentially occurring in responders and non-responders. They also investigated the TCR repertoire clonality of 42 samples and did not find any association with response. However, it is important to mention that times of sample acquisition were not immediately before and after treatment. The timing was quite broad, the study allowed the inclusion of samples collected over 12 months before start of treatment and also after treatment initiation. When the analysis was performed only on samples obtained within 4 months of treatment initiation, the non-responder group was only represented by five samples, including one potential outlier. Nonetheless, they noticed a trend toward higher clonality at baseline in patients who eventually responded to the therapy.

Inoue et al. analyzed the TCR repertoire of 10 pre- and postanti-PD-1 tumor samples (51). They noticed that the clonotypes with a read frequency >0.5% at baseline significantly increased after treatment in responders. The calculation of the diversity index highlighted a slight decrease in tumors of responders compared to non-responders, which suggests oligoclonal expansion of certain TCR clones.

More recently, Roh et al. published a complementary analysis on a cohort of patients for which they performed TCR sequencing (38). They analyzed tumor samples from melanoma patients treated sequentially with anti-CTLA-4 and anti-PD-1 *via* WES and TCR sequencing. The TCR clonality assay revealed that there was no significant difference between responders and non-responders, pre- or on-treatment with anti-CTLA-4. A subpopulation of the patients (*n* = 8) received anti-CTLA-4 followed by anti-PD-1 after progression. All three responders to anti-CTLA-4 followed by anti-PD-1 showed an increase in TCR clonality during anti-CTLA-4 treatment. In addition, higher TCR clonality was seen in the responders prior to treatment and on treatment with anti-PD-1.

Riaz et al. investigated the evolution of melanoma tumors and their microenvironment under anti-PD-1 therapy (52). Patients who had previously progressed on anti-CTLA-4 and were naïve to anti-CTLA-4 were included in the study. They performed TCR sequencing on 34 samples pre- and 4 weeks post- anti-PD-1 treatment. There were no statistically significant differences in the baseline samples of either group. On anti-PD-1 therapy, the anti-CTLA-4-pretreated group had increased TCR richness associated with response, whereas the anti-CTLA-4-naïve group had decreased TCR evenness associated with response. In line with Roh et al., pretreatment with anti-CTLA-4 seems to increase the expansion of tumor-specific T-cell cells, which are additionally expanded during anti-PD-1 treatment.

# MASS CYTOMETRY (CyTOF)

The advent of CyTOF has allowed a more comprehensible analysis of the whole immune system and will be an important asset for immune oncology (53). The basic principle of CyTOF is similar to conventional flow cytometry. The assay quantifies multiple protein expression markers at the single-cell level. In contrast to flow cytometry, the detection is not achieved by fluorophore excitation, but by stable mass isotope quantification. The transition isotope bound to the antibodies are analyzed by a time of flight mass spectrometer. CyTOF has some advantages over flow cytometry, namely, the high purity of the metal isotopes reduces background noise, eliminating spectral spillover and cellular autofluorescence associated with conventional flow cytometry. It also enables the detection of more markers in the same experiment, theoretically up to a hundred. Multiple samples can be analyzed at the same time thanks to a barcoding strategy (up to 20), and therefore reduce inter-sample variation. CyTOF has primarily been used to analyze peripheral blood from patients undergoing immunotherapy. A better characterization of the precise mode of action of those drugs is crucial to help overcoming and predicting resistance as well as contributing to optimal development of future combination therapies.

In 2015, Das et al. analyzed peripheral blood from melanoma patients undergoing immunotherapy with anti-CTLA-4, anti-PD-1, or the combination of the two (54). Samples were collected at baseline and after 3 weeks of treatment. In this early study, CyTOF was mainly used to further characterize the cell population of interest previously identified by flow cytometry. The analysis revealed that the Ki67+ cells, increasing after combination treatment, have a transitional memory T-cell-like phenotype. Additional experiments were performed using other techniques than CyTOF, which lead the authors to conclude that anti-PD-1 and anti-CTLA-4 have distinct effects on the immune system.

In the context of a clinical trial assessing the safety of combining radiotherapy and immunotherapy in melanoma patients, Hiniker et al. analyzed baseline and follow-up PBMC samples from 9 patients (3 progressive disease, 6 complete response/ partial response) (55). CyTOF analysis revealed that the level of CD8+ T-cells expressing IL-2 were higher at baseline and in the follow-up samples of responding patients. The same was true for central memory CD8+ T-cell levels. However, the cytokine production was not significantly different from the population seen in non-responding patients, thereby suggesting that the cells are not functionally different from the non-responders.

The first study to use CyTOF as a main technique for analyzing human melanoma patient samples was performed by Wistuba-Hamprecht et al. in early 2017 (56). The analysis consisted in performing CyTOF on 28 PBMC samples from stage IV melanoma patients who received different courses of treatments. A higher frequency in the APC-like population had a positive association with OS, whereas a higher frequency in the MDSClike population showed negative association with OS. Overall, an equal abundance of MDSC- and APC-like cells is associated with better survival. The analysis of the T-cell compartment revealed that there was a clear interpatient heterogeneity in the CD4+ and CD8+ T-cell compartments compared to the other compartments which have more homogenous frequencies between patients. Only one αβT-cell population had some prognostic potential: a higher level of early differentiated CD4+ T-cell was correlated with poorer OS. In the natural killer cell compartment, a highly cytotoxic cell population tends to correlate with better OS. Finally, a comprehensive analysis of immune signatures of all the melanoma-associated phenotypes identified a specific cluster with high prognostic capacity, performing even better than LDH. This cluster is significantly associated with poor OS and represented by an overall lower diversity across all the compartments, and especially in the myeloid compartment.

Takeuchi et al. investigated the effect of immunotherapy in melanoma patients by comparing PBMCs from 4 different patients receiving anti-PD-1 (2 responders and 2 non-responders) (57). The panel was composed of 35 markers and they used highdimensional clustering to analyze the data. The main finding in this paper is that CD4+ and CD4+CD8+ cell populations increase during therapy. CD4+CD27+FAS− central memory T-cell were shown to expand in a higher proportion in responders than in non-responders. These results were validated in a separate cohort (*n* = 4).

More recently, Krieg et al. performed a comprehensive analysis, assessing the correlation between baseline peripheral immune signature and response to anti-PD-1 in melanoma patients (58). The cohort was composed of 20 patients from whom baseline and on treatment samples were obtained. They used an optimized immune marker panel and a customized, interactive bioinformatics pipeline in order to identify potential predictive biomarkers. Three different CyTOF panels were used: one for the phenotypic characterization of lymphocytes, one to assess the T-cell functions, and the third one to characterize monocytes, which consisted of 30, 26, and 25 markers, respectively. By performing hierarchical clustering of all the samples pooled together, they identified a differential marker expression in responders compared to non-responders. Further analysis, and validation in an independent, blinded cohort by conventional flow cytometry, revealed that, at baseline, responders had a higher frequency of classical monocytes and lower frequency of lymphocytes compared to the non-responders.

# OUTLOOK

The development of high-throughput technologies such as NGS and CyTOF have allowed researchers and clinicians to evaluate hundreds to tens of thousands of genes from a bulk tumor to a single-cell level. NGS is an invaluable tool for analyzing mutations and copy number profiles, gene expression changes and gene signatures, epigenetic alterations, the TCR repertoire, and single-cell gene expression changes. The recent development of CyTOF has also allowed the analysis of many markers at a single-cell level. In the context of immunotherapy, these high dimensional datasets will enhance the discovery of novel biomarkers, prognostic markers, and resistance mechanisms.

Next-generation sequencing biomarker discovery for anti-CTLA-4 treatment have uncovered that mutational load and neoantigen load are the most informative for response and OS, but they are not perfect biomarkers as some non-responders may also present with high mutational load. Aneuploidy could also help foresee response to anti-CTLA4 since it was highlighted as an independent predictor in a multivariate Cox model which included mutational load. Copy number analysis could as well be informative as loss in chromosome 10 was shown to be a poor prognostic marker in two studies. Many of these studies also analyzed tumor samples upon progression and found no recurrent genetic mutation, which could mean that resistance to anti-CTLA-4 is patient specific. In the context of anti-PD-1 treatment, mutational load is not a clear informative marker for response. As anti-PD-1 is a relatively new therapy, no large cohort studies with over 100 patients for NGS biomarker discovery have yet been performed. There are single patient examples showing that genes involved in the JAK–STAT pathway or antigen presentation could be predictive biomarkers for anti-PD-1 treatment. Loss of function mutations in JAK1, JAK2, and B2M are negative biomarkers for response and are involved in resistance to anti-PD-1 treatment in individual cases, but these mutations do not seem to be recurrent. RNAseq analysis from several studies suggest that tumors with high immune activity are more likely to respond to anti-PD-1. To better stratify patients for anti-CTLA-4 or anti-PD-1 treatment, a combinatorial approach investigating WES, copy number variation, and RNAseq would be needed.

Overall, most studies support that anti-CTLA-4 and anti-PD-1 modulate TCR repertoire clonality upon treatment. This strengthens the notion that tumor-specific T-cell populations are affected by CTLA-4 or PD-1 inhibition. In summary, most studies support that anti-CTLA-4 induces an expansion of clones in a non-specific manner and, therefore, broadens the TCR repertoire. On the other hand, anti-PD-1 seems to favor the proliferation of fewer specific clones giving rise to a more skewed repertoire, thereby suggesting that the baseline TCR repertoire of the patients plays a role in the response to the treatment. However, for the moment, those predictions arise mainly from early on-treatment evaluations that examined the evolution of the repertoire from baseline, as we are not yet able to precisely pinpoint the tumor-specific clones that, once clonally expanded, will facilitate tumor elimination. It is also important to highlight that the mode of action of the current immunotherapies are still debated and we do not fully comprehend their overall impact on different immune cell subpopulations. As a result, it is difficult to assess the global impact of the drugs on the immune response by investigating specific mechanisms individually. This is why high-throughput techniques discussed here are powerful emerging tools, which will allow us to elucidate this problem by looking at numerous markers simultaneously. The more we increase our knowledge of exact mechanisms, the better we will be able to exploit the therapies by using them in a targeted/ patient-specific manner. Interesting work by Twyman-Saint et al. combining anti-CTLA-4, anti-PD-1 and radiotherapy, underpins this assertion (59).

To our knowledge, despite the great potential held by CyTOF technology, to date, no research was published on the analysis of human melanoma tumor samples in the context of immunotherapies. One should however expect to see more forthcoming data, thanks to a novel exciting add-on technology that is starting to emerge. Indeed, a new laser system can be coupled to the CyTOF device which allows for imaging mass cytometry (60). That is, the detection of metal-labeled antibodies, as in standard CyTOF analysis, but performed on tissue sections by using multiplexed ion beam imaging. This state of the art technology will allow, not only to assess a high range of markers at the same time, but also to obtain spatial resolution and warrant a very comprehensive analysis of the cell–cell interaction in the tumor microenvironment. New developments of the system should soon facilitate

# REFERENCES

the analysis of tumor samples in a similar fashion, while gaining spatial resolution to better interrogate the role of spatial interactions in immunotherapy response (with high throughput) (61).