The circuitry of the tumor microenvironment in adult and pediatric Hodgkin lymphoma: cellular composition, cytokine profile, EBV, and exosomes

Abstract Background: Classical Hodgkin lymphoma (cHL) is a unique lymphoid malignancy with a tumor microenvironment (TME) consisting of a small number of neoplastic—Hodgkin and Reed‐Sternberg (H‐RS) cells (<1%), surrounded by a large number of nonneoplastic infiltrating immune cells (>90%). The TME of cHL critically depends on immune cells to support tumor growth as H‐RS cells cannot survive and proliferate in isolation. Recent Findings: Programmed cell death protein 1 (PD‐1) ligand expressed on H‐RS cells inhibits the clearance of tumor by causing T‐cell exhaustion. Nivolumab and pembrolizumab, PD‐1 inhibitors, have been proven to be effective in treating adult and pediatric patients with R/R cHL. Tumor‐associated macrophages (TAMs) are a central component of TME and are known to cause poor prognosis in adult HL. However, the prognostic impact of CD68+ TAMs in pediatric HL remains ambiguous. EBV modulates the tumor milieu of HL and plays a strategic role in immune escape by enrichment of the TME with T reg cells and associated immunosuppressive cytokines in adult HL. In contrast, EBV+ pediatric patients have increased infiltration of CD8+ T‐cells and show a better therapeutic response suggesting viral‐related TME is distinct in childhood HL. The role of CASP3 in apoptosis of H‐RS cells and its correlation with response prediction in adult and pediatric HL suggest it may serve as a potential biomarker. In cHL, CD30, EBV, and NF‐κB signaling employ exosomes for cell–cell communication that triggers the migration capacity of fibroblasts, stimulate to produce proinflammatory cytokines, and help to create a tumor‐supportive microenvironment. Conclusion: The cHL microenvironment is distinct in adult and pediatric HL. Future studies are required to understand the role of interplay between H‐RS cells and EBV‐associated microenvironment and their clinical outcome. They may present novel therapeutic targets for the development of antilymphoma therapy.

showed an overall response rate (ORR) of 87% and 66%, respectively. 1,2 Likewise, pembrolizumab therapy in R/R CHL patients was associated with high response rates and an acceptable safety profile. 3,4 In phase II study of R/R cHL patients who had failed ASCT, pembrolizumab was associated with PFS and OS of 82% and 100%, respectively, at 18-months. 5 In pediatric HL patients also, nivolumab and pembrolizumab have proven to be safe and well-tolerated. In the phase I trial of R/R pediatric HL patients, nivolumab and pembrolizumab showed an objective response rate of 30% and 60%, respectively. 6,7 Further investigations of PD-1 inhibitors combined with other therapeutic agents may offer an effective cure in pediatric HL patients.
Current strategies in first-line treatment aim to improve the outcome and prevent treatment-related toxicity, including reproductive infertility, cardiopulmonary toxicity, and secondary malignancy. Pediatric patients may be overtreated by using unnecessary aggressive regimens. Pediatric HL is different from adult HL in terms of the relative incidence of specific histological subtypes and cellular composition of the TME thereby causing a distinct immune profile against H-RS cells. 8 There is considerable variability in the TME across various histological subtypes. Therefore, it is imperative to understand the biology of the TME in adult and pediatric HL to tailor the treatment accordingly and to prevent long-term side effects in children. 9   HL-AFs release growth factors and proinflammatory cytokines such as IL-1α, IL-6, and TNF-α into the TME to support tumor growth and maintenance ( Figure 1). 18 Tumor-associated macrophages (TAMs) in the TME are largely M2-polarized and are activated by Th2 antiinflammatory cytokines such as IL-4, IL-10, and IL-13 and macrophage migration inhibitory factor (MIF). 19 TAMs have been shown to promote an antiinflammatory response, proliferation, angiogenesis, matrix remodeling, tumor growth, and metastasis. Steidl et al, 20 first assessed the prognostic impact of TAMs and showed that a macrophage gene expression signature is associated with inferior outcome in cHL patients. This analysis was further validated in a patient cohort with increased CD68 expression, a characteristic marker of TAMs. Subsequently, many studies have confirmed the association of TAMs with poor prognosis of adult HL patients (Table 1). [21][22][23][24] A couple of studies investigated the impact of TAMs on prognosis of pediatric patients. In contrast to adult HL patients, TAMs failed to predict disease outcome in pediatric patients. 8,25,26 However, Barros et al showed that high numbers of CD163+ macrophages were associated with worse progression-free survival in EBV-cases but not in EBV+ cases (discussed below). 27 Possibly, in EBV-associated TME of pediatric HL, macrophages are M1-polarized and therefore may mediate effective immune surveillance. TAMs may have hormetic rather than linear relationship to outcome in HL.
Recently, it is shown that a small number of TAMs may have a moderate growth promoting effect on cHL, while with increasing numbers, macrophages display an inhibitory effect and only become supportive of tumor growth above a certain threshold. 28 Further investigation may provide insight into relationship of TAMs with treatment outcome in adult Hodgkin lymphoma and other tumors.

| Cytokines
Cytokines, produced by H-RS cells, contribute to HL pathogenesis, in an autocrine and paracrine manner, and help to recruit and sustain reactive T-cells. These cytokines are inducers of HL milieu-IL-4, 29 IL-5 30 ; growth factors-IL-6, IL-9, 31 IL-13 32 ; and anti-inflammatory-IL-10, 33,34 tumor growth factor β (TGF-β). 35 Additionally, H-RS cells produce Th2 and T reg chemoattractants such as TARC (CCL17), 34,36 CCL5/ RANTES, 37 and macrophage-derived chemokine (MDC/CCL22). 38 TARC levels increase in the sera of cHL patients and reflect tumor status since elevated levels decrease during treatment in most clinical responders. In a recent study of combined adult and pediatric HL, early reduction in TARC1 levels in combination with interim PET scan was predicted as the success of response (Table 1). 39 Tumor-infiltrating Tcells also express T-cell homing receptors such as CCR3, CXCR4, CCR5, and CCR7, corresponding to ligands expressed by H-RS cells. 13  Thus, CASP3 can serve as a potential biomarker for response prediction of HL. Moreover, CASP3 gene expression is associated with other TME-associated genes such as GrB and lysozyme (LYZ). Cytokine profiling in pediatric HL revealed that CD30, IL-10, IL-6, ICAM-1, VEGF, and TARC are independent prognostic markers (Table 1)

| EBV
EBV is present in tumor cells in about 40% of cHL patients in developed countries and plays a crucial role in cHL pathogenesis. EBV positivity is frequently observed in childhood (<10 years) and in older adults (>60 years) HL patients and is highest in mixed cellularity (~75%) type. 59 H-RS cells positive for EBV reveal type II latency phenotype, expressing a limited number of latency genes, latent membrane protein (LMP)1, LMP2A, EBNA1, and EBER-1/2. LMP1, an oncogene product, mimics CD40 that stimulates NF-κB pathway activation, 60 and LMP2A can substitute the function of BCR, 61 thereby immortalizing B-cells that are otherwise destined to undergo apoptosis. The presentation of peptides from these EBV proteins is mediated through human leukocyte antigens (HLA) classes I and/or II. Both CTL and CD4+ T-cells that recognize HLA class I and class II, respectively, are involved in an antitumor immune response against EBV. 62 Of note, HLA class I region polymorphism is known to have consistent susceptibility effects on sporadic and familial EBV-positive childhood and adult HL across different geographical locations. [63][64][65][66][67][68][69] The classic case study of familial HL demonstrated that clustering of EBV and certain identical HLA genotypes in pediatric patients with HL can occur in a single-family 66

| Exosomes
Exosomes are the smallest type of extracellular vesicles (EVs)

CONFLICT OF INTEREST
The authors declare no potential conflict of interest.

ETHICAL STATEMENT
Not applicable.

DATA AVAILABILITY STATEMENT
Data sharing is not applicable to this article as no new data were created or analyzed in this study.