Hodgkin lymphoma (HL) encompasses a group of neoplastic entities with unique histopathologic features, predominantly affecting lymph nodes. Unlike most tumors, HLs are distinct in that they are composed of rare neoplastic disguised B-cells scattered within a much more abundant background of mixed inflammatory non-neoplastic cells, including variable numbers of T-lymphocytes and B-lymphocytes, eosinophils, plasma cells, macrophages, mast cells, and neutrophils.1 It has been recognized that HL encompasses a cluster of diverse lymphomas, and can be divided into nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) and classic Hodgkin lymphoma (cHL) based upon differences in morphologic, immunophenotypic, molecular, and prognostic features in addition to response to therapy. CHL is further subdivided into several subtypes: nodular sclerosis (NS), mixed cellularity, lymphocyte-rich, and lymphocyte-depleted subtypes.
The neoplastic cells of interest in cHL were first described in 1902 by Dorothy Reed and Carl Sternberg2 and are recognized as large binucleated Reed Sternberg (RS) cells with abnormal nuclei, prominent eosinophilic nucleoli, and abundant cytoplasm. Mononuclear variants called Hodgkin (H) cells, lacunar cells with formalin-induced cytoplasmic retraction, and apoptotic “mummified” cells with condensed cytoplasm and pyknotic nuclei can also be seen. The origin of these neoplastic B-cells was long unknown, given their perplexing loss of a B-cell phenotype, common expression of other hematopoietic lineage markers, and highly atypical appearance. The neoplastic cells of NLPHL, formerly termed lymphocytic and histiocytic cells, are currently referred to as lymphocyte predominant (LP) or “popcorn” cells due to their irregularly lobed nuclei. Unlike the Hodgkin/Reed Sternberg (HRS) cells seen in cHL, LP cells often display markers of preserved B-cell programming.
Although the clinical behavior of HL is relatively predictable in its contiguous lymphatic spread, the paucity of neoplastic cells (accounting for 0.1% to 10% of all cells in the tumor) creates challenges in biological study of HL. Advances in tumor microdissection and single-cell sorting have supplied researchers with tools to further understand the cellular origin and genetic abnormalities of malignant HRS cells and the regulatory contribution of the surrounding inflammatory milieu. Here we review recent insights into the genetic hits driving the neoplastic cells of HL, including the oncogenic contribution of Epstein-Barr virus (EBV). We further discuss intercellular signaling between HRS cells and other inflammatory cells forming the bulk of the tumor microenvironment. Finally, we consider novel immunophenotypic diagnostic tools to aid in the identification of HL, and distinguish these lymphomas from a subset of other overlapping pathologic entities.
GENETIC ABNORMALITIES IN HODGKIN LYMPHOMAS
Single-cell polymerase chain reaction of HRS cell immunoglobulin genes has aided in the establishment of the cellular origin and clonality of HRS cells. Although the canonical B-cell program is often lost in HRS cells, largely a consequence of self-protection, clonal immunoglobulin rearrangements are seen in virtually all cases, indicating that they are of B-cell origin. The additional presence of somatic hypermutation indicates that they have encountered the germinal center (Fig. 1). Recent genetic studies into HL have been additionally informative in shedding light onto the chromosomal and molecular abnormalities driving tumorigenesis.
Historically, detection of recurrent chromosomal abnormalities by conventional karyotyping in HL has been difficult due to relative scarcity of HRS cells in the tumor microenvironment, and the low mitotic rate of the neoplastic cells. Recent use of comparative genomic hybridization and array comparative genomic hybridization has facilitated a more sensitive detection of individual chromosomal aberrations.3,4 Gains of chromosomes 2p (28% to 54% of cases), 9p (24% to 40%), 12q (37% to 40%), 16p (24% to 30%), 17p (27 % to 40%), 17q (20% to 70%), and 20q (15% to 23%) have been described in HRS cells derived from multiple subtypes of cHL.5–9 Loss of chromosomes 13q (22% to 35%) may be seen in HRS cells as well. Many of these aberrations encompass focal gains or losses of key oncogenes or tumor suppressors, respectively, that may play important roles in the pathogenesis of HL. For example, amplification of the REL oncogene was seen in cases of chromosome 2p gain, suggesting possible involvement of noncanonical nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) hyperactivation in HRS cell growth and proliferation.6,10 Similarly, chromosomal 9p24.1 gain has been demonstrated to lead to the overexpression of JAK2, PDL1, and PDL2.8,10 Gains of STAT6 (12q13), NOTCH1 (9q34), and JUNB (19p13) have also been described.11,12
Interestingly, it has been well established that RS cells have a low proliferative capacity, largely exceeded by that of smaller mononuclear Hodgkin cells.13,14 This has led to the proposal that a population of RS precursor cells exist in cHL, and that RS cells represent terminally differentiated tumor cells. Congruent with the possibility that RS cells may not represent the entirely of the tumor cell population, previous evidence has suggested that numerical chromosomal aberrations are likely not restricted to RS cells in the HL microenvironment.15 Smaller tumor-associated lymphocytes and circulating B-cells may carry similar genetic abnormalities.15–17 Indeed, in an earlier study, circulating mononuclear cells of a patient with HL displayed complex cytogenetic abnormalities, and strikingly formed intralymphatic tumors when injected into immunodeficient mice.18
In addition to copy number abnormalities, chromosomal translocations involving the immunoglobulin gene loci have been identified in cHL.19 Putative HRS cells identified by their large nuclear size and hyperdiploidy have been demonstrated to commonly contain breakpoints in the immunoglobulin heavy chain and immunoglobulin lambda and immunoglobulin kappa loci. Fusion partners of the immunoglobulin genes are heterogenous, and may include 2p16 (REL), 3q27 (BCL6), 8q24.1 (MYC), and 19q13.2 (BCL3/RELB) in addition to other unknown partners encoded on chromosomal loci 14q24.3, 16p13.1, 17q12, and 19q13.2.19,20 Intriguingly, while such immunoglobulin gene translocations typically lead to the overexpression of the partner gene, some of those in cHL lead to downregulation of the partner gene.
Advances in single-cell sorting and genomic sequencing have further increased opportunities for characterizing the genetic landscapes of HRS-enriched tumor sections and primary cHL cell lines. Here we discuss recurrent mutational variants that may impact several pathways in neoplastic cells.
Janus Kinase/Signal Transducers and Activators of Transcription (JAK/STAT) Signaling
The JAK/STAT pathway is a ubiquitous signaling pathway in humans downstream of multiple cytokines and growth factors, leading to regulation of cell processes including proliferation, apoptosis, differentiation, and migration.21 In addition to JAK2 amplification events,7,12,22 activating STAT3, STAT5, and STAT6 mutations have been recurrently identified in cHL (Table 1).23–28 Increased expression and activation of STAT proteins may also be due to upstream NF-κB signaling and interleukin-21 overexpression.26,27 In one study, both pharmacologic and RNA interference directed against STAT3 blocked HRS cell proliferation in vitro, suggesting the importance of STAT3 signaling in cHL.45 It should be noted however, that given the low proliferation rate of HRS cells in vivo, the clinical significance of this finding is unclear. Inactivating mutations in negative regulators of the JAK/STAT pathway may also propagate tumor cell proliferation (Table 1).
Constitutive activation of the NF-κB pathway that controls broad DNA transcriptional events, cytokine production, and cell proliferation and survival, may also be highly relevant in the pathogenesis of cHL.46 Recurrent inactivating mutations in NF-κB inhibitory genes, including IKK, TRAF3, and TNFAIP3 have been described (Table 1).29,32–35 In contrast, NF-κB activating protein MAP3K14 (NIK) and the cleaved activated p52 NF-κB2 subunit have been shown to be strongly expressed in cHL cell lines in vitro.25,34,47 Overactivation of the NOTCH signaling pathway, characterized by higher expressions of NOTCH1, NOTCH2, Jagged2, and coactivator MAML2 may also augment NF-κB signaling.48
Genetic Drivers of Tumor Immune Evasion
An emerging body of evidence has additionally implicated genetic alterations impacting HRS immune evasion (Table 1, Fig. 2). In light of the therapeutic success with immunomodulatory drugs such as programmed cell death protein 1 (PD1) and programmed cell death protein ligand 1 (PDL1) inhibitors, these data are of particular interest. Recurrent gains of chromosome 9p24.1 have been shown in HRS cells, leading to associated overexpression of PDL1 and PDL2, as well as JAK2, which may further contribute to activation of PD1/PDL1 signaling.12,49 Furthermore, mutations in B2M (encoding beta-2-microglobulin) and subsequent decrease in major histocompatibility complex (MHC) class I protein expression may be common in HRS cells, leading to decreased tumor antigen presentation.36–38,41 Diminished MHC class II expression on HRS cells has also been proposed secondary to the prevalence of in-frame gene fusions with a promiscuous partner, MHC class II transactivator (CIITA).50 In addition to MHC downregulation, CIITA gene fusions may lead to overexpression of PDL1 and PDL2, further obstructing the antitumor immune response.
Growth factor signaling through the PI3K/Akt pathway leads to a multistep intracellular activation process that interacts with the mammalian target of rapamycin pathway to regulate protein synthesis, cell metabolism, growth, proliferation, and survival.51 Few studies have demonstrated dysregulation of Akt activation in cHL. Of the recurrent mutations identified in small studies, truncating and missense mutations of both GNA13, a G-protein coupled receptor engaging as an Akt pathway inhibitor, and ITPKB, a kinase that converts the Akt-activating second messenger PIP3 to IP4, a soluble antagonist of IP3 have been identified (Table 1).23,36,41
Variants of gene products involved in other pathways may be similarly important, including mutations in exportin 1 (XPO1) and histone modifying genes CREBBP and EP300.42,43 Mutations in the tumor suppressor gene TP53 occurs in up to 25% of cases, particularly in chemorefractory cHL, and may contribute to the chromosomal instability that leads to complex cytogenetic abnormalities seen in HRS cells.25,43
Interestingly, loss of heterozygosity for KLHDC8B, a gene encoding an actin-binding midbody protein expressed during cytokinesis, has been characterized in families where multiple individuals have developed cHL.52 Subsequent KLHDC8B knock-down experiments led to the discovery of an increase in binucleated cells, suggesting this protein may promote the formation of the signature binucleated RS cells present in cHL (Fig. 1).44 It is possible that cytokinesis defects secondary to haploinsufficiency of KLHDC8B lead to the chromosomal instability that further drives HRS formation and cHL pathogenesis.
It should be noted that genetics studies of NLPHL have been less commonly performed, given the same challenges as cHL as well as its rarer incidence. Although the pathogenesis of NLPHL is generally believed to be quite distinct from cHL, similar JAK/STAT pathway perturbations may be present, including SOCS1 silencing mutations.53 In addition, nonsynonymous mutations of DUSP2, SGK1, and JUNB have been described in a small study of primary NLPHL cases.54
Given the paucity of tumor cells in HL and the associated difficulties in measurable residual disease monitoring by mutational analysis, interest has arisen in analyzing the utility of circulating tumor DNA (ctDNA). In a retrospective study, a deep next-generation sequencing approach for ctDNA in HL patients was described, the results of which reflected microdissected tumor DNA and known cHL mutational variants previously reported.55 In addition, ctDNA quantification appeared to mirror radiographic evidence of decreased lymphoma burden following chemotherapy treatment, suggesting the utility of ctDNA in residual disease monitoring. A subsequent analysis of ctDNA from pediatric HL patients demonstrated similar genetic variants as described in adults, and correlation between ctDNA levels and disease burden, indicating potential clinical utility.56 HL-related chromosomal abnormalities have also been detected in ctDNA.57
EPSTEIN-BARR VIRUS-MEDIATED PATHOGENESIS
A subset of cHLs is thought to be driven by EBV, particularly in patients from underdeveloped nations, in the setting of immunodeficiency, and in pediatric cases. These cases are more often associated with mixed cellularity and lymphocyte-depleted subtypes. In EBV-associated cases, the monoclonal viral genomes express a latency II pattern in HRS cells, generating intracellular EBNA1, viral-encoded RNAs EBER1 and EBER2, BART miRNAs, and the latent membrane proteins LMP1, LMP2A, and LMP2B (Fig. 3). Much of the focus surrounding EBV-driven tumorigenesis has been on the role of LMP1 in oncogenesis, given its function as a ligand-independent CD40 receptor mimic, leading to constitutive activation of many downstream B-cell regulatory pathways including NF-κB, JAK/STAT, and PI3K/Akt pathways described earlier. In addition, LMP2A mimics B-cell receptor activity, blocking apoptosis and promoting survival of HRS cells.58,59 EBV-positive cases are particularly associated with B-cell receptor defective HRS cells, hinting at the likelihood that EBV can rescue these cells from apoptosis.60 Moreover, LMP1 signaling via the PI3K/Akt/mammalian target of rapamycin pathways may induce aberrant expression of CD137 (4-1BB), a costimulatory molecule typically expressed by T-cells.61 In the case of cHL, CD137 signaling in HRS cells likely leads to release of cytokines such as IL-6, tumor necrosis factor alpha, and IL-13 that favor Th2-type T-cell enrichment, promoting HRS immune escape.
Interestingly, EBV-driven cHLs appear to harbor fewer cytogenetic and molecular abnormalities compared with EBV-negative cases, suggesting that latent viral persistence may serve as a “first hit” in HL tumorigenesis.62 Emerging evidence also supports the influence of EBV-modified extracellular vesicles in regulating important signaling pathways within the tumor microenvironment.63,64
As alluded to above, the abundant tumor microenvironment in HL is composed of non-neoplastic cells, including T-lymphocytes and B-lymphocytes, plasma cells, macrophages, eosinophils, neutrophils, mast cells, and fibroblasts, generating the possibility of complex cell-to-cell crosstalk in the establishment and maintenance of the lymphoma.
Lymphocytes and Plasma Cells
T-cells compose the vast majority of reactive cells in cHL, most of which are CD4-positive helper T-cells or regulatory T-cells. Although initially believed to contain predominately tumor-tolerant Th2 cell populations, later studies have supported the idea that cHL microenvironments may be rather enriched with antigen-presenting and antitumor Th1 cell subsets that display impaired function.65 It is likely that many of the genetic mechanisms driving immune evasion in HRS cells discussed in the above sections contribute to this impaired T-cell function. In addition, regulatory T-cells within the tumor may inhibit Th1 effector function.
Recent studies have described the enrichment of T-cells in cHL expressing cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), an immune checkpoint protein distinct from PD1.66 The CTLA-4 ligand, CD86 is present on HRS cells in addition to a subset of tumor-associated macrophages (TAMs), suggesting an additional mechanism of tumor immune evasion. Single-cell expression profiling of T-cells in cHL has also revealed prominent LAG3 expression, an inhibitory receptor on antigen-activated T-cells, to be often coexpressed with CTLA-4, both functionally supporting an immunosuppressive function.67 Expression of inhibitory ligands human leukocyte antigen-E, HLA-G, and PDL1 on HRS cells also likely inhibits NK cell cytolytic activity (Fig. 2).68
HLs also contain numerous non-neoplastic B-cells that may greatly outnumber HRS or HRS precursor cells, yet the roles of these B-cells are not well understood. It is possible that reactive B-cells may compete with tumor cells for growth signals and thereby be protective against lymphoma progression.
Finally, emerging data has suggested a possible role for plasma cells in cHL. High levels of tumor-infiltrating plasma cells may confer a worse prognosis.69 These plasma cell infiltrates, in particular may be composed of immunoglobulin G4-positive cells, occasionally mimicking an immunoglobulin G4-related disease.70
TAMs are also present abundantly in cHL. TAM and HRS cell interaction likely drives macrophages toward a CD163-positive M2 phenotype, promoting anti-inflammatory cytokine release and subsequent tumor growth and immune evasion.71 Using multiplex immunofluorescence and digital image analysis, a topographical analysis of PD1-positive and PDL1-positive cells in cHL showed that the majority of PDL1-positive cells were TAMs interacting with PD1-positive T-cells.72 These studies suggest TAMs may play a prominent role in the formation of an immune-tolerant microenvironment for HRS cells. Furthermore, these findings support the success of PD1/PDL1 targeting checkpoint blockade in relapsed and refractory cHL patients.73
Production of IL-5 by Th2 cells and mast cells may be responsible for abundant eosinophil infiltration into cHL tumors.74 Functional expression of CD30L and CD40L on eosinophils may enable these cells to partake in cell-contact dependent activation of HRS cells.75 The functions of neutrophils in the lymphoma microenvironment is largely unknown, but may contribute to an immunosuppressive effect, particularly in the context of chemotherapy.76 HL patients also have an increased population of granulocytic myeloid-derived suppressor cells, which are thought to exert suppressive activity on tumor T-cells.77 As a surrogate marker, higher absolute neutrophil to lymphocyte counts in the peripheral blood have been used to predict poorer prognosis of cHL.78
Stromal cells, particularly fibroblasts are necessary for formation of the tumor-embedded extracellular matrix. In addition, fibroblasts likely contribute to HRS proliferation by secretion of prosurvival factors, such as stem cell factor, IL-7, and IL-6.79 In return, HRS cells may stimulate fibroblast growth by CD40L-CD40 interaction and by secretion of extracellular vesicles that stimulate a cancer-associated fibroblast phenotype.80
IMMUNOPHENOTYPE OF HODGKIN LYMPHOMA
Immunohistochemistry studies are central to the diagnosis of HL in tissue sections. Expression of CD30 (TNFRSF8) and CD15 (Lewis X antigen) combined with the lack of expression of leukocyte common antigen CD45, the B-cell marker CD20, and T-cell co-receptor CD3 in HRS cells are often used in clinical diagnosis. HRS cells also classically dimly express the B-cell transcription factor PAX5 as well as MUM1, while lacking OCT2 and BOB1, reflective of partial downregulation of the B-cell program in HRS cells. In contrast, LP cells in NLPHL retain B-cell factors CD20, PAX5, OCT2, and BOB1, and lack CD15 and CD30 expression. Despite the utility of these biomarkers in establishing a diagnosis of HL, it is increasingly recognized that a number of non-Hodgkin B-cell and T-cell lymphomas as well as some reactive lymphoproliferations may contain scattered or rare Hodgkin-like cells. In addition, cHL and NLPHL share overlapping morphologic characteristics, and due to the nature of common presenting sites, are often sampled by small core-needle biopsies with limited nodal architecture present and relatively few evaluable tumor cells for immunohistochemical staining. Furthermore, the markers discussed above are not universally consistent across all cases of cHL or NLPHL. Because of these diagnostic limitations, recent studies have examined the utility of additional potential biomarkers for more robust distinction (Table 2).
Co-expression of multiple nonspecific antigens on HRS cells certainly enhances the sensitivity and specificity of protein analytics in HL. Traditionally, flow cytometric detection of HL has been hindered primarily by the rarity of HRS cell events. Innovations in flow cytometry instrumentation, including increased number of lasers and detectors allowing simultaneous interrogation of at least 10 antigens, and rapid digital event acquisition allowing enhanced event detection, have also made recognition of HL by flow cytometry possible. Newer advances in cell preparation strategies that decrease HRS cell lysis and allow analysis of rosetted cells in cytometric analysis may pave the way for future clinical diagnostic utility.88
Greater understanding of antigen co-expression in HL has also proved efficacious in the development of new therapeutic strategies. Recently, a bispecific antibody targeting CD30 and CD37, both members of the tumor necrosis factor receptor family overexpressed on HRS cells, has been proposed to increase specificity of HRS cell killing in comparison to anti-CD30 therapy currently available.89 Finally, the potential of shared antigens among HRS cells and the supportive tumor microenvironment may create opportunities to target malignant and immunosuppressive cells simultaneously. Chimeric antigen T-cell therapy targeting CD123, an IL-3 receptor component expressed on HRS cells and TAMs, has been reported to eradicate HL and establish long-term tumor immunity in mouse models.90
MIMICS OF HODGKIN LYMPHOMA
The morphologic and genetic overlap of HL with other closely related lymphoma entities has been increasingly recognized. Here we provide a brief overview of a subset of similar tumors.
Gray-zone Lymphoma (GZL)
GZL, also termed as “B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and cHL” is characterized by a spectrum of morphology spanning features of primary mediastinal large B-cell lymphoma (PMBL) and cHL. PMBL generally displays diffuse sheets of large mature B-cells with clear cytoplasm and expression of CD19, CD20, CD79A, PAX5, OCT2, and BOB1. Lymphoma cells often weakly express CD30. The features of GZLs vary significantly but often involve discordance between the morphologic and immunophenotypic findings suggestive of overlap between cHL and PMBL.91 For instance, lymphomas may contain dense tumor cell growth, but cells may be larger and display much more pleomorphism than often seen in typical PMBL with a sparse mixed inflammatory infiltrate in the background. Tumor cells with PMBL-like cytology may show HRS cell-like immunophenotype with loss of B-cell antigens and expression of CD30 and CD15. In contrast, other cases may show cHL cytology, but with a preserved B-cell program. Adding to the difficulties in GZL distinction, gene expression studies have demonstrated commonalities between cHL, PMBL, and GZL,92–94 although differences in methylation and mutational profiles have been described.94,95 Not surprisingly, due to these shared features, pathologic consensus of GZLs is challenging, and many cases may be subsequently re-classified.96 Advances in novel biomarkers, future understanding of distinct and recurrent genetic patterns, and refined diagnostic criteria will be necessary to reliably distinguish this entity from cHL.
T-Cell/Histiocyte-rich Large B-Cell Lymphoma (THRLBCL)
Early phenotypic characterization of THRLBCL revealed similar phenotypic features compared with some forms of NLPHL including rare neoplastic cells embedded in a background of T-cells and macrophages.97,98 However, given the more aggressive clinical course, bone marrow involvement, and hepatosplenomegaly, it is important to distinguish THRLBCL from NLPHL. Although focal areas of the large-cell lymphoma may appear indistinguishable from NLPHL, the neoplastic cells of THRLBCL generally show greater size variation than LP cells, and the background typically lacks the reactive small B-cell infiltrate seen in NLPHL.99 Subsequent genomic studies demonstrated NLPHL tumor cells to harbor significantly more numerous chromosomal aberrations in comparison to the neoplastic cells in THRLBCL, suggesting that NLPHL may not be a precursor of THRLBCL, as had been theorized previously.100 It is possible that THRLBCL and NLPHL may share a common cell of origin or original transforming event, and the 2 distinct entities could represent divergent neoplastic processes. It is also likely that the less well-studied reactive cellular microenvironment of both lymphomas may play a large role in their individual pathogeneses.101 Future genetic studies of these two entities using innovative sequencing strategies will be important for both diagnostic purposes and biological understanding.
Iatrogenic Lymphoproliferative Disorders
Finally, it may be increasingly important to recognize the prevalence of iatrogenic immunodeficiency-associated lymphoproliferative disorders (IILPDs), a subtype of which may fulfill the criteria for cHL. These cases may occur in patients treated with a variety of immunosuppressive drugs for autoimmune disorders, including but not limited to methotrexate, thiopurines, and a number of newer immunomodulatory agents such as anti-TNFα monoclonal antibodies.102 IILPDs resembling cHL are often associated with EBV positivity (~80%).103 It may be necessary to distinguish cHL from Hodgkin-like lymphoproliferations, both of which may occur in the setting of immunosuppressive therapy, particularly methotrexate. In some cases, Hodgkin-like lymphoproliferations may be carefully distinguished by the presence of a more homogenous background composed of mostly B-cells that are also EBV-positive.102,104 Similar to posttransplant lymphoproliferative disorders, IILPDs are likely to regress following withdrawal of the immunosuppressive drug, and are therefore important to recognize.105
HL encompasses unique neoplasms characterized by rare neoplastic cells surrounded by a communicative cellular microenvironment. Recent advances in understanding recurrent cytogenetic and molecular aberrations driving tumorigenesis, and in characterizing cell-to-cell cross-talk within the tumor microenvironment have driven the utility of novel therapeutic agents. Future studies will continue to unravel biological mechanisms important for diagnosis, classification and treatment.
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