- Email: [email protected]
histiocyte rich large B cell lymphoma
Pathology – Research and Practice 211 (2015) 901–904
Contents lists available at ScienceDirect
Pathology – Research and Practice journal homepage: www.elsevier.com/locate/prp
Review
From a pathologist’s point of view: Histiocytic cells in Hodgkin lymphoma and T cell/histiocyte rich large B cell lymphoma Sylvia Hartmann Dr. Senckenberg Institute of Pathology, Goethe University Hospital Frankfurt, Theodor-Stern-Kai 7, D-60590 Frankfurt, Germany
a r t i c l e
i n f o
Keywords: Macrophages Reactive bystander cells Hodgkin lymphoma T cell/histiocyte rich large B cell lymphoma Metallothionein 2A
a b s t r a c t While tumor cells were the focus of research for many years, only recently have attempts been made to understand the role of the reactive bystander cells in malignant lymphomas. In certain types of lymphomas, such as Hodgkin lymphoma and T cell/histiocyte rich large B cell lymphoma, more than 90% of the infiltrate represent non-neoplastic cells, and these have important functions for the development and progression of the tumor. Among the bystander cells are histiocytes of particular importance, which vary largely in number, shape and quality among different patients. In the present review, recent findings on the prognostic impact of histiocytic bystander cells in these lymphomas, as well as their molecular characteristics, are discussed. A better understanding of the role of these histiocytes may provide new concepts for diagnosis and treatment. © 2015 Elsevier GmbH. All rights reserved.
Contents 1. 2. 3.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 901 Macrophage subtypes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 902 Macrophages in Hodgkin lymphoma and T cell/histiocyte rich large B cell lymphoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 902 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 903
1. Introduction For many years, lymphoma pathology has focused on the main players in these tumors, the malignant tumor cells. Different efforts have been made to classify lymphomas according to their growth patterns or the cytology of the tumor cells [1,2]. However, not only the tumor cells, but also the microenvironment contributes significantly to the malignant phenotype observed. Whereas in some lymphomas this microenvironment is so abundant that it has been included as part of the diagnosis [3], in other entities, such as diffuse large B cell lymphoma (DLBCL) and follicular lymphoma, the importance of the reactive bystander cells has only recently been recognized [4,5]. For several lymphoma entities, it was not possible to establish cell lines due to the strong dependency of the tumor cells on the reactive bystander cells, which were not viable under cell culture conditions. Therefore, malignant lymphomas can be divided into more and less microenvironmentdependent types. Lymphomas which are strongly dependent on
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their microenvironment are e.g. follicular lymphoma, marginal zone lymphoma, Hodgkin lymphoma (HL), T cell/histiocyte rich large B cell lymphoma, as well as angioimmunoblastic or peripheral T cell lymphoma. Surprisingly, both B- and T cell neoplasias can equally depend on their microenvironment. In contrast, Burkitt lymphoma and some cases of DLBCL are rather independent of their microenvironment, which is also reflected by the usually low content of bystander cells in the lymphoma infiltrate and the availability of a large number of corresponding cell lines. HL represents the most extreme relationship between tumor cell content (frequently <1% of the infiltrate) and reactive bystander cells. Surprisingly, HL cell lines could be established which grow in culture without the respective bystander cells. However, these were established from relapsed or refractory patients and frequently from pleural effusions, in which the tumor cells had already gained independency from their environment and were able to grow autonomously in suspension [6–8]. The reactive microenvironment of HL usually contains a huge spectrum of cells, ranging from non-neoplastic B cells and special T cell subsets to dendritic cells, macrophages, mast cells, eosinophils
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S. Hartmann / Pathology – Research and Practice 211 (2015) 901–904
and stromal cells. The present review will focus on histiocytic cell types in the reactive microenvironment.
2. Macrophage subtypes While pathologists described different types of histiocytes a long time ago, histiocytic surface markers have only recently been characterized by immunologists. Pathologists define macrophages by their shape and phagocytic function, as well as epithelioid cells, which primarily secrete cytokines. In contrast to this morphologic description, immunologists classify all macrophages in connective tissue as histiocytes or tissue macrophages [9]. One of the most important features of tissue macrophages is the take up and presentation of antigens [10]. Originally, two subsets of classically activated proinflammatory (M1) and alternatively activated (M2) macrophages have been described [11,12] which participate in Th1 and Th2 triggered immune responses, respectively. Morphologically recognizable histiocyte subsets have so far not been well characterized by surface markers. However, there are certain reactions like tuberculoid granulomas, which have been shown to express inducible nitrogen oxide (NO) synthetase [13], corresponding to M1-polarized macrophages. Secretion of NO and reactive oxygen species (ROS) is involved in tissue injury and necrosis as it is typically observed in caseous necrosis in the center of tuberculoid granulomas, consistent with a M1 phenotype of these epithelioid cells. With reference to the complex situation in vivo, Qian and Pollard [14] described six types of macrophages, the activated and the angiogenic type, but also an invasive, metastasis-associated, perivascular and immunosuppressive subtype. These different types secrete unique, but also partly overlapping cytokines. The invasive type of macrophages is found at the outer borders of solid tumors, since this represents the localization where malignant tumors grow fastest. These invasive macrophages were characterized in mice to have an active Wnt signaling [15]. Whereas it was previously hypothesized that all tissue macrophages derive from circulating peripheral blood monocytes [16], recent studies have shown that mature macrophages can proliferate in the tissue in response to specific stimuli without loss of functional differentiation [17,18]. These tissue-resident and self-renewing macrophages were shown to have an M2-like phenotype, whereas monocytederived macrophages, which are recruited to the tissue during an inflammatory response, are supposed to be M1-polarized [19].
3. Macrophages in Hodgkin lymphoma and T cell/histiocyte rich large B cell lymphoma In 2010, the important impact of macrophages in the microenvironment of classical HL on event free and overall survival was first remarked [20]. Many other studies conducted since then have examined the impact of macrophages on clinical behavior. Whereas some could confirm previous observations and found a correlation between a high number of macrophages, mixed cellularity subtype and Epstein–Barr virus (EBV) infection of the Hodgkinand Reed–Sternberg (HRS) cells [21–23], others could not confirm the prognostic impact of a high number of tissue macrophages [24,25]. However, the methods how the macrophage content was counted (CD68- versus CD163-staining) and the thresholds differed between different studies and so far no general applicable test is available. A whole tissue gene expression study identified a 23-gene outcome predictor, which included several macrophageand M1-related genes like CXCL10, STAT1, IFNG, LYZ, CD68 and CD14 [26]. It may therefore be easier and more precise to quantify macrophages according to specific transcripts than counting numbers of macrophages. Particularly, the presence of special subtypes
of polarized macrophages can better be assessed by the quantification of different transcripts than by pure morphology. HIV-positive individuals with HL take a special position concerning the macrophage content in the HL infiltrate: they can present a particularly high macrophage content in the HL microenvironment when the CD4 count in the peripheral blood is low [27]. Therefore, the extent of macrophage infiltration in the lymphoma tissue seems to be related to the availability of CD4 T cells and monocytes in the blood, and monocytes/macrophages can replace CD4 T cells under certain conditions [27]. CD4 T cells get attracted to the microenvironment of HL by several cytokines like TARC and RANTES [28], which are secreted by the HRS cells. In HIV patients, CD4 blood counts are regularly tested, and the development of HL can therefore well be monitored in these patients. Already one year before HL becomes manifest, CD4 cells start to decrease in the peripheral blood, indicating recruitment of CD4 cells to the lymph nodes with HL infiltrates [29]. Interestingly, not only a high content of macrophages in the lymph nodes affected by HL was related to a worse clinical outcome: also a low ratio between lymphocyte and monocyte count in the peripheral blood was related with an adverse prognosis in classical and nodular lymphocyte predominant HL (in HIV-negative individuals) [30,31]. Therefore, in contrast to recent findings indicating that macrophages can renew in the tissue [17], the reactive HL microenvironment in the lymph node seems to reflect the availability of different cell types in the peripheral blood. The molecular mechanisms how macrophages contribute to the dissemination and advanced stage of classical HL have only allusively been characterized. Applying only a limited number of immunohistochemical markers macrophage polarization cannot definitely be determined. CD163, which was originally described as M2 marker, is e.g. negative in all forms of epithelioid cells (Fig. 1), which are particularly found in mixed cellularity subtype and nodular lymphocyte predominant HL (NLPHL), although epithelioid cells can express other M2-markers [32]. In contrast, most subsets of macrophages express CD163 and even classically (M1) activated macrophages can be CD163-positive [33]. The information obtained by approaches, applying a restricted set of immunostainings is therefore very limited and the complexity of macrophage subsets cannot be sufficiently subdivided in the respective types [34]. Furthermore it is unclear in how far the molecular characterized macrophage subsets can be translated into the morphological variety of histiocytic cells as can be demonstrated in histologic slides. This is mainly due to the difficulties which are encountered by the isolation of the cells by laser microdissection and the availability of frozen tissue. In gene expression profiling, laser microdissected spindle shaped epithelioid cells from granulomas of sarcoidosis, [32] showed an enrichment of the M1 signature described by Martinez et al. [11], comparable to tuberculoid granulomas. However, all other types of epithelioid cells tested (round epithelioid cells from Piringer lymphadenitis as well as NLPHL) expressed both M1and M2-related genes (Fig. 2). This confirms that the gene expression profiles of histiocytes are more complex than the division into M1 and M2 subtypes. In T cell/histiocyte rich large B cell lymphoma usually a high content of histiocytes is encountered in the infiltrate. However, to date it is unclear why such high numbers of histiocytic cells accumulate in this tumor. Gene expression profiling revealed a strong expression of metal-binding proteins like Metallothionein 2A (MT2A) [32], which can scavenge ROS [35] and which can be induced by oxidative stress [36]. The presence of this particular type of MT2A-positive macrophages is to our knowledge restricted to this lymphoma and is additionally only found in T cell/histiocyte rich large B cell lymphoma-like variants of NLPHL. Epithelioid cells in typical NLPHL, as well as other forms of classical HL and reactive lesions, are usually MT2A-negative or only present a weak or
S. Hartmann / Pathology – Research and Practice 211 (2015) 901–904
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Fig. 1. Expression of CD163 and Metallothionein (MT) 2A in epithelioid cells and macrophages in lymphomas and under reactive conditions. Both macrophages (a and b) and epithelioid cells (c–f) can show a round (b and c–e) or spindle shape (a and f). While all types of epithelioid cells are negative for CD163 (i–l) and MT2A (o–r), both round and spindle shapes macrophages express CD163 (g and h). Only the typical round macrophages in THRLBCL show and expression of MT2A (n), whereas spindle shaped macrophages in HIV-associated HL are usually negative (m). cHL: classical Hodgkin lymphoma, NLPHL: nodular lymphocyte predominant Hodgkin lymphoma, THRLBCL: T cell/histiocyte rich large B cell lymphoma.
Fig. 2. “Macrophage” polarization of epithelioid cells and macrophages by gene expression [32] with respect to M1 and M2 profiles as described by Martinez et al. [11]. Only the spindle shaped epithelioid cells of sarcoidosis show a pure enrichment of M1genes, whereas round epithelioid cells of nodular lymphocyte predominant Hodgkin lymphoma, Piringer lymphadenitis and histiocytes of T cell/histiocyte rich large B cell lymphoma express both M1- and M2-related genes. NLPHL: nodular lymphocyte predominant Hodgkin lymphoma, THRLBCL: T cell/histiocyte rich large B cell lymphoma.
scattered MT2A-expression (Fig. 1). It can only be speculated that MT2A-positive macrophages also contribute to tumor dissemination and advanced stage as it is usually observed in T cell/histiocyte rich large B cell lymphoma and T cell/histiocyte rich large B cell lymphoma-like variants of NLPHL [37–39]. Interestingly, MT2A
expression has also been documented in the tumor cells of several other tumors [35,40], including the HRS cells of classical HL [32,41]. This may suggest that in T cell/histiocyte rich large B cell lymphoma macrophages take over some functions of the tumor cells. This may also hold true for classical HL, since several genes, like CSF1R, SOD2, CXCL9 and TXN, which are usually expressed in macrophages and also related to ROS scavenging, can also be expressed in HRS cells [32,42,43], particularly in cases with a low macrophage content. PD-L1, which is currently used as therapeutic target, is also expressed both in HRS cells, as well as in macrophages [44,45]. The survival of HRS cells is therefore linked to a complex interplay between tumor cells and different subpopulations of macrophages. In conclusion, although first studies could shed light on the molecular characteristics of histiocytic populations in HL, the detailed functions and interactions have only been clarified to very limited extent. Further studies on this issue are warranted, which may lead to a better understanding of these histiocytes in tumor progression and dissemination and may provide new concepts for treatment.
References [1] K. Lennert, Histopathology of Non-Hodgkin Lymphomas, Springer, Berlin/Heidelberg/New York, 1981 (based on the Kiel classification edn). [2] W.W. Sheehan, H. Rappaport, Morphological criteria in the classification of the malignant lymphomas, Proc. Natl. Cancer Conf. 6 (1970) 59–71.
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[3] E. Patsouris, H. Noel, K. Lennert, Histological and immunohistological findings in lymphoepithelioid cell lymphoma (Lennert’s lymphoma), Am. J. Surg. Pathol. 12 (1988) 341–350. [4] S.S. Dave, G. Wright, B. Tan, A. Rosenwald, R.D. Gascoyne, W.C. Chan, et al., Prediction of survival in follicular lymphoma based on molecular features of tumor-infiltrating immune cells, N. Engl. J. Med. 351 (2004) 2159–2169. [5] G. Lenz, G. Wright, S.S. Dave, W. Xiao, J. Powell, H. Zhao, et al., Stromal gene signatures in large-B-cell lymphomas, N. Engl. J. Med. 359 (2008) 2313–2323. [6] H. Kanzler, M.L. Hansmann, U. Kapp, J. Wolf, V. Diehl, K. Rajewsky, et al., Molecular single cell analysis demonstrates the derivation of a peripheral blood-derived cell line (L1236) from the Hodgkin/Reed–Sternberg cells of a Hodgkin’s lymphoma patient, Blood 87 (1996) 3429–3436. [7] M. Schaadt, V. Diehl, H. Stein, C. Fonatsch, H.H. Kirchner, Two neoplastic cell lines with unique features derived from Hodgkin’s disease, Int. J. Cancer 26 (1980) 723–731. [8] A. Mader, S. Bruderlein, S. Wegener, I. Melzner, S. Popov, H.K. Muller-Hermelink, et al., U-HO1, a new cell line derived from a primary refractory classical Hodgkin lymphoma, Cytogenet. Genome Res. 119 (2007) 204–210. [9] P. Italiani, D. Boraschi, From monocytes to M1/M2 macrophages: phenotypical vs functional differentiation, Front. Immunol. 5 (2014) 514. [10] K. Ley, The second touch hypothesis: T cell activation, homing and polarization, F1000Research 3 (2014) 37. [11] F.O. Martinez, A. Sica, A. Mantovani, M. Locati, Macrophage activation and polarization, Front. Biosci. 13 (2008) 453–461. [12] S. Gordon, Alternative activation of macrophages, Nat. Rev. Immunol. 3 (2003) 23–35. [13] F. Facchetti, W. Vermi, S. Fiorentini, M. Chilosi, A. Caruso, M. Duse, et al., Expression of inducible nitric oxide synthase in human granulomas and histiocytic reactions, Am. J. Pathol. 154 (1999) 145–152. [14] B.Z. Qian, J.W. Pollard, Macrophage diversity enhances tumor progression and metastasis, Cell 141 (2010) 39–51. [15] L.S. Ojalvo, C.A. Whittaker, J.S. Condeelis, J.W. Pollard, Gene expression analysis of macrophages that facilitate tumor invasion supports a role for Wnt-signaling in mediating their activity in primary mammary tumors, J. Immunol. 184 (2010) 702–712. [16] R. van Furth, Origin and kinetics of monocytes and macrophages, Semin. Hematol. 7 (1970) 125–141. [17] L.C. Davies, M. Rosas, P.J. Smith, D.J. Fraser, S.A. Jones, P.R. Taylor, A quantifiable proliferative burst of tissue macrophages restores homeostatic macrophage populations after acute inflammation, Eur. J. Immunol. 41 (2011) 2155–2164. [18] D. Hashimoto, A. Chow, C. Noizat, P. Teo, M.B. Beasley, M. Leboeuf, et al., Tissue-resident macrophages self-maintain locally throughout adult life with minimal contribution from circulating monocytes, Immunity 38 (2013) 792–804. [19] B. Ajami, J.L. Bennett, C. Krieger, K.M. McNagny, F.M. Rossi, Infiltrating monocytes trigger EAE progression, but do not contribute to the resident microglia pool, Nat. Neurosci. 14 (2011) 1142–1149. [20] C. Steidl, T. Lee, S.P. Shah, P. Farinha, G. Han, T. Nayar, et al., Tumor-associated macrophages and survival in classic Hodgkin’s lymphoma, N. Engl. J. Med. 362 (2010) 875–885. [21] K.L. Tan, D.W. Scott, F. Hong, B.S. Kahl, R.I. Fisher, N.L. Bartlett, et al., Tumor-associated macrophages predict inferior outcomes in classic Hodgkin lymphoma: a correlative study from the E2496 Intergroup trial, Blood 120 (2012) 3280–3287. [22] A. Tzankov, M.S. Matter, S. Dirnhofer, Refined prognostic role of CD68-positive tumor macrophages in the context of the cellular micromilieu of classical Hodgkin lymphoma, Pathobiology 77 (2010) 301–308. [23] P. Kamper, K. Bendix, S. Hamilton-Dutoit, B. Honore, J.R. Nyengaard, F. d’Amore, Tumor-infiltrating macrophages correlate with adverse prognosis and Epstein–Barr virus status in classical Hodgkin’s lymphoma, Haematologica 96 (2011) 269–276. [24] D. Azambuja, Y. Natkunam, I. Biasoli, I.S. Lossos, M.W. Anderson, J.C. Morais, et al., Lack of association of tumor-associated macrophages with clinical outcome in patients with classical Hodgkin’s lymphoma, Ann. Oncol. 23 (2012) 736–742. [25] J.A. Harris, S. Jain, Q. Ren, A. Zarineh, C. Liu, S. Ibrahim, CD163 versus CD68 in tumor associated macrophages of classical Hodgkin lymphoma, Diagn. Pathol. 7 (2012) 12. [26] D.W. Scott, F.C. Chan, F. Hong, S. Rogic, K.L. Tan, B. Meissner, et al., Gene expression-based model using formalin-fixed paraffin-embedded biopsies
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
[43]
[44]
[45]
predicts overall survival in advanced-stage classical Hodgkin lymphoma, J. Clin. Oncol. 31 (2013) 692–700. S. Hartmann, C. Jakobus, B. Rengstl, C. Doring, S. Newrzela, H.R. Brodt, et al., Spindle-shaped CD163+ rosetting macrophages replace CD4+ T-cells in HIV-related classical Hodgkin lymphoma, Mod. Pathol. 26 (2013) 648–657. Y. Ma, L. Visser, H. Roelofsen, M. de Vries, A. Diepstra, G. van Imhoff, et al., Proteomics analysis of Hodgkin lymphoma: identification of new players involved in the cross-talk between HRS cells and infiltrating lymphocytes, Blood 111 (2008) 2339–2346. J. Bohlius, K. Schmidlin, F. Boue, G. Fatkenheuer, M. May, A.M. Caro-Murillo, et al., HIV-1-related Hodgkin lymphoma in the era of combination antiretroviral therapy: incidence and evolution of CD4(+) T-cell lymphocytes, Blood 117 (2011) 6100–6108. L.F. Porrata, K. Ristow, J.P. Colgan, T.M. Habermann, T.E. Witzig, D.J. Inwards, et al., Peripheral blood lymphocyte/monocyte ratio at diagnosis and survival in classical Hodgkin’s lymphoma, Haematologica 97 (2012) 262–269. L.F. Porrata, K. Ristow, T.M. Habermann, T.E. Witzig, J.P. Colgan, D.J. Inwards, et al., Peripheral blood lymphocyte/monocyte ratio at diagnosis and survival in nodular lymphocyte-predominant Hodgkin lymphoma, Br. J. Haematol. 157 (2012) 321–330. S. Hartmann, T. Tousseyn, C. Doring, P. Fluchter, H. Hackstein, A. Herreman, et al., Macrophages in T cell/histiocyte rich large B cell lymphoma strongly express metal-binding proteins and show a bi-activated phenotype, Int. J. Cancer 133 (2013) 2609–2618. J. Fuentes-Duculan, M. Suarez-Farinas, L.C. Zaba, K.E. Nograles, K.C. Pierson, H. Mitsui, et al., A subpopulation of CD163-positive macrophages is classically activated in psoriasis, J. Invest. Dermatol. 130 (2010) 2412–2422. M.H. Barros, P. Segges, G. Vera-Lozada, R. Hassan, G. Niedobitek, Macrophage polarization reflects T cell composition of tumor microenvironment in pediatric classical Hodgkin lymphoma and has impact on survival, PLOS ONE 10 (2015) e0124531. N. Thirumoorthy, A. Shyam Sunder, K. Manisenthil Kumar, M. Senthil Kumar, G. Ganesh, M. Chatterjee, A review of metallothionein isoforms and their role in pathophysiology, World J. Surg. Oncol. 9 (2011) 54. M. Yang, C.R. Chitambar, Role of oxidative stress in the induction of metallothionein-2A and heme oxygenase-1 gene expression by the antineoplastic agent gallium nitrate in human lymphoma cells, Free Radic. Biol. Med. 45 (2008) 763–772. S. Hartmann, C. Doring, C. Jakobus, B. Rengstl, S. Newrzela, T. Tousseyn, et al., Nodular lymphocyte predominant Hodgkin lymphoma and T cell/histiocyte rich large B cell lymphoma – endpoints of a spectrum of one disease? PLOS ONE 8 (2013) e78812. S. Hartmann, D.A. Eichenauer, A. Plutschow, A. Mottok, R. Bob, K. Koch, et al., The prognostic impact of variant histology in nodular lymphocyte-predominant Hodgkin lymphoma: a report from the German Hodgkin Study Group (GHSG), Blood 122 (2013) 4246–4252. R. Achten, G. Verhoef, L. Vanuytsel, C. De Wolf-Peeters, T-cell/histiocyte-rich large B-cell lymphoma: a distinct clinicopathologic entity, J. Clin. Oncol. 20 (2002) 1269–1277. J.M. Arriaga, E.M. Levy, A.I. Bravo, S.M. Bayo, M. Amat, M. Aris, et al., Metallothionein expression in colorectal cancer: relevance of different isoforms for tumor progression and patient survival, Hum. Pathol. 43 (2012) 197–208. M. Penkowa, B.L. Sorensen, S.L. Nielsen, P.B. Hansen, Metallothionein as a useful marker in Hodgkin lymphoma subclassification, Leuk. Lymphoma 50 (2009) 200–210. B. Lamprecht, K. Walter, S. Kreher, R. Kumar, M. Hummel, D. Lenze, et al., Derepression of an endogenous long terminal repeat activates the CSF1R proto-oncogene in human lymphoma, Nat. Med. 16 (2010) 571–579 (571p following 579). C. Steidl, A. Diepstra, T. Lee, F.C. Chan, P. Farinha, K. Tan, et al., Gene expression profiling of microdissected Hodgkin Reed Sternberg cells correlates with treatment outcome in classical Hodgkin lymphoma, Blood (2012). S. Paydas, E. Bagir, G. Seydaoglu, V. Ercolak, M. Ergin, Programmed death-1 (PD-1), programmed death-ligand 1 (PD-L1), and EBV-encoded RNA (EBER) expression in Hodgkin lymphoma, Ann. Hematol. (2015). M. Rodriguez-Garcia, F. Porichis, O.G. de Jong, K. Levi, T.J. Diefenbach, J.D. Lifson, et al., Expression of PD-L1 and PD-L2 on human macrophages is up-regulated by HIV-1 and differentially modulated by IL-10, J. Leukoc. Biol. 89 (2011) 507–515.
Contents lists available at ScienceDirect
Pathology – Research and Practice journal homepage: www.elsevier.com/locate/prp
Review
From a pathologist’s point of view: Histiocytic cells in Hodgkin lymphoma and T cell/histiocyte rich large B cell lymphoma Sylvia Hartmann Dr. Senckenberg Institute of Pathology, Goethe University Hospital Frankfurt, Theodor-Stern-Kai 7, D-60590 Frankfurt, Germany
a r t i c l e
i n f o
Keywords: Macrophages Reactive bystander cells Hodgkin lymphoma T cell/histiocyte rich large B cell lymphoma Metallothionein 2A
a b s t r a c t While tumor cells were the focus of research for many years, only recently have attempts been made to understand the role of the reactive bystander cells in malignant lymphomas. In certain types of lymphomas, such as Hodgkin lymphoma and T cell/histiocyte rich large B cell lymphoma, more than 90% of the infiltrate represent non-neoplastic cells, and these have important functions for the development and progression of the tumor. Among the bystander cells are histiocytes of particular importance, which vary largely in number, shape and quality among different patients. In the present review, recent findings on the prognostic impact of histiocytic bystander cells in these lymphomas, as well as their molecular characteristics, are discussed. A better understanding of the role of these histiocytes may provide new concepts for diagnosis and treatment. © 2015 Elsevier GmbH. All rights reserved.
Contents 1. 2. 3.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 901 Macrophage subtypes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 902 Macrophages in Hodgkin lymphoma and T cell/histiocyte rich large B cell lymphoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 902 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 903
1. Introduction For many years, lymphoma pathology has focused on the main players in these tumors, the malignant tumor cells. Different efforts have been made to classify lymphomas according to their growth patterns or the cytology of the tumor cells [1,2]. However, not only the tumor cells, but also the microenvironment contributes significantly to the malignant phenotype observed. Whereas in some lymphomas this microenvironment is so abundant that it has been included as part of the diagnosis [3], in other entities, such as diffuse large B cell lymphoma (DLBCL) and follicular lymphoma, the importance of the reactive bystander cells has only recently been recognized [4,5]. For several lymphoma entities, it was not possible to establish cell lines due to the strong dependency of the tumor cells on the reactive bystander cells, which were not viable under cell culture conditions. Therefore, malignant lymphomas can be divided into more and less microenvironmentdependent types. Lymphomas which are strongly dependent on
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their microenvironment are e.g. follicular lymphoma, marginal zone lymphoma, Hodgkin lymphoma (HL), T cell/histiocyte rich large B cell lymphoma, as well as angioimmunoblastic or peripheral T cell lymphoma. Surprisingly, both B- and T cell neoplasias can equally depend on their microenvironment. In contrast, Burkitt lymphoma and some cases of DLBCL are rather independent of their microenvironment, which is also reflected by the usually low content of bystander cells in the lymphoma infiltrate and the availability of a large number of corresponding cell lines. HL represents the most extreme relationship between tumor cell content (frequently <1% of the infiltrate) and reactive bystander cells. Surprisingly, HL cell lines could be established which grow in culture without the respective bystander cells. However, these were established from relapsed or refractory patients and frequently from pleural effusions, in which the tumor cells had already gained independency from their environment and were able to grow autonomously in suspension [6–8]. The reactive microenvironment of HL usually contains a huge spectrum of cells, ranging from non-neoplastic B cells and special T cell subsets to dendritic cells, macrophages, mast cells, eosinophils
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and stromal cells. The present review will focus on histiocytic cell types in the reactive microenvironment.
2. Macrophage subtypes While pathologists described different types of histiocytes a long time ago, histiocytic surface markers have only recently been characterized by immunologists. Pathologists define macrophages by their shape and phagocytic function, as well as epithelioid cells, which primarily secrete cytokines. In contrast to this morphologic description, immunologists classify all macrophages in connective tissue as histiocytes or tissue macrophages [9]. One of the most important features of tissue macrophages is the take up and presentation of antigens [10]. Originally, two subsets of classically activated proinflammatory (M1) and alternatively activated (M2) macrophages have been described [11,12] which participate in Th1 and Th2 triggered immune responses, respectively. Morphologically recognizable histiocyte subsets have so far not been well characterized by surface markers. However, there are certain reactions like tuberculoid granulomas, which have been shown to express inducible nitrogen oxide (NO) synthetase [13], corresponding to M1-polarized macrophages. Secretion of NO and reactive oxygen species (ROS) is involved in tissue injury and necrosis as it is typically observed in caseous necrosis in the center of tuberculoid granulomas, consistent with a M1 phenotype of these epithelioid cells. With reference to the complex situation in vivo, Qian and Pollard [14] described six types of macrophages, the activated and the angiogenic type, but also an invasive, metastasis-associated, perivascular and immunosuppressive subtype. These different types secrete unique, but also partly overlapping cytokines. The invasive type of macrophages is found at the outer borders of solid tumors, since this represents the localization where malignant tumors grow fastest. These invasive macrophages were characterized in mice to have an active Wnt signaling [15]. Whereas it was previously hypothesized that all tissue macrophages derive from circulating peripheral blood monocytes [16], recent studies have shown that mature macrophages can proliferate in the tissue in response to specific stimuli without loss of functional differentiation [17,18]. These tissue-resident and self-renewing macrophages were shown to have an M2-like phenotype, whereas monocytederived macrophages, which are recruited to the tissue during an inflammatory response, are supposed to be M1-polarized [19].
3. Macrophages in Hodgkin lymphoma and T cell/histiocyte rich large B cell lymphoma In 2010, the important impact of macrophages in the microenvironment of classical HL on event free and overall survival was first remarked [20]. Many other studies conducted since then have examined the impact of macrophages on clinical behavior. Whereas some could confirm previous observations and found a correlation between a high number of macrophages, mixed cellularity subtype and Epstein–Barr virus (EBV) infection of the Hodgkinand Reed–Sternberg (HRS) cells [21–23], others could not confirm the prognostic impact of a high number of tissue macrophages [24,25]. However, the methods how the macrophage content was counted (CD68- versus CD163-staining) and the thresholds differed between different studies and so far no general applicable test is available. A whole tissue gene expression study identified a 23-gene outcome predictor, which included several macrophageand M1-related genes like CXCL10, STAT1, IFNG, LYZ, CD68 and CD14 [26]. It may therefore be easier and more precise to quantify macrophages according to specific transcripts than counting numbers of macrophages. Particularly, the presence of special subtypes
of polarized macrophages can better be assessed by the quantification of different transcripts than by pure morphology. HIV-positive individuals with HL take a special position concerning the macrophage content in the HL infiltrate: they can present a particularly high macrophage content in the HL microenvironment when the CD4 count in the peripheral blood is low [27]. Therefore, the extent of macrophage infiltration in the lymphoma tissue seems to be related to the availability of CD4 T cells and monocytes in the blood, and monocytes/macrophages can replace CD4 T cells under certain conditions [27]. CD4 T cells get attracted to the microenvironment of HL by several cytokines like TARC and RANTES [28], which are secreted by the HRS cells. In HIV patients, CD4 blood counts are regularly tested, and the development of HL can therefore well be monitored in these patients. Already one year before HL becomes manifest, CD4 cells start to decrease in the peripheral blood, indicating recruitment of CD4 cells to the lymph nodes with HL infiltrates [29]. Interestingly, not only a high content of macrophages in the lymph nodes affected by HL was related to a worse clinical outcome: also a low ratio between lymphocyte and monocyte count in the peripheral blood was related with an adverse prognosis in classical and nodular lymphocyte predominant HL (in HIV-negative individuals) [30,31]. Therefore, in contrast to recent findings indicating that macrophages can renew in the tissue [17], the reactive HL microenvironment in the lymph node seems to reflect the availability of different cell types in the peripheral blood. The molecular mechanisms how macrophages contribute to the dissemination and advanced stage of classical HL have only allusively been characterized. Applying only a limited number of immunohistochemical markers macrophage polarization cannot definitely be determined. CD163, which was originally described as M2 marker, is e.g. negative in all forms of epithelioid cells (Fig. 1), which are particularly found in mixed cellularity subtype and nodular lymphocyte predominant HL (NLPHL), although epithelioid cells can express other M2-markers [32]. In contrast, most subsets of macrophages express CD163 and even classically (M1) activated macrophages can be CD163-positive [33]. The information obtained by approaches, applying a restricted set of immunostainings is therefore very limited and the complexity of macrophage subsets cannot be sufficiently subdivided in the respective types [34]. Furthermore it is unclear in how far the molecular characterized macrophage subsets can be translated into the morphological variety of histiocytic cells as can be demonstrated in histologic slides. This is mainly due to the difficulties which are encountered by the isolation of the cells by laser microdissection and the availability of frozen tissue. In gene expression profiling, laser microdissected spindle shaped epithelioid cells from granulomas of sarcoidosis, [32] showed an enrichment of the M1 signature described by Martinez et al. [11], comparable to tuberculoid granulomas. However, all other types of epithelioid cells tested (round epithelioid cells from Piringer lymphadenitis as well as NLPHL) expressed both M1and M2-related genes (Fig. 2). This confirms that the gene expression profiles of histiocytes are more complex than the division into M1 and M2 subtypes. In T cell/histiocyte rich large B cell lymphoma usually a high content of histiocytes is encountered in the infiltrate. However, to date it is unclear why such high numbers of histiocytic cells accumulate in this tumor. Gene expression profiling revealed a strong expression of metal-binding proteins like Metallothionein 2A (MT2A) [32], which can scavenge ROS [35] and which can be induced by oxidative stress [36]. The presence of this particular type of MT2A-positive macrophages is to our knowledge restricted to this lymphoma and is additionally only found in T cell/histiocyte rich large B cell lymphoma-like variants of NLPHL. Epithelioid cells in typical NLPHL, as well as other forms of classical HL and reactive lesions, are usually MT2A-negative or only present a weak or
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Fig. 1. Expression of CD163 and Metallothionein (MT) 2A in epithelioid cells and macrophages in lymphomas and under reactive conditions. Both macrophages (a and b) and epithelioid cells (c–f) can show a round (b and c–e) or spindle shape (a and f). While all types of epithelioid cells are negative for CD163 (i–l) and MT2A (o–r), both round and spindle shapes macrophages express CD163 (g and h). Only the typical round macrophages in THRLBCL show and expression of MT2A (n), whereas spindle shaped macrophages in HIV-associated HL are usually negative (m). cHL: classical Hodgkin lymphoma, NLPHL: nodular lymphocyte predominant Hodgkin lymphoma, THRLBCL: T cell/histiocyte rich large B cell lymphoma.
Fig. 2. “Macrophage” polarization of epithelioid cells and macrophages by gene expression [32] with respect to M1 and M2 profiles as described by Martinez et al. [11]. Only the spindle shaped epithelioid cells of sarcoidosis show a pure enrichment of M1genes, whereas round epithelioid cells of nodular lymphocyte predominant Hodgkin lymphoma, Piringer lymphadenitis and histiocytes of T cell/histiocyte rich large B cell lymphoma express both M1- and M2-related genes. NLPHL: nodular lymphocyte predominant Hodgkin lymphoma, THRLBCL: T cell/histiocyte rich large B cell lymphoma.
scattered MT2A-expression (Fig. 1). It can only be speculated that MT2A-positive macrophages also contribute to tumor dissemination and advanced stage as it is usually observed in T cell/histiocyte rich large B cell lymphoma and T cell/histiocyte rich large B cell lymphoma-like variants of NLPHL [37–39]. Interestingly, MT2A
expression has also been documented in the tumor cells of several other tumors [35,40], including the HRS cells of classical HL [32,41]. This may suggest that in T cell/histiocyte rich large B cell lymphoma macrophages take over some functions of the tumor cells. This may also hold true for classical HL, since several genes, like CSF1R, SOD2, CXCL9 and TXN, which are usually expressed in macrophages and also related to ROS scavenging, can also be expressed in HRS cells [32,42,43], particularly in cases with a low macrophage content. PD-L1, which is currently used as therapeutic target, is also expressed both in HRS cells, as well as in macrophages [44,45]. The survival of HRS cells is therefore linked to a complex interplay between tumor cells and different subpopulations of macrophages. In conclusion, although first studies could shed light on the molecular characteristics of histiocytic populations in HL, the detailed functions and interactions have only been clarified to very limited extent. Further studies on this issue are warranted, which may lead to a better understanding of these histiocytes in tumor progression and dissemination and may provide new concepts for treatment.
References [1] K. Lennert, Histopathology of Non-Hodgkin Lymphomas, Springer, Berlin/Heidelberg/New York, 1981 (based on the Kiel classification edn). [2] W.W. Sheehan, H. Rappaport, Morphological criteria in the classification of the malignant lymphomas, Proc. Natl. Cancer Conf. 6 (1970) 59–71.
904
S. Hartmann / Pathology – Research and Practice 211 (2015) 901–904
[3] E. Patsouris, H. Noel, K. Lennert, Histological and immunohistological findings in lymphoepithelioid cell lymphoma (Lennert’s lymphoma), Am. J. Surg. Pathol. 12 (1988) 341–350. [4] S.S. Dave, G. Wright, B. Tan, A. Rosenwald, R.D. Gascoyne, W.C. Chan, et al., Prediction of survival in follicular lymphoma based on molecular features of tumor-infiltrating immune cells, N. Engl. J. Med. 351 (2004) 2159–2169. [5] G. Lenz, G. Wright, S.S. Dave, W. Xiao, J. Powell, H. Zhao, et al., Stromal gene signatures in large-B-cell lymphomas, N. Engl. J. Med. 359 (2008) 2313–2323. [6] H. Kanzler, M.L. Hansmann, U. Kapp, J. Wolf, V. Diehl, K. Rajewsky, et al., Molecular single cell analysis demonstrates the derivation of a peripheral blood-derived cell line (L1236) from the Hodgkin/Reed–Sternberg cells of a Hodgkin’s lymphoma patient, Blood 87 (1996) 3429–3436. [7] M. Schaadt, V. Diehl, H. Stein, C. Fonatsch, H.H. Kirchner, Two neoplastic cell lines with unique features derived from Hodgkin’s disease, Int. J. Cancer 26 (1980) 723–731. [8] A. Mader, S. Bruderlein, S. Wegener, I. Melzner, S. Popov, H.K. Muller-Hermelink, et al., U-HO1, a new cell line derived from a primary refractory classical Hodgkin lymphoma, Cytogenet. Genome Res. 119 (2007) 204–210. [9] P. Italiani, D. Boraschi, From monocytes to M1/M2 macrophages: phenotypical vs functional differentiation, Front. Immunol. 5 (2014) 514. [10] K. Ley, The second touch hypothesis: T cell activation, homing and polarization, F1000Research 3 (2014) 37. [11] F.O. Martinez, A. Sica, A. Mantovani, M. Locati, Macrophage activation and polarization, Front. Biosci. 13 (2008) 453–461. [12] S. Gordon, Alternative activation of macrophages, Nat. Rev. Immunol. 3 (2003) 23–35. [13] F. Facchetti, W. Vermi, S. Fiorentini, M. Chilosi, A. Caruso, M. Duse, et al., Expression of inducible nitric oxide synthase in human granulomas and histiocytic reactions, Am. J. Pathol. 154 (1999) 145–152. [14] B.Z. Qian, J.W. Pollard, Macrophage diversity enhances tumor progression and metastasis, Cell 141 (2010) 39–51. [15] L.S. Ojalvo, C.A. Whittaker, J.S. Condeelis, J.W. Pollard, Gene expression analysis of macrophages that facilitate tumor invasion supports a role for Wnt-signaling in mediating their activity in primary mammary tumors, J. Immunol. 184 (2010) 702–712. [16] R. van Furth, Origin and kinetics of monocytes and macrophages, Semin. Hematol. 7 (1970) 125–141. [17] L.C. Davies, M. Rosas, P.J. Smith, D.J. Fraser, S.A. Jones, P.R. Taylor, A quantifiable proliferative burst of tissue macrophages restores homeostatic macrophage populations after acute inflammation, Eur. J. Immunol. 41 (2011) 2155–2164. [18] D. Hashimoto, A. Chow, C. Noizat, P. Teo, M.B. Beasley, M. Leboeuf, et al., Tissue-resident macrophages self-maintain locally throughout adult life with minimal contribution from circulating monocytes, Immunity 38 (2013) 792–804. [19] B. Ajami, J.L. Bennett, C. Krieger, K.M. McNagny, F.M. Rossi, Infiltrating monocytes trigger EAE progression, but do not contribute to the resident microglia pool, Nat. Neurosci. 14 (2011) 1142–1149. [20] C. Steidl, T. Lee, S.P. Shah, P. Farinha, G. Han, T. Nayar, et al., Tumor-associated macrophages and survival in classic Hodgkin’s lymphoma, N. Engl. J. Med. 362 (2010) 875–885. [21] K.L. Tan, D.W. Scott, F. Hong, B.S. Kahl, R.I. Fisher, N.L. Bartlett, et al., Tumor-associated macrophages predict inferior outcomes in classic Hodgkin lymphoma: a correlative study from the E2496 Intergroup trial, Blood 120 (2012) 3280–3287. [22] A. Tzankov, M.S. Matter, S. Dirnhofer, Refined prognostic role of CD68-positive tumor macrophages in the context of the cellular micromilieu of classical Hodgkin lymphoma, Pathobiology 77 (2010) 301–308. [23] P. Kamper, K. Bendix, S. Hamilton-Dutoit, B. Honore, J.R. Nyengaard, F. d’Amore, Tumor-infiltrating macrophages correlate with adverse prognosis and Epstein–Barr virus status in classical Hodgkin’s lymphoma, Haematologica 96 (2011) 269–276. [24] D. Azambuja, Y. Natkunam, I. Biasoli, I.S. Lossos, M.W. Anderson, J.C. Morais, et al., Lack of association of tumor-associated macrophages with clinical outcome in patients with classical Hodgkin’s lymphoma, Ann. Oncol. 23 (2012) 736–742. [25] J.A. Harris, S. Jain, Q. Ren, A. Zarineh, C. Liu, S. Ibrahim, CD163 versus CD68 in tumor associated macrophages of classical Hodgkin lymphoma, Diagn. Pathol. 7 (2012) 12. [26] D.W. Scott, F.C. Chan, F. Hong, S. Rogic, K.L. Tan, B. Meissner, et al., Gene expression-based model using formalin-fixed paraffin-embedded biopsies
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
[43]
[44]
[45]
predicts overall survival in advanced-stage classical Hodgkin lymphoma, J. Clin. Oncol. 31 (2013) 692–700. S. Hartmann, C. Jakobus, B. Rengstl, C. Doring, S. Newrzela, H.R. Brodt, et al., Spindle-shaped CD163+ rosetting macrophages replace CD4+ T-cells in HIV-related classical Hodgkin lymphoma, Mod. Pathol. 26 (2013) 648–657. Y. Ma, L. Visser, H. Roelofsen, M. de Vries, A. Diepstra, G. van Imhoff, et al., Proteomics analysis of Hodgkin lymphoma: identification of new players involved in the cross-talk between HRS cells and infiltrating lymphocytes, Blood 111 (2008) 2339–2346. J. Bohlius, K. Schmidlin, F. Boue, G. Fatkenheuer, M. May, A.M. Caro-Murillo, et al., HIV-1-related Hodgkin lymphoma in the era of combination antiretroviral therapy: incidence and evolution of CD4(+) T-cell lymphocytes, Blood 117 (2011) 6100–6108. L.F. Porrata, K. Ristow, J.P. Colgan, T.M. Habermann, T.E. Witzig, D.J. Inwards, et al., Peripheral blood lymphocyte/monocyte ratio at diagnosis and survival in classical Hodgkin’s lymphoma, Haematologica 97 (2012) 262–269. L.F. Porrata, K. Ristow, T.M. Habermann, T.E. Witzig, J.P. Colgan, D.J. Inwards, et al., Peripheral blood lymphocyte/monocyte ratio at diagnosis and survival in nodular lymphocyte-predominant Hodgkin lymphoma, Br. J. Haematol. 157 (2012) 321–330. S. Hartmann, T. Tousseyn, C. Doring, P. Fluchter, H. Hackstein, A. Herreman, et al., Macrophages in T cell/histiocyte rich large B cell lymphoma strongly express metal-binding proteins and show a bi-activated phenotype, Int. J. Cancer 133 (2013) 2609–2618. J. Fuentes-Duculan, M. Suarez-Farinas, L.C. Zaba, K.E. Nograles, K.C. Pierson, H. Mitsui, et al., A subpopulation of CD163-positive macrophages is classically activated in psoriasis, J. Invest. Dermatol. 130 (2010) 2412–2422. M.H. Barros, P. Segges, G. Vera-Lozada, R. Hassan, G. Niedobitek, Macrophage polarization reflects T cell composition of tumor microenvironment in pediatric classical Hodgkin lymphoma and has impact on survival, PLOS ONE 10 (2015) e0124531. N. Thirumoorthy, A. Shyam Sunder, K. Manisenthil Kumar, M. Senthil Kumar, G. Ganesh, M. Chatterjee, A review of metallothionein isoforms and their role in pathophysiology, World J. Surg. Oncol. 9 (2011) 54. M. Yang, C.R. Chitambar, Role of oxidative stress in the induction of metallothionein-2A and heme oxygenase-1 gene expression by the antineoplastic agent gallium nitrate in human lymphoma cells, Free Radic. Biol. Med. 45 (2008) 763–772. S. Hartmann, C. Doring, C. Jakobus, B. Rengstl, S. Newrzela, T. Tousseyn, et al., Nodular lymphocyte predominant Hodgkin lymphoma and T cell/histiocyte rich large B cell lymphoma – endpoints of a spectrum of one disease? PLOS ONE 8 (2013) e78812. S. Hartmann, D.A. Eichenauer, A. Plutschow, A. Mottok, R. Bob, K. Koch, et al., The prognostic impact of variant histology in nodular lymphocyte-predominant Hodgkin lymphoma: a report from the German Hodgkin Study Group (GHSG), Blood 122 (2013) 4246–4252. R. Achten, G. Verhoef, L. Vanuytsel, C. De Wolf-Peeters, T-cell/histiocyte-rich large B-cell lymphoma: a distinct clinicopathologic entity, J. Clin. Oncol. 20 (2002) 1269–1277. J.M. Arriaga, E.M. Levy, A.I. Bravo, S.M. Bayo, M. Amat, M. Aris, et al., Metallothionein expression in colorectal cancer: relevance of different isoforms for tumor progression and patient survival, Hum. Pathol. 43 (2012) 197–208. M. Penkowa, B.L. Sorensen, S.L. Nielsen, P.B. Hansen, Metallothionein as a useful marker in Hodgkin lymphoma subclassification, Leuk. Lymphoma 50 (2009) 200–210. B. Lamprecht, K. Walter, S. Kreher, R. Kumar, M. Hummel, D. Lenze, et al., Derepression of an endogenous long terminal repeat activates the CSF1R proto-oncogene in human lymphoma, Nat. Med. 16 (2010) 571–579 (571p following 579). C. Steidl, A. Diepstra, T. Lee, F.C. Chan, P. Farinha, K. Tan, et al., Gene expression profiling of microdissected Hodgkin Reed Sternberg cells correlates with treatment outcome in classical Hodgkin lymphoma, Blood (2012). S. Paydas, E. Bagir, G. Seydaoglu, V. Ercolak, M. Ergin, Programmed death-1 (PD-1), programmed death-ligand 1 (PD-L1), and EBV-encoded RNA (EBER) expression in Hodgkin lymphoma, Ann. Hematol. (2015). M. Rodriguez-Garcia, F. Porichis, O.G. de Jong, K. Levi, T.J. Diefenbach, J.D. Lifson, et al., Expression of PD-L1 and PD-L2 on human macrophages is up-regulated by HIV-1 and differentially modulated by IL-10, J. Leukoc. Biol. 89 (2011) 507–515.