Pediatric Hematology and Oncology, Early Online:1–10, 2014Copyright C© Informa Healthcare USA, Inc.ISSN: 0888-0018 print / 1521-0669 onlineDOI: 10.3109/08880018.2014.954071

ORIGINAL ARTICLE

Pediatric Hodgkin Lymphoma in a South IndianRegional Cancer Center: Its Immunomorphology,Tumor-Associated Macrophages, and Associationwith Epstein–Barr Virus

Mohammed Abdul Lateef Zameer, MD,1 Chennagiri S. Premalata, MD,1

Bandagadde Arunakumari, DM,2 Lingappa Appaji, DM,2

and Clementina Rama Rao, MD1

1Department of Pathology, Kidwai Memorial Institute of Oncology, Bangalore, India;2Department of Pediatric Oncology, Kidwai Memorial Institute of Oncology, Bangalore,India

Pediatric Hodgkin lymphoma (HL) comprises approximately a fifth of all patients with HL inIndia. Seventy-four cases of pediatric classical Hodgkin Lymphoma (cHL) from a regional cancercenter in southern India were analyzed on a tissue microarray (TMA) for the stage of B-celldifferentiation of the Hodgkin/Reed Sternberg (HRS) cell by immunohistochemistry (IHC) usingCD10, bcl6, MUM1/IRF4, and CD 138. Fifty-two of seventy-four (70.3%) cases were of late germinalcenter/early post-germinal center phenotype (CD10−/bcl6−/MUM1+/CD138−). Epstein–Barrvirus (EBV) association using Epstein-Barr virus encoded RNA (EBER) RISH and EBV-LMP1 im-munohistochemistry (IHC) revealed an EBV association of 93%. Tumor-associated macrophages(TAM) in the microenvironment were also assessed on the TMA by CD68 IHC, and most cases(59.7%) showed >25% TAMs, with no case showing ≤5%. These findings indicate that pediatriccHL in India is a tumor, predominantly, of late germinal center/early post-germinal center B cells,is almost invariably EBV associated, and with a high number of TAMs in the microenvironment.This latter finding suggests that criteria other than TAM scores need to be developed for riskstratification of pediatric EBV-associated HL especially in developing countries.

Keywords EBV, Hodgkin lymphoma, pediatric, markers for germinal center B cells, markers forpost-germinal center B cells, tumor-associated macrophages

INTRODUCTION

Childhood Hodgkin lymphoma (HL) is an enigmatic disease with characteristic epi-demiologic and clinical features and pathology [1]. In India, pediatric HL constitutes afifth of all cases of HL [2], with its incidence exceeding that of non-Hodgkin lymphoma(NHL) [3, 4]. Furthermore, pediatric patients in developing countries tend to presentwith marked male predominance, more advanced disease, and a mixed cellularitymorphology [5]. The Hodgkin/Reed–Sternberg (HRS) cell of classical Hodgkin lym-phoma (cHL) is now known to be a mature pre-apoptotic B cell that has participated in

Received 20 May 2014; accepted 8 August 2014.Address correspondence to Dr Clementina Rama Rao, B-305, Wilson Manor Apartments, 11/3,13th Cross Wilson Garden, Bangalore, 560027, Karnataka, India. E-mail: rao.tina@gmail.com

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immune responses involving the germinal center (GC) [6, 7]. A few studies have beendone using markers for GC and post-GC B cells to investigate the stage of B-cell dif-ferentiation of the HRS cell [8–12]. These include CD10, bcl6, and MUM1/IRF4, all ofwhich have been used in algorithms to subtype diffuse large B-cell lymphomas intoGC and activated B-cell/nongerminal center phenotypes [13]. Bai et al. used CD10,Bcl6, MUM1, and CD138 to subtype cHL into late GC/early post-GC, post-GC, and in-determinate phenotypes [12].To the best of our knowledge, this is the first study to havespecifically looked at the stage of B-cell differentiation of HRS cell in pediatric cHL. Themicroenvironment of cHL plays an important role, both in pathogenesis and prognos-tication. Recent studies by gene expression profiling and immunohistochemistry havehighlighted the importance of tumor-associated macrophages (TAMs) in risk stratifi-cation of cHL, especially in early-stage disease [14]. Many studies have shown that anincreased number of macrophages in the microenvironment indicate a worse progno-sis [14–16]. However, Epstein–Barr virus (EBV) positivity is associated with an increasein the number of macrophages in the tumor microenvironment [15, 17]. This is espe-cially important in pediatric cHL which is almost always EBV-associated in developingcountries, but responds well to therapy, has a good prognosis [18, 19], and, therefore,different criteria would need to be developed for risk stratification of pediatric cHL[20].

The aim of our study was to investigate the morphologic spectrum and EBV asso-ciation of pediatric cHL at our institute, which is a tertiary referral cancer center insouthern India. Using a panel of four immunohistochemistry (IHC) markers (CD10,bcl6, MUM1, and CD138), we have also investigated the stage of B-cell differentiationof the HRS cell in pediatric cHL. The proportion of CD68-positive macrophages in themicroenvironment was also determined and correlated with EBV positivity.

MATERIALS AND METHODS

Tissue microarray was constructed from 74 immunohistochemically proven cases ofpediatric cHL using the manual Beecher tissue microarrayer (Beecher instruments,USA). One-milimeter diameter cores in triplicate from each case were removed fromrepresentative areas on the paraffin blocks. Two marker cores were placed asymmetri-cally for orientation, and six internal control cores from known positive cases for eachof the antibodies to be tested were added to each TMA.

ImmunohistochemistryImmunohistochemical staining for CD10 (clone 56C6, Biogenex; dilution 1:50), bcl6(clone LN22, Novocastra; dilution 1:100), MUM1/IRF4 (clone EAU32, Novocastra; di-lution 1:100), CD138 (clone M115, Dako: dilution 1:60), CD68 (clone KP1, Novocastra;dilution 1:60), and EBV-LMP1 (clone CS1-4, Novocastra; dilution1:40) was carried outon the TMAs according to a standard protocol using polymer–horse radish peroxidase(HRP)-conjugated secondary antibody (Biogenex). Known positive controls were in-cluded with each run. A cut-off of 30% was used to assign positivity for all the antibod-ies. For CD68, the entire core was scanned for positively stained cells and percentagesassigned and scored: Score 1, <5%; Score 2, 5–25%; Score 3, 26–50%; and Score 4, >50%[14].

In Situ Hybridization for EBEREBV was demonstrated using the RISH EBER detection kit (Biocare) according to themanufacturers’ instructions. A known case of EBV-positive HL was used as a positivecontrol.

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Statistical analysis was carried out using the Fisher’s exact test, and there were nostatistically significant correlations between any of the parameters analyzed.

RESULTS

Of the 74 cases of pediatric cHL for which TMAs were constructed, 38 cases were ofmixed cellularity, 32 were of nodular sclerosis, two were lymphocyte-rich, and twowere lymphocyte-depleted type (Figure 1, A, B). GC markers (CD10 and bcl6) and lateGC/post-GC markers (MUM1 and CD138) were analyzed on the TMAs.

All cases showed strong nuclear positivity in more than 30% of the HRS cellsfor MUM1/IRF4. Both CD10 and CD 138 were negative in all cases. Bcl 6(>30%)was positive in 22 cases (29.7%) of which 12 cases were of mixed cellular-ity, nine were of nodular sclerosis, and one was lymphocyte rich. Bcl6 positiv-ity was equally distributed among different subtypes. Using the algorithm of Baiet al., 52/74 (70.3%) cases belonged to the late GC/early post-GC B-cell-like im-munophenotype (bcl6−/CD10−/MUM1+/CD138−) whereas 22/74 (29.7%) casesshowed an indeterminate immunophenotype (bcl6+/CD10−/MUM1+/CD138−;Figure 2). None of the cases was of the post-GC B-cell-like immunophenotype(bcl6−/CD10−/MUM1+/CD138+).

IHC for CD68All cases showed >5% CD68-positive macrophages in the tumor microenvironment.Most cases (39 cases, 52.7%) showed a macrophage population ranging from 26% to50%–Score 3. Twenty-five cases (33.8%) showed >50% positivity–Score 4, and 10 cases(13.5%) showed 5–25% macrophages–Score 2, in the microenvironment (Figure 1, G–I)There was a trend toward higher CD68 scores in EBV-positive cases. There were nocases with a macrophage percentage of <5%.

EBV-LMP1IHC with EBV-LMP1 was positive in 62/74 cases (83.8%; Figure 1D). Five cases eachof mixed cellularity and nodular sclerosis and both cases of lymphocyte-depletedHL were negative for EBV-LMP1. Of the 12 negative cases, 10 patients (83.3%) were>10 years of age.

In Situ Hybridization for EBERSixty-seven of 74 (90.5%) cases analyzed for EBER by in situ hybridization on the tissuemicroarrays showed strong nuclear positivity in HRS cells, some with nucleolar spar-ing (Figure 1C). Of the 74 cases analyzed, 37 patients (50%) were less than 10 yearsof age and 37 cases were in the age range of 10–15 years. Thirty-six of the 37 children<10 years of age were EBER positive (97.3%), whereas 31/37 children 10–15 years ofage were EBER positive (83.7%; Figure 3A). Of the seven EBER-negative cases, threewere of mixed cellularity subtype, three were of nodular sclerosis, and one was of lym-phocyte depletion. Of the 67 EBER-positive cases, 35 were of mixed cellularity, 29 wereof nodular sclerosis, two were lymphocyte-rich, and one was of lymphocyte depletion(Figure 3B). In 53 cases, the small lymphoid cells also showed EBV positivity by EBER(71.6%). Most cases showed occasional to less than 10% positive cells.

EBER and EBV-LMP1EBV-LMP1 was less sensitive than EBER, with five cases which were positive with EBERbeing negative with LMP1. However, two cases which were EBER negative were posi-tive with LMP1, giving an overall EBV positivity of 93%.

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FIGURE 3 (A) Age and EBV association in pediatric Hodgkin lymphoma; (B) shows morphologictypes and EBV association. MC, mixed cellularity; NS, nodular sclerosis; LD, lymphocyte depletion;and LR, lymphocyte rich.

DISCUSSION

The incidence of HL equals or exceeds NHL in Indian children—a pattern which dif-fers from the developed world [4, 21]. EBV is considered to be an important candidateagent for HL, particularly in developing countries [22]. In India, EBV was found in 78%of HL of all age groups and in 91% to 100% of children. This is in contrast to Westerncountries where EBV was detected in a much lower percentage (30–50%) of patients.Another important observation was that EBV-associated HL is more frequent in chil-dren younger than 10 years of age [22–26]. Childhood HL may be heterogeneous, withcases occurring in very young children being a universally EBV-associated diseasewhereas those occurring in children ages 10–15 years may be two types—one simi-lar to very young childhood HL and the other, which is EBV negative, being similar toyoung adult HL [26, 27]. Our results are similar to those of other Indian studies, with93% of childhood HL being positive for EBV by EBER in situ hybridization and LMP1.Only one of 37 children younger than 10 years was EBV negative.

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The expression of GC and post-GC markers CD 10, bcl6, MUM1/IRF4, and CD138in combination with other markers was studied in HL by various investigators. Faliniet al. reported consistent expression of MUM1/IRF4 in HL [28]; MUM1 is a marker forthe transition from bcl6 positivity (GCB cells) to CD138 expression (post-GC B cells)[29]. CD10 and bcl6 are markers of follicular center B-cell differentiation. Unlike CD10which is consistently negative, bcl6 can be positive in up to 50% of cHL; however, bcl6does not appear to be relevant to the pathogenesis of cHL, and absence of bcl6 can beinterpreted as indicative of late GC and post-GC B cells, which are largely bcl6 negative[8, 11]. In our cases of pediatric cHL, we found universal expression of MUM1(74/74)and complete absence of CD10(0/74) expression. Bcl6 was positive in 29.7% (22/74)of cases.

It has been suggested that plasma cell differentiation in HRS cells may downregu-late B-cell antigen expression. There has been wide variation in expression of CD138,a marker of plasma cell differentiation, by HRS cells in different studies, from 0% to100% [9, 30–34]. Buettner et al. reported complete absence of CD138 expression andrestriction of Blimp1, a key regulator of plasma cell differentiation, to a small propor-tion of HRS cells, suggesting that plasma cell differentiation may be initiated in a smallsubset of HRS cells but remains abortive [30]. Complete absence of CD138 expressionin HRS cells was seen in our childhood cases as well.

Three immunophenotypes were distinguished by Bai et al. on the basis of theimmunohistochemical positivity of HRS cells: (1) late GC/early post-GC B-cell-likeimmunophenotype (bcl6−/CD10−/MUM1+/CD138−) in 59/101 cases (59%);(2) post-GC B-cell-like immunophenotype (bcl6−/CD10−/MUM1+/CD138+)in 24/101 cases (24%); and (3) indeterminate immunophenotype(bcl6+/CD10−/MUM1+/CD138−) in 14 cases and (bcl6+/CD10−/MUM1+/CD138+) in four cases, 18/101 cases (18%) [12]. IHC expression of CD10, bcl6, andMUM1 in our patients was similar to that reported in other studies. However, all ourcases were CD138 negative. Thus, the majority (70.3%) belonged to the late GC/earlypost-GC B-cell-like immunophenotype, whereas 29.7% showed an indeterminateimmunophenotype (bcl6+/CD10−/MUM1+/CD138−). None of the cases was of thepost-GC B-cell-like immunophenotype.

In the setting of immunodeficiency, as exemplified by HIV-positive HL, and using apanel of bcl6/MUM1/CD138, Carbone et al. found the immunophenotype of HIV HLto be of post-GC B cells (bcl6−/MUM1+/CD138+). Moreover, they found that LMP1antigen expression among EBV-positive AIDS NHL and HIV HL associates preferen-tially with the post-GC B-cell-like immunophenotype, suggesting that this phenotypeis permissive for LMP 1 expression or that LMP1 induces post-GC maturation in theselymphomas [35]. As it has been postulated that HL in the pediatric population oc-curs in the background of a developing immune system [25], we were interested tosee if the immunophenotype in our cases of EBV-positive childhood HL was similarto that of HIV HL. However, we did not observe a post-GC immunophenotype in anyof our cases, 93% of which were EBV positive and most of which also expressed LMP1,suggesting that EBV-LMP1 expression is independent of the immunophenotype of theHRS cell. There were no HIV-positive children in our study.

Increasingly, the importance of the tumor microenvironment in the pathogenesisand prognosis of lymphomas is being recognized. In a study by Steidl et al., an increasein TAMs in the microenvironment of cHL was associated with an inferior prognosis,and a significant treatment failure was found in patients with >5% TAMs in the mi-croenvironment [14]. In a study by Kamper et al., higher levels of CD68 and CD163were seen in EBV-positive cHL and there was no correlation with disease outcome[15]. In pediatric patients, higher scores for CD68 and CD163 were not associated withpoor outcome [36]. Barros et al., showed that the prognostic role of macrophages in

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pediatric cHL is affected by the EBV status of HRS cells. EBV-positive cases showeda higher number of CD68, CD163, and CD14-positive cells with no adverse effect onoutcome, suggesting that, in pediatric cHL, the macrophage composition is numeri-cally and functionally distinct from adults and the value of macrophages as prognosticindicators may depend on EBV status [20]. This is especially relevant to pediatric cHLin developing countries, where a majority of patients are EBV positive. In our pediatricpatients, 93% of whom were EBV positive, 100% of patients had >5% of CD68-positivemacrophages, with 86% exhibiting >25% macrophages in the tumor microenviron-ment. This indicates that different criteria for risk stratification would be needed inEBV-positive pediatric cHL. Keegan et al. have shown that, in children, EBV presencein neoplastic cells was suggestively associated with favorable survival [37]. Plasma EBVDNA levels have been used to prognosticate adult cHL both at baseline and after ther-apy [38]. The possibility of using plasma EBV DNA levels as a biomarker for risk strati-fication and follow-up of pediatric EBV-positive HL needs to be investigated [39].

CONCLUSIONS

Pediatric cHL in developing countries is almost invariably EBV associated across mor-phologic subtypes, especially in children younger than 10 years of age.

Although pediatric cHL develops in the background of a developing immune sys-tem, the stage of B-cell differentiation of the HRS cell is usually of late GC/early post-GC phenotype unlike the post-GC B-cell phenotype found in HIV-HL and EBV-LMP1that is uniformly expressed, independent of immunophenotype.

In spite of a high number of TAMs in the microenvironment, probably associatedwith the presence of EBV, these children generally have a good prognosis, indicatingthat criteria other than TAM scores would be required for risk stratification.

Declaration of Interest

The authors declare they have no conflicts of interest and have not received any finan-cial assistance for the study.

REFERENCES

[1] Dinand V, Arya LS. Epidemiology of Childhood Hodgkin’s disease: is it different in developing coun-tries? Indian Pediatr. 2006;43:141–147.

[2] Dinshaw K, Pande S, Advani S, et al. Pediatric Hodgkin’s disease in India. J Clin Oncol.1985;3(12):1605–1612.

[3] Manipadam MT, Nair S, Viswabandya A, et al. Non-Hodgkin lymphoma in childhood and adoles-cence: frequency and distribution of immunomorphological types from a tertiary care center inSouth India. World J Pediatr. 2011;7(4):318–325.

[4] Arora RS, Eden TO, Kapoor G. Epidemiology of childhood cancer in India. Indian J Cancer.2009;46(4):264–273.

[5] Correa P, O’Conor GT. Epidemiologic patterns of Hodgkin’s disease. Int J Cancer. 1971;8(2):192–201.[6] Re D, Kuppers R, Diehl V. Molecular pathogenesis of Hodgkin’s lymphoma. J Clin Oncol.

2005;23(26):6379–6386.[7] Kuppers R, Schwering I, Brauninger A, et al. Biology of Hodgkin’s lymphoma. Ann Oncol. 2002;13

Suppl 1:11–18.[8] Dogan A, Bagdi E, Munson P, Isaacson PG. CD10 and BCL-6 expression in paraffin sections of normal

lymphoid tissue and B-cell lymphomas. Am J Surg Pathol. 2000;24(6):846–852.[9] Carbone A, Gloghini A, Gaidano G, et al. Expression status of BCL-6 and syndecan-1 identifies dis-

tinct histogenetic subtypes of Hodgkin’s disease. Blood. 1998;92:2220–2228.[10] Carbone A, Gloghini A, Aldinucci D, et al. Expression pattern of MUM1/IRF4 in the spectrum of

pathology of Hodgkin’s disease. Br J Haematol. 2002;117:366–372.

Pediatric Hematology and Oncology

Pedi

atr

Hem

atol

Onc

ol D

ownl

oade

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om in

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ahea

lthca

re.c

om b

y U

nive

rsity

of

Nor

th C

arol

ina

on 0

9/25

/14

For

pers

onal

use

onl

y.

Pediatric Hodgkin lymphoma

[11] Natkunam Y, Hsi ED, Aoun P, et al. Expression of the human germinal center-associated lymphoma(HGAL) protein identifies a subset of classic Hodgkin lymphoma of germinal center derivation andimproved survival. Blood. 2007;109(1):298–305.

[12] Bai M, Panoulas V, Papoudou-Bai A, et al. B-cell differentiation immunophenotypes in classicalHodgkin lymphomas. Leuk Lymphoma. 2006;47(3):495–501.

[13] Hans CP, Weisenburger DD, Greiner TC, et al. Confirmation of the molecular classificationof diffuse large B-cell lymphoma by immunohistochemistry using a tissue microarray. Blood.2004;103(1):275–282.

[14] Steidl C, Lee T, Shah SP, et al. Tumor- associated macrophages and survival in classic Hodgkin’slymphoma. N Engl J Med. 2010;362:875–885.

[15] Kamper P, Bendix K, Hamilton-Dutoit S, et al. Tumor-infiltrating macrophages correlate with ad-verse prognosis and Epstein-Barr virus status in classical Hodgkin’s lymphoma. Haematologica.2011;96(2):269–276.

[16] Yoon DH, Koh YW, Kang HJ, et al. CD68 and CD163 as prognostic factors for Korean patients withHodgkin lymphoma. Eur J Haematol. 2012;88:292–305.

[17] Azambuja D, Natkunam Y, Biasoli I, et al. Lack of association of tumor-associated macrophages withclinical outcome in patients with classical Hodgkin’s lymphoma. Ann Oncol. 2012;23(3):736–742.

[18] Arya LS, Dinand V, Thavaraj V, et al. Hodgkin’s disease in Indian children: outcome with chemother-apy alone. Pediatr Blood Cancer. 2006;46(1):26–34.

[19] Trehan A, Singla S, Marwaha RK, et al. Hodgkin lymphoma in children: experience in a tertiary carecentre in India. J Pediatr Hematol Oncol. 2013;35(3):174–179.

[20] Barros MHM, Hassan R, Niedobitek G. Tumor-associated macrophages in pediatric classicalHodgkin lymphoma: association with Epstein–Barr virus, lymphocyte subsets, and prognostic im-pact. Clin Cancer Res. 2012;18(14):3762–3771.

[21] Parkin DM, Stiller CA, Draper GJ, Bieber CA. The international incidence of childhood cancer. Int JCancer. 1988;42:511–520.

[22] Glaser SL, Lin RJ, Stewart SL, et al. Epstein-Barr virus associated Hodgkin’s disease: epidemiologiccharacteristics in international data. Int J Cancer. 1997;70:375–382.

[23] Naresh KN, Johnson J, Srinivas V, et al. Epstein-Barr virus association in classical Hodgkin’s diseaseprovides survival advantage to patients and correlates with higher expression of proliferation mark-ers in Reed-Sternberg cells. Ann Oncol. 2000;11:91–96.

[24] Dinand V, Dawar R, Arya LS, et al. Hodgkin’s lymphoma in Indian children: prevalence and sig-nificance of Epstein-Barr virus detection in Hodgkin’s and Reed Sternberg cells. Eur J Cancer.2007;43:161–168.

[25] Rao CR, Franklin J, Lalitha N, et al. Frequency of Epstein-Barr virus(EBV) association and p53 ex-pression in childhood and adult Hodgkin’s Disease in India. Int J Paed Hematol Oncol. 2000;6:377–386.

[26] Armstrong AA, Alexander FE, Pinto Paes R, et al. Association of Epstein-Barr virus with pediatricHodgkin’s disaese. Am J Pathol. 1993;142(6):1683–1688.

[27] Medeiros L J, Greiner TC. Hodgkin’s disease. Cancer. 1995;75:357–369.[28] Falini B, Fizzotti M, Pucciarini A, et al. A monoclonal antibody (MUM1p) detects expression of the

MUM1/IRF4 protein in a subset of germinal center B cells, plasma cells, and activated T cells. Blood.2000;95(6):2084–2092.

[29] Gaidano G, Carbone A. MUM1: a step ahead toward the understanding of lymphoma histogenesis.Leukemia. 2000;14:563–566.

[30] Buettner M, Greiner A, Avramidou A, et al. Evidence of abortive plasma cell differentiation inHodgkin and Reed-Sternberg cells of classical Hodgkin lymphoma. Hematol Oncol. 2005;23:127–132.

[31] Valsami S, Pappa V, Rontogianni D, et al. A clinicopathological study of B-cell differentiationmarkers and transcription factors in classical Hodgkin’s lymphoma: a potential prognostic role ofMUM1/IRF4. Haematologica. 2007;92(10):1343–1350.

[32] Tzankov A, Zimpfer A, Pehrs AC, et al. Expression of B-cell markers in classical hodgkin lymphoma:a tissue microarray analysis of 330 cases. Mod Pathol. 2003;16(11):1141–1147.

[33] Watanabe K, Yamashita Y, Nakayama A, et al. Varied B-cell immunophenotypes ofHodgkin/Reed–Sternberg cells in classic Hodgkin’s disease. Histopathology. 2000;36:353–361.

[34] Gharbaran R, Goy A, Tanaka T, et al. Fibroblast growth factor-2 (FGF2) and syndecan-1 (SDC1) arepotential biomarkers for putative circulating CD15+/CD30 +cells in poor outcome Hodgkin lym-phoma patients. J Hematol Oncol. 2013;6:62.

[35] Carbone A, Gloghini A, Larocca LM, et al. Expression profile of MUM1/IRF4, BCL-6, andCD138/syndecan-1 defines novel histogenetic subsets of human immunodeficiency virus-relatedlymphomas. Blood. 2001;97(3):744–751.

[36] Gupta S, Yeh S, Chami R, et al. The prognostic impact of tumour-associated macrophages and Reed-Sternberg cells in paediatric Hodgkin lymphoma. Eur J Cancer. 2013;49:3255–3261.

Copyright C© Informa Healthcare USA, Inc.

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[37] Keegan THM, Glaser SL, Clarke CA, et al. Epstein-Barr virus as marker of survival after Hodgkin’slymphoma: a population-based study. J clin Oncol. 2005;23:7604–7613.

[38] Kanakry JA, Li H, Gellert LL, et al. Plasma Epstein-Barr virus DNA predicts outcome in advancedHodgkin lymphoma: correlated analysis from a large North American cooperative group trial. Blood.2013;121(18):3547–3553.

[39] Sinha M, Rao CR, Shaffiulla M, et al. Cell-free Epstein-Barr viral loads in childhood Hodgkin lym-phoma: a study from South India. Pediatr Hematol Oncol. 2013;30(6):537–543.

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