The role of tumour-infiltrating eosinophils, mast cells and macrophages in Classical and Nodular Lymphocyte Predominant Hodgkin Lymphoma in children

O R I G I N A L A R T I C L E

The role of tumour-infiltrating eosinophils, mast cells and macrophages in Classical and Nodular Lymphocyte

Predominant Hodgkin Lymphoma in children

Annika Englund ¹ , Daniel Molin ² , Gunilla Enblad ² , Jonas Karl
en ³ , Ingrid Glimelius ² , Gustaf Ljungman ^1, *, Rose-Marie Amini ^4, *

1

Department of Women’s and Children

s Health, Paediatric Oncology, Uppsala University, Uppsala;

Department of Immunology, Genetics and Pathology, Experimental and Clinical Oncology, Uppsala University, Uppsala;

Department of Women’s and Children’s Health, Karolinska Institutet, Stockholm;

Department of Immunology, Genetics and Pathology, Unit of Pathology, Uppsala University, Uppsala, Sweden

Abstract

Objectives: To study Hodgkin lymphoma (HL) microenvironment in a Swedish paediatric population and its relation to clinical parameters. Methods: Tumour tissue from classical HL (cHL) (n = 87) and nodular lymphocyte predominant HL (NLPHL) (n = 11) was investigated for Epstein-Barr Virus (EBV) and analysed for eosinophils, mast cells and macrophages. Results: In cHL, EBV positivity was more common in low age (P < 0.001) and in mixed cellularity (MC) (P < 0.001). Higher mast cell infiltration was seen in stage III- IV (P < 0.001), and with presence of B-symptoms (P = 0.01). Cases with high mast cell counts displayed higher erythrocyte sedimentation rate (ESR), lower haemoglobin and albumin levels. Higher macrophage infiltration was seen in stage III-IV (P = 0.02) and there was elevated ESR and neutrophil count. All NLPHL cases were EBV negative, had lower rates of inflammatory cells and lower degree of inflammatory reaction in laboratory parameters. There was no difference in survival estimates with regard to infiltration of inflammatory cells. Conclusions: Higher levels of mast cells and macrophages in cHL tumours reflected the clinical presentation in laboratory parameters, B-symptoms and more advanced stages. NLPHL differs from cHL in numbers of inflammatory cells in the tumour, and in laboratory parameters.

Key words paediatric; Hodgkin; Microenvironment; mast cells; eosinophils; macrophages

Correspondence Annika Englund, Department of Women’s and Children’s Health, Uppsala University, Akademiska sjukhuset, 751 85 Uppsala, Sweden. Tel: +46186115858; Fax: +4618508680; e-mail: annika.englund@kbh.uu.se

*Both last authors contributed equally.

Accepted for publication 10 February 2016 doi:10.1111/ejh.12747

Hodgkin lymphoma in adults and children is in most cases a curable disease using contemporary treatment protocols.

Unfortunately, the treatment can lead to severe late side- effects, such as secondary malignancies, lung and cardiovas- cular disease, infertility and muscular atrophy (1). Thus, a remaining challenge is to find markers to identify those who will benefit from less intensive treatment and those in need of more intensive treatment. A better understanding of the microenvironment may provide additional information to support this work.

HL tumours are characterised by very few tumour cells, the Hodgkin and Reed-Sternberg cells (HRS cells) in cHL,

lymphocyte-predominant (LP) cells in NLPHL, surrounded by an intense inflammatory response. The inflammatory cells (e.g. lymphocytes, eosinophils, mast cells, macrophages, fibroblasts, plasma cells) interact with the tumour cells through different cytokine pathways. The composition of the microenvironment is probably caused by cytokine production of the tumour cells acting through autocrine and paracrine loops involving both the malignant cells and the benign inflammatory cells (2, 3).

A hallmark of HRS cells in cHL is the expression of

CD30, a receptor that is a member of the tumour necrosis

factor (TNF) receptor superfamily. CD30 can be stimulated

knowledge, not been evaluated previously. The role of macrophage infiltration has been described in cHL in adults, and associated with worse prognosis (15–26), although there are also studies reporting no prognostic signi ficance (27, 28). Only a few studies have investigated their role in chil- dren. One study (29) has described an association to shorter progression-free survival (PFS) and one has described no effect on survival (30). A third study (31) did not present prognostic data, but a large proportion of EBV positivity and high macrophage rates.

A proportion of cHL tumours express markers for EBV in the tumour cells. The expression is monoclonal, indicat- ing that the infection occurred before the malignant trans- formation (32). The tumour cells express latency membrane protein 1 and 2 (LMP1, LMP2) and EBV nuclear antigen-1 (EBNA-1). The incidence varies with age, gender, histology geographical regions and socioeco- nomic status (33). NLPHL is generally considered to be EBV negative in the tumour cells (6), but has in a few cases, both in adults and in children, been reported to stain EBV positive (34).

NLPHL has a different clinical presentation than cHL, with less B-symptoms and more cases presenting in earlier stages, and is receiving different treatment (35). NLPHL also differ with regard to the inflammatory infiltrate where LP cells are surrounded by CD4+/CD57+ T-cells in a distinct nodular pattern of many small B-cells and follicular dendritic cells where eosinophils are sparse and macrophages may be found with epitheloid appearance in the margins of the nod- ules, whereas the presence of mast cells has previously not been investigated.

The aim of this study was to investigate the EBV status and the relation of eosinophils, mast cells and macro- phages in the microenvironment to clinical parameters in a population-based paediatric Swedish cohort of cHL and NLPHL.

Materials and methods Patients

Taken from the Swedish Childhood Cancer Register, all patients 0–17 yr of age and diagnosed with HL in three

Methods

Clinical data, including laboratory parameters prior to start of treatment, were retrieved from the Swedish Childhood Cancer Register and from patient charts. EBV serology was available in 80 cases and analysed routinely at time of diag- nosis.

All biopsies were taken before initiation of treatment at primary diagnosis. The paraffin-embedded tumour tissue was sectioned in 3–5 lm sections. To determine EBV status LMP1 staining was performed in all cases (anti LMP1, Dako M0897, dilution 1 : 50, pretreatment PT Link Envision Flex Target Retrieval Solution High pH, K8000). The LMP1 neg- ative cases were then analysed with EBER (in situ hybridisa- tion for EBV-encoded small RNAs) to fully differentiate between EBV-positive and EBV-negative cases. All cases were stained with haematoxylin-eosin for eosinophils and by immunohistochemistry with a monoclonal antibody against tryptase for mast cells (G3 Chemicon International, Temec- ula, CA, USA, pretreatment Proteinase K, S3020; Dako, Glostrup, Danmark) and a monoclonal antibody against CD68 for macrophages (PG-M1 M 0896 Dako, dilution 1 : 200, pretreatment as for LMP1). Eosinophils and mast cells were counted in 10 randomly selected high power fields (HPF) in 4009 and 2009 magnification respectively.

The mast cells were analysed at a lower magnification to

cover a larger amount of the tumour and to allow compar-

ison with another study previously performed by our group

on adults (38). The ocular on the microscope was equipped

with a lattice square net and the absolute numbers of posi-

tive cells within the net area were counted. For the CD68

staining, three different regions of the tumour were counted

in 4009 and the percentage of CD68 positivity in the cyto-

plasm was scored. All cases were counted by one of the

authors (AE) with a high intra-individual reproducibility by

intra-class correlation (ICC); eosinophils 0.99 (95% confi-

dence interval (CI):0.97, 0.99), P < 0.001, mast cells 0.87

(95% CI: 0.7, 0.95), P < 0.001. For the CD68 for macro-

phages staining, 35 cases were also counted with a high

inter-individual reproducibility (ICC 0.88 95% CI: 0.77,

0.94), P < 0.001 by an experienced haematopathologist

(RMA).

Statistical methods

cHL and NLPHL were analysed separately. Eosinophils, mast cells and macrophages were analysed as continuous or cate- gorical variables. For eosinophils the cut-off points for cate- gorisation were set at 0 –9, 10–199 and ≥200, respectively, based on an earlier finding of poorer prognosis in adults in cases with ≥200 eosinophils in tissue (7). The categorisation of mast cells was based on the distribution of mast cells in the material and analysed as 0–23 vs. ≥24 mast cells per 10 HPF and 0–61 vs. ≥62 (median and upper quartile as cut-off points, respectively). Macrophages were counted as a quote of CD68-positive cells relative to overall cellularity and scored as <5%, 5–24%, 25–49% and more than 50% to allow

comparison with results from other groups. An illustration of cases with high and low numbers of each respective in filtrat- ing cells is displayed in Fig. 1. The Mann–Whitney U-test was used for non-parametrical analyses to compare continu- ous and categorical variables. Dichotomous variables were analysed with Pearson’s Chi squared-test or Fishers exact test, when applicable. Students t-test was used to compare means between groups. The Kaplan–Meier method was used for sur- vival estimates and the log rank test to compare difference between groups. The level of significance was set at 0.05 in two-tailed tests. Disease-free survival was defined as the time from diagnosis to relapse or death from HL. Patients who were disease-free at the last follow-up and patients who had

Figure 1 Images illustrating high and low

infiltration of (A) eosinophils, (B) mast cells and

(C) macrophages.

died due to reasons other than HL in CR were censored. The statistical analyses were performed using SPSS 20.0 for Windows (SPSS Inc., Chicago, IL, USA).

Results

Eighty-seven (89%) presented with cHL and 11 (11%) with NLPHL (Table 1). Boys were overrepresented among NLPHL cases (P = 0.02). Patients with NLPHL were more likely to present in earlier stages (stage I–II) than cHL (P = 0.03) and cHL patients presented with B-symptoms to a higher extent than NLPHL cases (P = 0.03). All NLPHL cases had EBV negative tumour cells. In cHL there were 12 relapses and five deaths and in NLPHL there were no relapses and all patients are still alive. Laboratory parameters are displayed in Table S1.

Classical HL

EBV: 25% (n = 22) of the cHL patients were EBV positive in the tumour cells. The proportion of EBV positivity varied significantly by age and subgroup (Fig. 2). Mean age in EBV positive cases was lower (EBV positive 10 yr, EBV negative 14 yr, P = 0.01) and MC histology was more com- mon among the youngest (0 –9 yr) and more likely to be EBV positive than NS (77% vs. 16% P < 0.001). EBV posi- tivity was somewhat more common among males when comparing all cases, although it did not reach statistical sig- nificance (P = 0.06). Thirteen patients were under 10 yr of age (one case < 5 yr), nine males and four females. Among those, eight had MC histology (seven males, one female) all of which were EBV positive. Five patients had NS histol- ogy, two were EBV positive (one male, one female) and three were EBV negative (one male, two females).

EBV positive (% of male) 15 (34) 0

Female 43 (49) 1 (9)

EBV positive (% of female) 7 (16) 0

Stage (%)

I 9 (10) 7 (64) 0.03

(I –II vs. III–IV)

II 50 (57) 4 (36)

III 17 (20) 0

IV 11 (13) 0

B-symptoms (%)

Yes 29 (33) 0 0.03 n.s.

No 58 (67) 11 (100)

Subclass (%)

NScHL 68 (69) NScHL vs. MCcHL P < 0.001

EBV positive (% of NScHL) 9 (13)

MCcHL 18 (18)

EBV positive (% of MCcHL) 13 (72)

LRcHL 1 (1)

EBV positive (% of LRcHL) 0 (0)

LDcHL –

NLPHL 11 (11)

EBV positive 0

Relapse (%)

Yes 12 (14) 0 n.s.

No 75 (86) 11 (100)

Alive (%)

Yes 82 (94) 11 (100) n.s.

No 5 (6) 0

cHL, Classical Hodgkin Lymphoma; EBV, Epstein Barr Virus; NLPHL, Nodular Lymphocyte Predominant Hodgkin Lymphoma; NScHL, Nodular

Sclerosis cHL; MCcHL, Mixed Cellularity cHL; LRcHL, Lymphocyte Rich cHL; LDcHL, Lymphocyte, Depleted cHL; n.s.=not significant.

Accordingly, the majority of male cases <10 yr were EBV- positive (8/9), although not statistically signi ficant (P = 0.2), in females two out of four. EBV serology from time of diag- nosis was available in 70 cHL patients and was positive in 50 (71%), with no significant difference in patients over or under 10 yr of age. Seventeen patients were negative for EBV in both serum and tumour.

Eosinophils: Sex, age (under or over 10 yr of age), stage, B-symptoms, EBV status and laboratory parameters did not differ significantly with regard to the number of eosinophils.

A trend towards a higher eosinophil count was observed in more advanced stages (stage III–IV, mean 137, vs. I–II, mean 70, P = 0.08) and towards higher thrombocyte count when comparing 0–199 vs. ≥200 eosinophils per 10 HPF (355 vs. 422, P = 0.07).

Mast cells: Stage and B-symptoms differed with regard to the number of mast cells in the tumours, with a higher in filtration in advanced stages (stage III–IV, mean n = 81, vs. I–II, mean n = 28, P < 0.001) and with the presence of B-symptoms (mean n = 63 vs. n = 31 in patients without B-symptoms, P = 0.01) (Fig. 3). In cases with high mast cell counts (≥62 per 10 HPF), haemoglobin levels were lower, ESR elevated and albumin levels lower (Table S1).

In cases with mast cell counts over median (≥24 per 10 HPF), ESR and CRP were elevated (P < 0.001 and

P = 0.02 respectively), and there was an increase in leuko- cyte and neutrophil count with borderline signi ficance (P = 0.05) (data not shown). No difference was observed with respect to gender, age or EBV status.

Macrophages: Ten cases (12%) had <5% macrophages, 71 cases (82%) had 5–24%, six cases (7%) had 25–49% and no cases had >50%. Higher counts of macrophages in the tumours were seen in more advanced stages (stage III–IV, mean 16%, vs. I–II, mean 10%, P = 0.02) (Fig. 4), but not in patients with B-symptoms (mean 13% in both groups).

Cases with >25% macrophages presented with higher ESR, higher C-reactive protein (CRP) and higher neutrophil counts (Table S1). No difference was observed with respect to gen- der, age or EBV status.

cHL vs. NLPHL

All NLPHL cases were EBV negative in the tumours. The inflammatory response in cHL vs. NLPHL differed. Cases with cHL presented with higher levels of infiltrating mast cells, eosinophils and macrophages (Fig. 5, Table S2).

NLPHL cases presented with higher haemoglobin and albu- min levels, lower ESR, higher thrombocyte counts and lower leukocyte counts (lower neutrophil but higher lymphocyte count), Table S2.

Figure 2 Proportion EBV positive tumours in different histological subgroups and different age groups; cHL ( *only one case <5 yr of age).

Figure 3 Mast cell distribution according to

stage (P < 0.001) and B-symptoms (P = 0.01)

respectively; cHL.

Survival analyses

We could not detect any differences in DFS or OS when comparing the above groups (gender, age (under or over 10 yr), stage, B-symptoms, rate of mast cell/eosinphil/macro- phage infiltration, cHL vs. NLPHL). However, there were few events (five deaths, 12 relapses). In cHL cases with low mast cell counts (<24 per 10 HPF) there was a trend towards fewer relapses (4/43) compared to those with ≥24 per 10 HPF (8/44), although this was not significant. When compar- ing <62 with ≥62/10 HPF there was no difference (9/65 vs.

3/22, log rank 0.9).

Discussion

In this study, we describe the variation of three types of inflammatory cells in the microenvironment in a population- based cohort of paediatric cHL and NLPHL with respect to EBV status, clinical status and laboratory parameters. Param- eters associated with more advanced disease were more common in patients with high counts of mast cells and macrophages. Survival analyses were performed and did not reveal any significant differences between the groups. How- ever, the power in this cohort with excellent treatment results is too low to draw any reliable conclusions on prog- nosis. cHL and NLPHL differ in the amount of infiltrating eosinophils, mast cells and macrophages, and in laboratory parameters, which might contribute to the different clinical behaviour and more favourable prognosis for NLPHL.

EBV status varied with age and histology, which is con- sistent with earlier findings (33, 39). Hjalgrim et al. (40) describe, in a recent study based on the Nordic registers, evidence of a distinct childhood HL entity, mainly affecting boys, occurring after a period of high incidence of mononu- cleosis among Danish boys (41). Our study supports their hypothesis that most of the childhood HL cases would be EBV-positive, especially in males.

In adults, eosinophilia has been associated with inferior prognosis. Eosinophilia in paediatric HL tumours has earlier been described as being associated with extra nodal disease, but not as affecting survival (11). In our material we could not find any association with extra nodal disease, nor with any of the other studied factors, although few patients pre- sented with extra nodal disease (n = 8). With contemporary treatment protocols the number of eosinophils is not an inde- pendent prognostic factor, despite the biological explanations for stimulating tumour growth (5).

Mast cell infiltration in paediatric HL has not, to our knowledge, been described before. We showed differences with regard to stage and B-symptoms, lower haemoglobin levels, higher ESR and lower albumin levels. The effect of high mast cell infiltration has previously been shown, by our group, to affect relapse-free survival in adults (12) and the number of mast cells were higher in the NS subgroup (38), which has also been shown by Andersen et al. (14) although the effect of high mast cell count on outcome in their mate- rial was restricted to the MC subgroup. In our material the number of relapses was higher in patients with higher mast

Figure 5 Distribution of eosinophils (P < 0.001),

mast cells (P < 0.001) and macrophages

(P < 0.001) respectively; cHL vs. NLPHL.

cell count, which possibly could re flect a stimulation of HRS cells by mast cells via CD30-CD30L interaction (4, 12), although no conclusions could be reached from these limited data. The number of mast cells was not higher in the NS subgroup in this paediatric cohort.

In adults, macrophage-rich tumours have been associated with inferior prognosis, both measured as CD68+ and CD163+ (15–26), but also with no significant difference (27, 28). Three studies have described macrophage infiltration in paediatric HL. Barros et al. (29) studied 100 cases of HL from a developing area in Brazil and in this material pro- gression-free survival (PFS) was lower in cases with high numbers of CD163+ cells, but CD68+ cases did not affect OS or PFS. Gupta et al. (30), presented a treatment-failure enriched cohort of 96 patients where no association between OS or event-free survival (EFS) could be found in macro- phages evaluated with either CD163 or CD68. High rates of HRS cells in the tumours were associated with worse EFS in univariate analysis, but did not reach significance in the multivariate analysis. Zameer et al.(31) presented a cohort from India, where they demonstrated a large proportion of EBV-positive cases (93% in total, <10 yr 97.3%, 10–15 yr 83.7%, measured with EBER) and many cases presented with high macrophage counts, detected with CD68 (86%

>25%, none <5%). Our patient cohort is, however, popula- tion-based on a Western population with an excellent treat- ment outcome and very few events. Thus, our study is not fully comparable to the others. The studies available on macrophages (children and adults) have used different tech- niques for analysing and calculating cells (manually or com- puter-assisted), some have calculated the absolute number of cells positive for each marker and some have calculated a quote with the positive cells related to the total cellularity.

Our finding with a co-variation between advanced stages and elevated ESR, factors considered to be associated with a more advanced disease, could imply that macrophages indeed affect the prognosis in paediatric HL.

In the light of this, and previously published studies on the tumour microenvironment, the cell count might have prognostic implications, but is not a strong prognostic factor.

However, this study adds to the small but growing body of literature on what the microenvironment and inflammation mean for tumour development and progression. The differ- ences in clinical presentation in children with cHL and NLPHL are of importance at the diagnostic stage because they receive different treatment, and in addition, it is impor- tant to further understand the differences in disease develop- ment between these two entities. The aetiology to HL, and the factor causing cHL or NLPHL is unknown, but our results imply that chronic inflammation has a more pro- nounced role in cHL than in NLPHL.

We are aware of the lack of power to perform prognostic analyses in this material, where the treatment protocols used provide excellent disease-free and overall survival. However,

the microenvironment in paediatric HL has only been partly described previously, and this study contains additional information contributing to the knowledge in this field. As suggested by Gupta et al., further studies on microenviron- ment and paediatric HL are needed, preferably multicenter, population-based, and with standardised calculating tech- niques.

In conclusion, EBV status varied with age, gender (bor- derline) and subclass, where MC is more common among the youngest and MC cases are more likely to be EBV posi- tive. This is consistent with earlier published findings.

Higher levels of inflammatory cells in cHL tumours reflected the clinical presentation in laboratory parameters, B-symp- toms and more advanced stages; factors associated with more advanced disease. In this cohort we found no effect on risk of relapse or death, although the number of relapses was higher in the group with many mast cells. However, the power to detect smaller differences is limited. NLPHL dif- fers from cHL, with lower numbers of inflammatory cells in the tumours, and in laboratory parameters.

Acknowledgements

Financial support was obtained from the Swedish Childhood Cancer Foundation, The Mary B
eve Foundation, The Selan- der Foundation and Lions Medical Research Foundation in the Uppsala- € Orebro region. G€oran Roos, professor and pathologist, Ume a, has re-evaluated and collected the mate- rial from Ume a, Rut-Inger Enryd, Umea and Yvonne Cope- land, Stockholm, both research nurses, have provided additional clinical information about the patients.

Potential conflicts of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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Supporting Information

Additional Supporting Information may be found in the online version of this article:

Table S1. Laboratory parameters according to cell count categories, cHL.

Table S2. Laboratory parameters and cell count; cHL vs.

NLPHL.