Tumor-associated macrophages predict inferior outcomes in classic Hodgkin lymphoma: a correlative study from the E2496 Intergroup trial

Abstract

Increased tumor-associated macrophages (TAMs) are reported to be associated with poor prognosis in classic Hodgkin lymphoma (CHL). We investigated the prognostic significance of TAMs in the E2496 Intergroup trial, a multicenter phase 3 randomized controlled trial comparing ABVD and Stanford V chemotherapy in locally extensive and advanced stage CHL. Tissue microarrays were constructed from formalin-fixed, paraffin-embedded tumor tissue and included 287 patients. Patients were randomly assigned into training (n = 143) and validation (n = 144) cohorts. Immunohistochemistry for CD68 and CD163, and in situ hybridization for EBV-encoded RNA were performed. CD68 and CD163 IHC were analyzed by computer image analysis; optimum thresholds for overall survival (OS) were determined in the training cohort and tested in the independent validation cohort. Increased CD68 and CD163 expression was significantly associated with inferior failure-free survival and OS in the validation cohort. Increased CD68 and CD163 expression was associated with increased age, EBV-encoded RNA positivity, and mixed cellularity subtype of CHL. Multivariate analysis in the validation cohort showed increased CD68 or CD163 expression to be significant independent predictors of inferior failure-free survival and OS. We demonstrate the prognostic significance of TAMs in locally extensive and advanced-stage CHL in a multicenter phase 3 randomized controlled clinical trial.

Introduction

Despite advances in the treatment of classic Hodgkin lymphoma (CHL), current therapies fail to cure 10%-15% of patients, and a similar proportion of patients may be overtreated, resulting in both short-term and long-term treatment-related complications. The International Prognostic Factors Project Score (IPS) is the current gold standard used to risk-stratify patients with advanced-stage CHL, but its power to identify patients in whom treatment is likely to fail in the modern treatment era has weakened.^1–3 Robust biomarkers are thus needed to better risk-stratify patients at diagnosis.

In CHL, the malignant Hodgkin-Reed-Sternberg (HRS) cells are greatly outnumbered by non-neoplastic cells in the tumor microenvironment, including macrophages, T cells, B cells, eosinophils, mast cells, and other stromal elements. Manipulation of the microenvironment by HRS cells through expression of a variety of cytokines and chemokines is thought to be the driving force for an abnormal immune response, perpetuated by additional factors secreted by recruited reactive cells in the microenvironment.⁴ Tumor-associated macrophages (TAMs) were shown to be associated with inferior outcomes in CHL.⁵ Steidl et al showed a macrophage gene expression signature to be associated with primary treatment failure in CHL and subsequently showed, using an independent validation cohort, that increased CD68 IHC expression was associated with inferior outcomes, including outcome after salvage treatment with autologous stem cell transplantation.⁶ Since then, most,^7–18 but not all,^12,18–20 subsequent studies have confirmed the inferior prognostic significance of TAMs in CHL using CD68 and/or CD163 IHC. In addition, early interim positron emission tomography analysis after 2 courses of chemotherapy has prognostic value in advanced-stage CHL, and increased CD68 IHC expression was recently shown to be associated with a higher rate of early positron emission tomography positivity.⁸ However, there has been variability in suggested threshold values for CD68 and CD163 IHC expression in the literature. This variability may reflect differences in IHC quantitation methodology between studies, the use of manual visual scoring techniques, and lack of subsequent validation of thresholds in their respective studies. In addition, studies thus far represent retrospective single institution experiences.

We address these current issues in our study by investigating the prognostic significance of TAMs using CD68 and CD163 IHC in the E2496 Intergroup trial, a large multicenter phase 3 randomized controlled clinical trial comparing ABVD (doxorubicin, bleomycin, vinblastine, and dacarbazine) and Stanford V (doxorubicin, vinblastine, bleomycin, vincristine, mechloroethamine, etoposide, and prednisone) chemotherapy. We use an objective method of quantitating CD68 and CD163 IHC expression with computer image analysis (Aperio Technologies) and establish optimum thresholds for CD68 and CD163 IHC expression using software X-tile (Version 3.6.1), which is based on the maximal χ² value of the log-rank test for overall survival (OS) in a training cohort. These thresholds are then tested in a separate independent validation cohort.

Methods

Patients and samples

A total of 287 patients diagnosed with CHL according to the World Health Organization 2008 classification²¹ and with tissue available were included in this study, conducted in accordance with the Declaration of Helsinki. This represents a subset of the main clinical trial based on the availability of diagnostic paraffin blocks following central pathology review and patient consent for correlative studies (supplemental Table 1, available on the Blood Web site; see the Supplemental Materials link at the top of the online article). These patients had locally extensive and advanced-stage CHL (stage 1 or 2 with bulky mediastinal disease, stage 3 and 4) enrolled in the E2496 ECOG/SWOG/NCIC/CALGB Intergroup trial, a phase 3 randomized controlled trial comparing ABVD and Stanford V chemotherapy treatment. All patients had complete data for CD68 and CD163 IHC, and in situ hybridization for EBV-encoded RNA (EBER ISH). The statistical software X-tile (Version 3.6.1)²² was used to randomly assign patients into training (n = 143) and validation (n = 144) cohorts. All participating sites received local institutional review board approval.

IHC

Tissue microarrays were constructed using duplicate 1.5-mm-diameter cores of formalin-fixed, paraffin-embedded tumor tissue. IHC performed on the tissue microarrays included CD68 (clone KP1, Dako North America; dilution 1:2000), CD163 (clone 10D6, Novocastra; dilution 1:100), and CD30 (clone BerH2, Dako North America; dilution 1:30). IHC stains were performed on a fully automated stainer (Ventana Benchmark XT) using a multimer detection kit (UltraView Universal DAB).

EBER ISH

EBER ISH was performed using the INFORM EBER probe (Ventana). Slides were also stained on an automated stainer (Ventana Benchmark XT) using the Ventana ISH/iView Blue detection kit. A known positive control was used. Nuclear staining in HRS cells was considered positive.

Immunohistochemistry scoring

CD68 and CD163 IHCs were analyzed by computer image analysis (Aperio Technologies) and pathologist scoring (visual, K.L.T.). Immunostained slides were scanned by Aperio ScanScope XT at 20× magnification. CD68 and CD163 IHCs were analyzed using the Positive Pixel Count algorithm with the Aperio ImageScope (Version 11) viewer. Every core of tissue on the TMA was checked by a pathologist (K.L.T.) to ensure that computer image analysis was performed correctly. Aperio was able to analyze tissue cores in their entirety. Only areas containing tumor were analyzed for IHC expression. Areas without tumor (eg, fibrosis, medium to large blood vessels, residual reactive lymph node) and areas with necrosis or significant artifact (eg, tissue folding and crush artifact) were deselected and excluded from analysis. Cores lacking CD30⁺ HRS cells were also excluded from analysis. For the positive pixel count algorithm, hue value of 0.1 and hue width of 0.5 were used, and any intensity of staining was considered positive. A color saturation threshold of 0.1 was used for most cores. The color saturation threshold was rarely increased to 0.15 in cases with nonspecific background staining, to minimize analysis of nonspecific background staining. The number of positive pixels was divided by the total number of pixels (negative and positive) in the analyzed area, and multiplied by 100, to derive the percentage of positive pixels. Scores from both cores of the same patient were averaged when possible (Figure 1).

Figure 1.

CD68 and CD163 IHC expression and accompanying computer image analysis (original magnification ×10). (A) CD68^low, 4.2%. (B) CD68^high, 19.5%. (C) CD163^low, 1.8%. (D) CD163^high, 26.9%.

Visual scoring was performed by estimating relative percentages of CD68⁺ and CD163⁺ cells in relation to overall cellularity in both tissue cores from the same patient where possible; scores were recorded in 10% increments. Visual and Aperio scores showed excellent correlation (supplemental Figure 1).

Statistical analysis

Failure free survival (FFS) was defined as the time from randomization to treatment arm until progression, relapse, or death from any cause. OS was defined as the date of randomization to treatment arm to death from any cause. Correlation between variables was analyzed by Pearson correlation coefficient (R). Differences in variables between groups were analyzed by Pearson χ² test, Student t test, and ANOVA. Survival estimates were calculated using the Kaplan-Meier method with differences assessed using the log-rank test. The Cox proportional hazards regression model was used for multivariate analysis. Statistical analyses were performed using SPSS software (Version 14.0). Two-sided P < .05 was considered statistically significant.

The statistical software X-tile (Version 3.6.1)²² was used to randomly assign patients into training and validation cohorts, as mentioned previously. X-tile was also used to determine the thresholds for CD68 and CD163 IHC expression, by selecting the maximal χ² values of the log-rank test for OS between 2 groups, designated as low and high risk. These thresholds were then carried forward and tested in the independent validation cohort.

Results

Patient characteristics

There were no significant differences between the subset of cases available for correlative studies (n = 287) and those not available from the total clinical trial cohort (n = 507, giving a total of 794 patients; supplemental Table 1), suggesting that these cases were representative of the entire patient population. There were also no significant differences in patient characteristics between training and validation cohorts (Table 1).

Table 1.

Comparison of patient characteristics in training and validation cohorts

	Training		Validation		P
	n	%	n	%
Total	143		144
Age (≥ 45 y)
No	118	83	122	85	.61
Yes	25	17	22	15
Sex
Female	63	44	63	44	.96
Male	80	56	81	56
Stage 4 disease
No	103	72	113	78	.21
Yes	40	28	31	22
Albumin (< 4 g/dL)*
No	45	31	42	29	.70
Yes	94	66	97	67
Unknown	4	3	5	3
Hemoglobin (< 10.5 g/dL)*
No	117	82	109	76	.15
Yes	18	13	27	19
Unknown	8	6	8	6
WBC count (≥ 15 000/mm3)*
No	114	80	112	78	.85
Yes	20	14	21	15
Unknown	9	6	11	8
Lymphocyte count (< 600/mm3 or < 8%)*
No	126	88	125	87	.81
Yes	9	6	10	7
Unknown	8	6	9	6
IPS (≥ 3)*
No	99	69	90	63	.26
Yes	44	31	53	37
Unknown	0	0	1	1
Histologic subtype*
Nodular sclerosis	115	80	108	75	.61
Mixed cellularity	18	13	20	14
Lymphocyte rich	3	2	5	3
Lymphocyte depleted	0	0	1	1
Unclassified	7	5	10	7
Treatment received
ABVD	74	52	70	49	.60
Stanford V	69	48	74	51
CD68 IHC expression
≤ 12.7% (CD68low)	89	62	89	62	.94
> 12.7% (CD68high)	54	38	55	38
CD163 IHC expression
≤ 16.8% (CD163low)	90	63	78	54	.13
> 16.8% (CD163high)	53	37	66	46
EBER ISH
Positive	25	17	24	17	.85
Negative	118	83	120	83

P values are for comparing training with validation cohorts.

^*Pearson χ² test was performed with unknown or unclassified cases excluded.

CD68 expression

Using the optimum threshold of 12.7% obtained with X-tile, 89 patients had low CD68 expression (≤ 12.7%, CD68^low) and 54 patients had high CD68 expression (> 12.7%, CD68^high) in the training cohort. Carrying this threshold forward, the validation cohort consisted of 89 CD68^low and 55 CD68^high patients. There were no significant differences in CD68 expression (P = .91) or in proportions of CD68^low and CD68^high patients between training and validation cohorts (P = .94).

In the training cohort, CD68^high patients had inferior outcomes, with the 5-year FFS rate being 50% versus 81% and 5-year OS rate being 76% versus 98%. In the validation cohort, CD68^high patients also had significantly inferior outcomes, with the 5-year FFS rate being 64% versus 78% (P = .04) and 5-year OS rate being 81% versus 94% (P < .01; Figure 2).

Figure 2.

Survival analysis based on macrophage content. (A) CD68 and (B) CD163 IHC expression and survival in training and validation cohorts.

CD163 expression

Using the optimum threshold of 16.8% obtained with X-tile, 90 patients had low CD163 expression (≤ 16.8%, CD163^low) and 53 patients had high CD163 expression (> 16.8%, CD163^high) in the training cohort. Carrying this threshold forward, the validation cohort consisted of 78 CD163^low and 66 CD163^high patients. There were no significant differences in CD163 expression (P = .48) and proportions of CD163^low and CD163^high patients between training and validation cohorts (P = .13).

In the training cohort, CD163^high patients had inferior outcomes, with the 5-year FFS rate being 56% versus 78% and the 5-year OS rate being 79% versus 96%. In the validation cohort, CD163^high patients also had significantly inferior outcomes with the 5-year FFS rate being 63% versus 82% (P < .01) and 5-year OS rate being 81% versus 96% (P < .01; Figure 2).

Correlation of increased CD68 and CD163 expression with clinical and pathologic characteristics

When considering the entire cohort, patients with increased CD68 expression (CD68^high) were significantly older (P < .01) and had increased proportions of mixed cellularity subtype of CHL (P < .01) and EBER⁺ cases (P < .01). Similarly, CD163^high patients were also significantly older (P = .04) and had increased proportions of mixed cellularity subtype of CHL (P < .01) and EBER⁺ cases (P < .01; Table 2).

Table 2.

Comparison of patient characteristics with CD68 and CD163 IHC expression in the entire cohort

	CD68low		CD68high		P	CD163low		CD163high		P
	n	%	n	%		n	%	n	%
Total	178		109			168		119
Age (≥ 45 y)
No	157	88	83	76	< .01	147	88	93	78	.04
Yes	21	12	26	24		21	13	26	22
Sex
Female	82	46	44	40	.35	76	45	50	42	.59
Male	96	54	65	60		92	55	69	58
Stage 4 disease
No	132	74	84	77	.58	129	77	87	73	.48
Yes	46	26	25	23		39	23	32	27
Albumin (< 4 g/dL)*
No	56	31	31	28	.62	45	27	42	35	.11
Yes	117	66	74	68		118	70	73	61
Unknown	5	3	4	4		5	3	4	3
Hemoglobin (< 10.5 g/dL)*
No	140	79	86	79	.97	136	81	90	76	.39
Yes	28	16	17	16		24	14	21	18
Unknown	10	6	6	6		8	5	8	7
WBC count (≥ 15 000/mm3)*
No	135	76	91	83	.10	129	77	97	82	.18
Yes	30	17	11	10		28	17	13	11
Unknown	13	7	7	6		11	7	9	8
Lymphocyte count (< 600/mm3 or < 8%)*
No	157	88	94	86	.19	148	88	103	87	.32
Yes	9	5	10	9		9	5	10	8
Unknown	12	7	5	5		11	7	6	5
IPS (≥ 3)*
No	120	67	69	63	.54	114	68	75	63	.45
Yes	58	33	39	36		54	32	43	36
Unknown	0	0	1	1		0	0	1	1
Histologic subtype*
Nodular sclerosis	152	85	71	65	< .01	141	84	82	69	< .01
Mixed cellularity	11	6	27	25		13	8	25	21
Lymphocyte rich	5	3	3	3		6	4	2	2
Lymphocyte depleted	0	0	1	1		0	0	1	1
Unclassified	10	6	7	6		8	5	9	8
Treatment received
ABVD	88	49	56	51	.75	82	49	62	52	.58
Stanford V	90	51	53	49		86	51	57	48
EBER ISH
Positive	15	8	34	31	< .01	20	12	29	24	< .01
Negative	163	92	75	69		148	88	90	76

P values compare CD68^low with CD68^high, and CD163^low with CD163^high patients.

^*Pearson χ² test was performed with unknown or unclassified cases excluded.

Both CD68^high and CD163^high were significantly associated with inferior outcomes in patients treated with either ABVD (CD68: FFS, P < .01; OS, P < .01; CD163: FFS, P = .03; OS, P = .04) or Stanford V chemotherapy (CD68: FFS, P < .01; OS, P = .02; CD163: FFS, P < .01; OS, P < .01; supplemental Figure 2).

EBER⁺ cases showed significantly higher CD68 and CD163 expression than EBER⁻ cases (P < .01; Table 3). In addition, EBER⁺ patients were significantly older (P = .01), more often male (P = .02), with lower white blood cell counts (P = .02), IPS ≥ 3 (P = .04), and increased proportions of mixed cellularity subtype of CHL (P < .01), CD68^high (P < .01), and CD163^high (P < .01; supplemental Table 2). It was thus not surprising to observe significantly higher CD68 and CD163 expression in mixed cellularity subtype of CHL, compared with nodular sclerosis and lymphocyte rich subtypes (P < .01; Table 3).

Table 3.

Comparison of CD68 and CD163 IHC expression in training and validation cohorts, in EBER⁺ and EBER⁻ cases, and selected subtypes of CHL in the entire cohort

	n	CD68 expression			CD163 expression
		Median	Range	P	Median	Range	P
Training	143	10.6	2.7-57.4	.91	13.0	0.4-79.5	.48
Validation	144	10.5	2.4-51.8		13.5	0.4-79.1
EBER+	49	15.7	3.1-57.4	< .01	29.9	1.5-79.5	< .01
EBER−	238	9.6	2.4-37.2		11.1	0.4-79.1
Nodular sclerosis	223	9.6	2.7-51.8	< .01	10.9	0.4-79.1	< .01
Mixed cellularity	38	16.1	2.8-57.4		28.9	0.4-79.5
Lymphocyte rich	8	7.1	2.8-26.7		8.9	1.4-41.4

No significant differences in outcome were seen between EBER⁺ and EBER⁻ patients (FFS, P = .66; OS, P = .44). However, CD163^high was significantly associated with inferior outcomes in both EBER⁺ (FFS, P < .01; OS, P = .02) and EBER⁻ (FFS, P = .01; OS, P < .01) patients. CD68^high was significantly associated with inferior outcomes in EBER⁻ cases (FFS, P < .01; OS, P < .01) but not EBER⁺ cases (FFS, P = .34; OS, P = .33; supplemental Figure 3).

Increased CD68 or CD163 expression is a significant independent predictor of inferior outcome

Univariate and multivariate analyses were performed on the validation cohort. On univariate analysis, stage 4 disease, low lymphocyte count, and increased CD68 and CD163 expression were significantly associated with inferior FFS. Increased age and increased CD68 and CD163 expression were significantly associated with inferior OS (Table 4).

Table 4.

Univariate analysis in the validation cohort

Factor	5-y FFS, %	P	5-y OS, %	P
Age (≥ 45 y)
No	74	.49	91	< .01
Yes	69		77
Sex
Female	77	.39	90	.95
Male	70		88
Stage 4 disease
No	77	.01	91	.12
Yes	57		84
Albumin (< 4 g/dL)
No	74	.96	87	.9
Yes	73		89
Hemoglobin (< 10.5 g/dL)
No	77	.17	91	.08
Yes	58		76
WBC count (≥ 15 000/mm3)
No	73	.85	89	.67
Yes	76		86
Lymphocyte count (< 600/mm3 or < 8%)
No	77	.02	89	.48
Yes	25		78
IPS (≥ 3)
No	74	.65	91	.34
Yes	70		86
CD68
≤ 12.7% (CD68low)	78	.04	94	< .01
> 12.7% (CD68high)	64		81
CD163
≤ 16.8% (CD163low)	82	< .01	96	< .01
> 16.8% (CD163high)	63		81
EBER
Negative	75	.24	90	.43
Positive	62		83

To determine whether CD68 or CD163 was independently associated with outcomes, respectively, 2 separate multivariate analyses were performed, including the factors significantly associated with FFS or OS in univariate analysis. These analyses demonstrated that increased CD68 or CD163 expression was a significant independent predictor of inferior FFS and OS (Table 5).

Table 5.

Multivariate analyses in the validation cohort

Factor	HR	95% CI	P
FFS
Lymphocyte count (< 600/mm3 or < 8%)	2.1	0.8-5.8	.14
Stage 4 disease	2.2	1.0-4.7	.04
CD68high	2.1	1.1-4.2	.04
Lymphocyte count (< 600/mm3 or < 8%)	2.1	0.8-5.9	.14
Stage 4 disease	1.8	0.8-3.9	.13
CD163high	2.5	1.2-5.3	.02
OS
Age (≥ 45 y)	2.5	0.9-7.1	.08
CD68high	3.5	1.2-10.2	.02
Age (≥ 45 y)	3.4	1.3-9.2	.02
CD163high	3.9	1.3-11.9	.02

HR indicates hazard ratio; and CI, confidence interval.

Discussion

We confirm the prognostic significance of TAMs in CHL in the E2496 ECOG/SWOG/NCIC/CALGB Intergroup trial, a multicenter phase 3 randomized controlled trial comparing ABVD and Stanford V chemotherapy. Increased CD68 and CD163 IHC expression was significantly associated with inferior outcomes in locally extensive and advanced-stage CHL. Multivariate analysis in the validation cohort showed CD68 or CD163 expression to be significant independent predictors of FFS and OS. All previous studies on TAMs and outcome in CHL have been based on retrospective single institution experiences. This work represents the first confirmation of the prognostic role of TAMs in CHL in a multicenter randomized controlled clinical trial setting.

Although most studies in the literature demonstrate an association between increased TAMs and inferior outcome in CHL, there has been variability in suggested threshold values for CD68 and/or CD163 IHC expression. This probably relates to differences in IHC quantitation methodology, with most using manual visual scoring techniques, and a lack of subsequent validation of thresholds in these studies. The lack of reproducibility and inconsistency of manual or visual IHC scoring has been identified as a potential pitfall regarding the use of IHC biomarkers in routine clinical practice.⁵ Azambuja et al showed poor interobserver agreement for low scores with CD68 IHC.²⁰ These issues are potentially overcome through use of computer image analysis to produce greater objectivity in scoring. Kamper et al used computer-assisted stereologic analysis and point grid counting methodology in assessing CD68 and CD163 IHC, and showed increased CD68 and CD163 IHC expression to be associated with inferior outcome in CHL.¹⁶ However, they reported this method to be labor intensive.¹⁶ We used Aperio Technologies for computer image analysis to assess CD68 and CD163 IHC. In addition, we attempted to produce robust thresholds for CD68 and CD163 IHC expression by developing optimum thresholds based on the maximal χ² values of the log-rank test for OS in a training cohort, and then testing these thresholds in a separate independent validation cohort. Aperio was able to analyze tissue cores in their entirety, with scores averaged from both cores of the same patient to provide a more representative score for each case, within the limitations of a tissue microarray. In addition, Aperio is a user friendly system and shows potential for application on whole tissue sections. Compared with CD68, CD163 appeared to show an overall crisper and stronger intensity of staining and a cleaner background in our hands, making it more ideal for analysis by Aperio's Positive Pixel Count algorithm. This experience with the quality of CD163 IHC is in agreement with other investigators.^19,20 In addition, the KP1 clone for CD68 has been reported to be a less specific marker for macrophages, as it is also known to react with myeloid and fibroblastic cells,²³ whereas clone 10D6 for CD163 has been reported to be more specific than both KP1 and PGM1 clones for CD68 in identifying macrophages.²⁴ For these reasons, CD163 may be a better marker for identifying TAMs than CD68.

Expression of CD163 is restricted to cells of the monocyte/macrophage lineage and is reported to be a more specific marker for alternatively activated (M2) macrophages in the M1/M2 macrophage polarization model.^24–27 We showed a correlation between CD68 and CD163 expression, suggesting that TAMs in CHL show features described for M2 macrophages, including promoting tumor growth and angiogenesis, tissue remodeling, and suppression of adaptive immune responses, contributing to immune evasion by tumor cells, and thus associated with poor prognosis.^28–32 However, the macrophage polarization model based on in vitro experiments is probably an oversimplification of macrophages in vivo, which probably display a spectrum of activated phenotypes and with the ability to switch from one functional phenotype to another in response to varied local microenvironmental signals.^30,33,34

We also showed increased CD68 and CD163 expression to be associated with increased age, EBER positivity, and mixed cellularity subtype of CHL.^16,20,35 Indeed, a gene expression profiling study showed overexpression of genes associated with either histiocytes or T cells, including CD68 and CD163, in EBV⁺ CHL compared with EBV⁻ CHL.³⁶ Despite an association with increased CD68 and CD163 expression in EBER⁺ cases, no survival differences were seen with regards to EBER status in our study. However, TAMs remained predictive of inferior outcomes in both EBER⁺ and EBER⁻ cases, suggesting that there are probably other mechanisms responsible for the recruitment and the poor prognostic impact of increased TAMs in the CHL microenvironment. CD163 may be a better marker than CD68 in predicting inferior outcomes in EBER⁺ cases; however, numbers of EBER⁺ patients in our study were small, and this requires further study. The precise biologic mechanisms underlying TAMs and the relationship between TAMs with EBV and tumor cells are currently not well understood, and further functional studies are required.

In conclusion, we confirm the prognostic significance of TAMs using CD68 and CD163 IHC in CHL in the E2496 Intergroup trial, a multicenter phase 3 randomized controlled clinical trial comparing ABVD and Stanford V chemotherapy. We demonstrate an objective method of quantitating CD68 and CD163 IHC expression using computer image analysis (Aperio Technologies) and established robust thresholds for CD68 and CD163 IHC expression that is trained and validated in independent cohorts. Our findings, in conjunction with other previous studies, firmly establish TAMs to be important in the CHL tumor microenvironment. Further functional studies are required to determine the precise biologic mechanisms associated with increased numbers of TAMs in the tumor microenvironment and the relationship with EBV in CHL. Evaluation of TAMs should be considered in prospective clinical trials, and patients with increased TAMs may benefit from more intensive chemotherapy or novel agents designed to disrupt the crosstalk between HRS cells and benign macrophages.

Authorship

Contribution: K.L.T. performed the research, analyzed data, and prepared the manuscript; D.W.S. analyzed data; F.H., B.S.K., R.I.F., N.L.B., R.H.A., R.B., L.M.R., J.M.C., C.S., L.I.G., and S.J.H. contributed data and materials; and R.D.G. designed the research, analyzed data, and prepared the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Randy D. Gascoyne, British Columbia Cancer Agency & British Columbia Cancer Research Centre, 675 W 10th Ave, Room 5-113, Vancouver, BC, V5Z 1L3, Canada; e-mail: rgascoyn@bccancer.bc.ca.