Expression of FOXP3, CD68, and CD20 at diagnosis in the microenvironment of classical Hodgkin lymphoma is predictive of outcome

Abstract

Purpose: The immune microenvironment is key to the pathophysiology of classical Hodgkin lymphoma (CHL). Twenty percent of patients experience failure of their initial treatment, and others receive excessively toxic treatment. Prognostic scores and biomarkers have yet to influence outcomes significantly. Previous biomarker studies have been limited by the extent of tissue analyzed, statistical inconsistencies, and failure to validate findings. We aimed to overcome these limitations by validating recently identified microenvironment biomarkers (CD68, FOXP3, and CD20) in a new patient cohort with a greater extent of tissue and by using rigorous statistical methodology.

Patients and methods: Diagnostic tissue from 122 patients with CHL was microarrayed and stained, and positive cells were counted across 10 to 20 high-powered fields per patient by using an automated system. Two statistical analyses were performed: a categorical analysis with test/validation set-defined cut points and Kaplan-Meier estimated outcome measures of 5-year overall survival (OS), disease-specific survival (DSS), and freedom from first-line treatment failure (FFTF) and an independent multivariate analysis of absolute uncategorized counts.

Results: Increased CD20 expression confers superior OS. Increased FOXP3 expression confers superior OS, and increased CD68 confers inferior FFTF and OS. FOXP3 varies independently of CD68 expression and retains significance when analyzed as a continuous variable in multivariate analysis. A simple score combining FOXP3 and CD68 discriminates three groups: FFTF 93%, 62%, and 47% (P < .001), DSS 93%, 82%, and 63% (P = .03), and OS 93%, 82%, and 59% (P = .002).

Conclusion: We have independently validated CD68, FOXP3, and CD20 as prognostic biomarkers in CHL, and we demonstrate, to the best of our knowledge for the first time, that combining FOXP3 and CD68 may further improve prognostic stratification.

Fig 1.

CD68 expression and outcome. Representative examples of (A) low and (B) high expression of CD68 in classical Hodgkin lymphoma (magnification ×20). CD68 expression is shown by horseradish peroxidase–diaminobenzidine (HRP-DAB) immunostaining. Time to (C) first-line treatment failure and (D) overall survival (OS) of patients based on area of low (< 5%), intermediate (INT; 5% to 15%) or high (> 15%) CD68 expression. FFTF, freedom from first-line treatment failure.

Fig 2.

FOXP3 expression and outcome. Representative examples of (A) low and (B) high FOXP3 expression in classical Hodgkin lymphoma (magnification ×20). FOXP3 expression is shown by horseradish peroxidase–diaminobenzidine (HRP-DAB) immunostaining. Time to (C) first-line treatment failure and (D) overall survival (OS) of patients on the basis of low (< 125/hpf), intermediate (125-500/hpf) or high (> 500 hpf) numbers of FOXP3 expressing cells. FFTF, freedom from first-line treatment failure.

Fig 3.

CD20 expression and outcome. Representative examples of (A) low and (B) high nonfollicular expression of CD20 in classical Hodgkin lymphoma (magnification ×20). CD20 expression is shown by horseradish peroxidase–diaminobenzidine (HRP-DAB) immunostaining. (C) Overall survival of patients based on low (< 250 cells/hpf [high-powered field]) or high (≥ 250 cells/hpf) CD20-expressing cells.

Fig 4.

Kaplan-Meier survival analysis stratifying by a combined score incorporating both FOXP3 and CD68 expression (FOX/Mac). Derivation of FOX/Mac is shown in Appendix Figure A2 (online only) using cut points for FOXP3 expression shown in Figure 2 and CD68 shown in Figure 1. (A) Time to first-line treatment failure on the basis of FOX/Mac score for entire cohort. (B) Overall survival (OS) on the basis of FOX/Mac score for entire cohort. (C) Time to first-line treatment failure on the basis of FOX/Mac score for patients with early-stage disease only. (D) Time to first-line treatment failure for patients with advanced-stage disease only. FFTF, freedom from first-line treatment failure.

Fig A1.

Variance of expression of FOXP3 and CD68. Linear regression analysis of percentage of expression of CD68 and number of cells expressing FOXP3 demonstrating no significant correlation (P = .684; F = 0.1666).

Fig A2.

Derivation of combined FOXP3/Mac score based on score for each marker showing (A) distribution of patients with cut points indicated by solid lines and sectors defined by (B) these cut points translated to risk groups (P = poor risk, Int = intermediate risk, G = good risk).