Tumor-associated macrophages and survival in classic Hodgkin's lymphoma

Abstract

Background: Despite advances in treatments for Hodgkin's lymphoma, about 20% of patients still die from progressive disease. Current prognostic models predict the outcome of treatment with imperfect accuracy, and clinically relevant biomarkers have not been established to improve on the International Prognostic Score.

Methods: Using gene-expression profiling, we analyzed 130 frozen samples obtained from patients with classic Hodgkin's lymphoma during diagnostic lymph-node biopsy to determine which cellular signatures were correlated with treatment outcome. We confirmed our findings in an independent cohort of 166 patients, using immunohistochemical analysis.

Results: Gene-expression profiling identified a gene signature of tumor-associated macrophages that was significantly associated with primary treatment failure (P=0.02). In an independent cohort of patients, we found that an increased number of CD68+ macrophages was correlated with a shortened progression-free survival (P=0.03) and with an increased likelihood of relapse after autologous hematopoietic stem-cell transplantation (P=0.008), resulting in shortened disease-specific survival (P=0.003). In multivariate analysis, this adverse prognostic factor outperformed the International Prognostic Score for disease-specific survival (P=0.003 vs. P=0.03). The absence of an elevated number of CD68+ cells in patients with limited-stage disease defined a subgroup of patients with a long-term disease-specific survival of 100% with the use of current treatment strategies.

Conclusions: An increased number of tumor-associated macrophages was strongly associated with shortened survival in patients with classic Hodgkin's lymphoma and provides a new biomarker for risk stratification.

Figure 1. Hierarchical Clustering of Gene-Expression Profiles in Hodgkin's Lymphoma, True and False Positive and Negative Rates for Three Models of Outcome Prediction, and the Importance of Individual Genes for Outcome Prediction

Panel A shows hierarchical clustering of 130 gene-expression profiles for patients with classic Hodgkin's lymphoma. Cluster A has been enriched with primary treatment successes, and Cluster B with both primary treatment successes and failures. Immediately below the cluster bars, the first multicolored bar indicates sex (red for male and black for female), the second bar indicates stage (yellow for limited disease and gray for advanced disease), the third bar indicates the type of treatment failure (green for no treatment failure, purple for refractory, dark blue for early relapse, and light blue for late relapse), and the fourth bar indicates the primary treatment outcome (black for failure and red for success). (For details, see Tables S2 and S3 in the Supplementary Appendix, available with the full text of this article at NEJM.org.) Panel B shows plots of true positive and false positive rates and true negative and false negative rates (receiver-operating-characteristic curves) for three models that were used for feature selection: gene-expression profiling (GEP), a model based on the International Prognostic Score (IPS) for clinical variables, and a model combining these two features. This comparison showed that the value for the area under the curve was highest for the GEP model, as compared with the clinical and combined models (0.837 vs. 0.625 and 0.821, respectively). For comparison with the established IPS, red asterisks indicate an IPS of more than 3, as calculated with the use of IPS thresholds. Panel C shows the relative importance of individual genes for outcome prediction. Relative importance is shown for 30 annotated probe sets (selected with the use of sparse multinomial logistic regression) that were more influential than Ann Arbor staging. Among the 27 individual genes exceeding the importance of the best clinical variable, age (shown in red), was MMP11.

Figure 2. Representative Immunohistochemical Analyses for CD68 in Lymph-Node Biopsy Samples from Two Patients

Panel A shows a sample obtained from a patient in the treatment-success group, with few CD68+ macrophages, and Panel B shows a sample from a patient in the treatment-failure group, with many CD68+ macrophages. Both patients had the nodular sclerosis subtype of Hodgkin's lymphoma.

Figure 3. Survival in a Validation Cohort of 166 Patients, According to the Number of Infiltrating CD68+ Macrophages in Pretreatment Lymph-Node Biopsy Specimens

The graphs show progression-free survival in all patients (Panel A) and disease-specific survival in all patients (Panel B) and in 41 patients with limited-stage disease (Panel C). According to the immunohistochemical scoring system that was used, a score of 1 indicates less than 5% CD68+ cells, a score of 2 indicates 5 to 25%, and a score of 3 indicates more than 25%. Clinically relevant biomarkers for predicting the outcome of treatment in patients with Hodgkin's disease have not been established. In this study, gene profiling and immunohistochemical analysis were used to find such a marker. A strong association was found between a poor outcome of treatment and an increased number of CD68+ cells in the microenvironment of Reed–Sternberg cells. CD68, a marker of macrophages, outperformed the conventional International Prognostic Score and can be immunohistochemically stained in diagnostic samples of Hodgkin's lymphoma.