Abstract 5580: Macrophage polarization and acquired resistance to rituximab in CNS lymphoma
Authors: Kadoch Cigall , Wong Valerie S. , Rambaldo Tara C. , Hyun William C. , Gao Hua Xin , Lowell Clifford A. , Rubenstein James L. 
Published: February 2011

Abstract
Macrophages are a critical component of anti-tumor immunity but may be subverted from an M1 phenotype, which mediates tumor elimination, to an M2 phenotype, which promotes tumor progression. IL-4 signaling is a pivotal regulator of macrophage polarization to the M2 phenotype; we have recently demonstrated the intratumoral expression of IL-4 in CNS lymphoma (Blood, 2006; CCR, 2009).
Rituximab efficacy appears to be mediated predominantly by ADCC, with tumor-associated macrophages being the dominant effector cell. We hypothesize that the phenotype of tumor-associated macrophages (M1 vs. M2) may be a determinant of rituximab efficacy and resistance.
There is an extreme paucity of information regarding the role and phenotype of macrophages in non-Hodgkin lymphoma (NHL), including CNS lymphoma. We have developed a novel flow-cytometry-based approach for the isolation and characterization of macrophages from the cerebrospinal fluid (CSF) of patients with CNS NHL. Our gene expression studies of CD14+ activated CSF macrophages suggested two candidate markers of M2 differentiation, CD206 and Factor XIII. M1 differentiation was identified using DAF-FM diacetate, a cell-permeable marker of nitric oxide synthase (iNOS). Our results suggest the presence of at least four distinct subpopulations of activated macrophages in CSF based upon the relative expression of these markers.
CSF macrophage subpopulations were characterized and sorted from 42 subjects: 22 immunocompetent patients with CNS NHL and 20 control subjects with non-neoplastic conditions. Our data suggest an association of M2 macrophages in the CSF with the pathogenesis of CNS NHL: a greater than six-fold increase in the proportion of macrophages with M2 features was detected in CSF from CNS NHL subjects compared to controls (p&lt;0.001). By contrast, the proportion of macrophages with M1 features was similar between NHL and controls. Intraventricular administration of rituximab was reproducibly associated with a greater than three-fold increase in the relative ratio of M2:M1 macrophages in the CSF of patients with recurrent CNS NHL (p&lt;0.001), usually within two weeks of administration. By contrast, intra-CSF administration of methotrexate (MTX) was associated with an increased ratio of M1:M2 macrophages (within 96h). In each of five patients participating in a Phase I clinical trial involving intrathecal rituximab plus MTX, increased M2 macrophage polarization anticipated the onset of rituximab resistance and tumor progression.
We believe this to be the first application of flow-cytometry to define the phenotypes of intratumoral macrophages in NHL as well as the first description of dynamic changes in macrophage phenotypes during the evolution of resistance to rituximab therapy. The elucidation of distinct macrophage subpopulations based upon the expression of candidate markers of M1 vs. M2 phenotypes may provide insight into tumor pathogenesis and prognosis.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5580.