There is an unmet need for effective biological therapies for relapsed central nervous system (CNS) lymphoma. Lenalidomide is active in activated B-cell type diffuse large B-cell lymphoma and rituximab is effective in CNS lymphoma. These observations are the basis for this first trial of an immunomodulatory drug as monotherapy in CNS lymphoma, and, in patients with inadequate responses to lenalidomide, with rituximab. In an independent cohort, we evaluated lenalidomide maintenance after salvage with high-dose methotrexate or focal irradiation in relapsed primary CNS lymphoma (PCNSL). We determined safety, efficacy, and cerebrospinal fluid (CSF) penetration of lenalidomide at 10-, 15-, and 20-mg dose levels in 14 patients with refractory CD20 CNS lymphoma. Nine subjects with relapsed, refractory CNS lymphoma achieved better than partial response with lenalidomide monotherapy, 6 maintained response ≥9 months, and 4 maintained response ≥18 months. Median progression-free survival for lenalidomide/rituximab was 6 months. In the independent cohort, response duration with lenalidomide maintenance after complete responses 2 through 5 were significantly longer than response durations after standard therapy. The CSF/plasma partition coefficient of lenalidomide was ≥20% at 15- and 20-mg dose levels. Change in CSF interleukin-10 at 1 month correlated with clinical response and response duration to lenalidomide. Metabolomic profiling of CSF identified novel biomarkers, including lactate, and implicated indoleamine-2,3 dioxygenase activity with CNS lymphoma progression on lenalidomide. We conclude that lenalidomide penetrates ventricular CSF and is active as monotherapy in relapsed CNS lymphomas. We provide evidence that maintenance lenalidomide potentiates response duration after salvage in relapsed PCNSL and delays whole brain radiotherapy (WBRT). This trial was registered at www.clinicaltrials.gov as #NCT01542918.

Background The mechanistic basis for neurocognitive deficits in CNS lymphoma and other brain tumors is incompletely understood. We tested the hypothesis that tumor metabolism impairs neurotransmitter pathways and neurocognitive function. Methods We performed serial cerebrospinal fluid (CSF) metabolomic analyses using liquid chromatography-electrospray tandem mass spectrometry to evaluate changes in the tumor microenvironment in 14 patients with recurrent CNS lymphoma, focusing on 18 metabolites involved in neurotransmission and bioenergetics. These were paired with serial mini-mental state examinations (MMSE) and MRI studies for tumor volumetric analyses. Patients were analyzed in the setting of the phase I trial of lenalidomide/rituximab. Associations were assessed by Pearson and Spearman correlation coefficient. Generalized estimating equation (gee) models were also established, adjusting for within-subject repeated measures. Results Of 18 metabolites, elevated CSF lactate correlated most strongly with lower MMSE score (p<8E-8, rho=-0.67). High lactate was associated with lower GABA, higher glutamate/GABA ratio and dopamine. Conversely, high succinate correlated with higher MMSE score. Serial analysis demonstrated a reproducible, time-dependent, reciprocal correlation between changes in lactate and GABA concentrations. While high lactate and low GABA correlated with tumor contrast enhancing volume, they correlated more significantly with lower MMSE scores than tumor volumes. Conclusions We provide evidence that lactate production and Warburg metabolism may impact neurotransmitter dysregulation and neurocognition in CNS lymphomas. We identify novel metabolomic biomarkers that may be applied in future studies of neurocognition in CNS lymphomas. Elucidation of mechanistic interactions between lymphoma metabolism, neurotransmitter imbalance and neurocognition may promote interventions that preserve cognitive function.

Background: The etiologic basis for neurocognitive and neuropsychological deficits in cancer patients and cancer survivors is poorly understood. Because of ongoing improvements in therapy and advances in survival, the problem of cancer-associated cognitive dysfunction is increasingly significant. While there is agreement that T2-weighted imaging abnormalities on MRI correlate with neurocognitive deficits, there has been limited insight into the neurochemical abnormalities associated with cognitive dysfunction in brain tumor patients. We are testing the hypothesis that tumor metabolism directly impairs neurotransmitter pathways and cognitive function, independent of anatomic extent of the cancer. Methods: Our approach has been to focus on a type of brain tumor, CNS lymphoma, in which we are able to simultaneously monitor cognitive function, as assayed by repeat Mini-Mental Status Examinations (MMSE), tumor volume, as assessed by MRI, and dynamic changes in the tumor metabolic microenvironment, as characterized by quantitative measurement of tumor-associated metabolites in correlation with neurotransmitters that we hypothesize to be linked to normal cognitive function. Our initial study was to evaluate 14 subjects with CNS lymphoma that were treated on a phase I trial of the immunomodulatory agent lenalidomide. Volumetric analysis of CNS lymphomas was performed using Smartbrush Software (Brainlab) on pre-and post-therapy MRI's conducted at baseline and at monthly restaging. Metabolomic analysis of cerebrospinal fluid (CSF), using GC/MS, was conducted at baseline and at timepoints within 1 week of corresponding MRI. MMSE tests were conducted in all subjects at baseline and at corresponding monthly restaging examinations. Results: Of 20 CSF metabolites analyzed, including 12 neurotransmitters, elevated CSF lactate correlated most strongly with impaired neurocognitive function as measured by MMSE score. (P=2.5e-6; rho= -0.66). Patients with high lactate had lower relative CSF concentration of the inhibitory neurotransmitter GABA, and higher concentrations of the excitotoxic glutamate. Notably, we determined that CSF lactate concentration more significantly correlated with lower MMSE score than size of the brain tumor, as quantified by volumetric analysis of tumor T2 hyperintensity and lesional contrast-enhancing volume. Conclusions: To our knowledge, this is the first data linking cancer metabolism, neurotransmitter dysregulation, and neurocognitive deficits in a brain tumor patient population. We anticipate that elucidation of the mechanistic basis between tumor lactate metabolism, neurotransmitter imbalance, and neurocognitive deficits will provide potential opportunities for pharmacologic intervention to preserve neurologic function and potentially minimize cognitive and neuropsychological deficits in cancer patients. Citation Format: Lakshmi Subbaraj, Huimin Geng, Jigyasa Sharma, Marisa LaFontaine, James L. Rubenstein. Tumor metabolism and cognitive dysfunction in CNS lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4975.

;'Insights into the molecular and immunologic pathogenesis of primary CNS lymphomas are essential for meaningful progress in therapy. Tumor-associated macrophages represent the dominant infiltrating leukocyte and there are few established insights into their phenotypes and role in this disease. While upregulation of Th2 cytokines IL-4 and IL-10 in the microenvironment has been demonstrated, the relative roles of M1 and M2 macrophages in contributing to CNS lymphoma pathogenesis has not been elucidated. To date, there is also no information regarding the relative contributions of brain resident microglia and infiltrating macrophages and their interactions with lymphoma. Additional key questions include the identification of factors that mediate both immune cell chemotaxis in CNS lymphomas, as well as the relationship between myeloid cell infiltration and T-cell mediated immune surveillance and immunosuppression. We combined analyses of clinical specimens and mechanistic studies using preclinical in vivo models and show evidence that infiltrating tumor-associated macrophages, derived from monocyte precursors, have a critical role in attenuating CNS lymphoma progression. Immunohistochemical analysis of the density and morphologic features of CD68+ tumor-associated macrophages in 62 diagnostic specimens of immunocompetent PCNSL demonstrated that smaller macrophage size and lower macrophage density correlated with significantly shorter OS. Evaluation of CD68 immunoreactivity using image analysis software (ImageJ) confirmed the heterogeneity of macrophage size and infiltrative density in PCNSL. A multivariate Cox model including age, IELSG score, receipt of consolidation and/or maintenance therapy demonstrated that tumor-associated macrophage density (both count and area) was a significant, independent predictor of favorable PFS and OS and that larger macrophage size a significant, independent predictor of OS in PCNSL treated with standard MTX-based induction (predominantly MTX, temozolomide, rituximab). Using a variety of syngeneic and non-syngeneic preclinical models, including patient-derived CNS lymphoma cells, as well as diagnostic clinical specimens, we characterized the phenotype of tumor-associated macrophages in PCNSL. Using flow-cytometry, we demonstrated that while CD45 high tumor-associated macrophages exhibit strong expression of the canonical M2 marker CD206, a scavenger receptor, these also displayed high co-expression of iNOS and MHC II, markers of classically-activated M1 macrophages. Pharmacologic inhibition of the CSF-1 receptor led to accelerated CNS lymphoma progression, attenuated T-cell infiltration and blocked rituximab efficacy. A flow-cytometric assay of phagocytosis, using Raji lymphoma transduced to express mCherry, demonstrated that infiltrating CD206+ macrophages are the dominant mediator of lymphoma phagocytosis. We applied 2P intravital imaging of a CNS lymphoma model using Cx3cr1GFP/+:Ccr2RFP/+ myeloid cell dual reporter mice and transcriptional studies to define the time-dependent infiltration and phenotypic changes in tumor-associated macrophages and microglia that correlate with disease progression. Using IFN-γ -/- mice we identified a critical role for IFN-γ in the regulation of CNS lymphoma, in the presence and absence of T-cells. We identified IFN-γ-regulated genes in tumor-associated macrophages that may contribute to direct lymphoma cytotoxicity as well as stimulation of T-cell chemotaxis and antigen processing, including TAP1 and TAP2. By IHC, we confirmed TAP1 expression in a subset of diagnostic specimens of PCNSL and determined, using Cox multivariate model, that strong TAP1 correlated with improved PFS (p<0.0006). Notably, independent of receipt of maintenance therapy, TAP1 also correlated with improved PFS in 38 patients that received only MTX-based induction, without dose-intensive chemotherapy consolidation. (Figure 1) Our results support a direct, immune-editing role for monocyte-derived macrophages in the regulation of CNS lymphoma progression, via several mechanisms, including antigenic processing and cross-presentation. We suggest that tumor-associated CD68 and TAP1 (and TAP2) be evaluated further as candidate biomarkers for risk stratification in PCNSL, particularly in trials that involve targeted immunotherapy. Supported by NCI and LLS. Figure 1 Figure 1. Disclosures Rubenstein: Kymera: Research Funding.