Expression of IDO1 in PCNSL and IDO activity and response to lenalidomide in relapsed CNS lymphoma. (A) Quantitative reverse transcription polymerase chain reaction demonstrated increased expression of IDO1 transcripts in 10 cases of PCNSL (DLBCL) compared with 3 cases of nonneoplastic brain (P < .014, Wilcoxon test). Immunohistochemistry using α-IDO-1 antibody (Abcam clone 55305, with diaminobenzidine detection, hematoxylin counterstain) demonstrated IDO-1 expression in 19/20 cases of newly diagnosed PCNSL (supplemental Table 4). In 12 of these, IDO1 immunoreactivity was exhibited by (B, ×1000 >40% of lymphoma cells) and (C, ×400) tumor-associated macrophages. These displayed dendritic morphology and coexpressed CD163, a marker of alternative activation (not shown). Elevated baseline CSF kynurenine concentration (1.5 μM) correlated with shorter overall survival among the 14 phase 1 trial subjects as well as shorter progression-free survival with lenalidomide. (D-E) We also detected increasing CSF kynurenine/tryptophan ratios in 3 patients who had low CSF kynurenine at baseline and in whom CSF kynurenine levels exceeded the high-risk benchmark of 1.5 μM at tumor progression. Serial quantification of the K/T ratio in CSF in lenalidomide/rituximab trial subjects demonstrated that changes in IDO activity correlated with early progression and/or response to lenalidomide in 10 of 13 evaluable patients. Representative examples are shown: axial-T1 postgadolinium MRI scan demonstrating CNS lymphoma progression during first month of lenalidomide in patient 2: pretreatment (F) at baseline and (G) 1 month. The CSF K/T ratio at 1 month increased by nearly threefold compared with baseline during this interval. (H) MRI scan showing progressive regression of CNS lymphoma in response to lenalidomide monotherapy in patient 5: pretreatment (I) at baseline, (J) at 1 month, and (K) 2 months. (L) The CSF K/T ratio declined by >50% during the first 2 months of lenalidomide therapy in this patient. mRNA, messenger RNA.