Protein biomarker identification in the CSF of patients with CNS lymphoma

Abstract

Purpose: Elucidation of the CSF proteome may yield insights into the pathogenesis of CNS disease. We tested the hypothesis that individual CSF proteins distinguish CNS lymphoma from benign focal brain lesions.

Methods: We used a liquid chromatography/mass spectrometry-based method to differentially quantify and identify several hundred CSF proteins in CNS lymphoma and control patients. We used enzyme-linked immunosorbent assay (ELISA) to confirm one of these markers in an additional validation set of 101 cases.

Results: Approximately 80 CSF proteins were identified and found to be present at significantly different concentrations, both higher and lower, in training and test studies, which were highly concordant. To further validate these observations, we defined in detail the expression of one of these candidate biomarkers, antithrombin III (ATIII). ATIII RNA transcripts were identified within CNS lymphomas, and ATIII protein was localized selectively to tumor neovasculature. Determination of ATIII concentration by ELISA was significantly more accurate (> 75% sensitivity; > 98% specificity) than cytology in the identification of cancer. Measurement of CSF ATIII levels was found to potentially enhance the ability to diagnose and predict outcome.

Conclusion: Our findings demonstrate for the first time that proteomic analysis of CSF yields individual biomarkers with greater sensitivity in the identification of cancer than does CSF cytology. We propose that the discovery of CSF protein biomarkers will facilitate early and noninvasive diagnosis in patients with lesions not amenable to brain biopsy, as well as provide improved surrogates of prognosis and treatment response in CNS lymphoma and brain metastasis.

Fig 1

(A) Differential expression of CSF proteins that distinguish CNS lymphoma from patients without CNS malignancy. Values are shown as a color scale with positive values represented by red, negative represented by green, and black corresponding to values close to 0. Missing values are shown in white. (B) The concordance between training and test set was exceptional, as shown by comparing the test statistics from the two data sets.

Fig 2

Intratumoral expression of antithrombin III (ATIII) in diagnostic tumor specimens of primary CNS lymphoma, confirmed by quantitative real-time polymerase chain reaction (n + 20). Intratumoral expression of ATIII RNA was highest in patients with the shortest survival in this cohort (< 6 months).

Fig 3

Immunohistochemistry demonstrating antithrombin III (ATIII) expression by tumor cells and neovasculature in CNS lymphomas. Magnification = 100× (A) and 400× (B). (C) von Willebrand Factor expression by all of the vessels. (D) Absent ATIII expression by vessels of healthy brain. compared CSF from eight patients with CNS lymphoma to nine controls without brain tumors. We identified more than 1,000 molecular ions corresponding to 196 CSF proteins expressed at different concentrations (FDR < 0.1), either higher or lower, in CNS lymphoma patients compared with control subjects without CNS malignancy.

Fig 4

CSF antithrombin III (ATIII) concentration distinguishes CNS lymphoma from non-neoplastic CNS conditions. Receiver operating characteristic analysis of ATIII concentration in cross-validation set (n + 101 patients).

Fig 5

Two patients with focal brain lesions not amenable to biopsy. (A) Diagnosis was ultimately established as neurosarcoid (CSF antithrombin III [ATIII] concentration was 0.5 μg/mL). (B) Diagnosis was lymphoma (CSF ATIII level was 2.1 μg/mL).

Fig 6

CSF antithrombin III (ATIII) concentration > 1.4 μg/mL is associated with shorter overall survival in patients with CNS non-Hodgkin’s lymphoma at first relapse (n = 11; Kaplan-Meier method). Karnofsky performance score < 70 also correlated with short survival (P < .009); however, neither elevated total CSF protein nor age > 60 years correlated with shortened survival at first CNS relapse.

Fig 7

Relationship between CNS lymphoma progression and CSF concentration of antithrombin III (ATIII). (A, B) Negative magnetic resonance imaging (MRI), positive CSF cytology. (C) Progression of disease. (D) Response after systemic chemotherapy. CNS lymphoma progression and therapeutic response are reflected by rise and fall in CSF concentrations of ATIII in specimens obtained at time of each MRI. Columns, mean; bars, standard deviation.

Fig 8

CSF antithrombin III (ATIII) concentration reflects persistent disease in patient with refractory CNS lymphoma (A) Remission. (B) Relapse of CNS lymphoma; remission was later achieved after brain irradiation. (C) Two months after irradiation, the patient exhibited new cranial nerve deficits and gait instability; magnetic resonance imaging of brain and spine as well as CSF cytology were negative. Columns, mean; bars, standard deviation.