PMC9878211_01
Brain magnetic resonance (MR) fluid-attenuated inversion recovery (FLAIR) images (A, B), gadolinium (Gd)-enhanced T1-weighted image (C), and MR spectroscopy (D) findings. Brain MRI FLAIR 7 days before admission revealed high-intensity lesions in the bilateral frontal lobes (A). MRI FLAIR (B) and Gd-enhanced T1-weighted image (C) on admission revealed that high-intensity area in the right frontal lobe on MRI FLAIR showed round shaped on Gd enhancement. MR spectroscopy demonstrated an increase in choline/creatine ratio and decrease in N-acetyl aspartate levels in the Gd-enhanced lesion (D).
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F1
MRI revealed high-intensity lesions in the right temporal and bilateral frontal lobes on FLAIR images, with the right frontal lesion demonstrating gadolinium (Gd) enhancement, which can be considered to indicate CNS inflammation or a high-grade brain tumor. Magnetic resonance spectroscopy of the right frontal lesion revealed an increase in the choline/creatine ratio and a normal N-acetyl aspartate level (Figure 1). In addition, 67Ga scintigraphy showed no abnormal uptake in the systemic organs and lymph nodes (Supplementary Figure 1). NR1/NR2-transfected HEK293 cells (BIOCHIP, Euroimmun, Labor Berlin) confirmed anti-NMDAR antibodies in the CSF alone (Figure 2, antibody titers were 1:32). No onconeural antibodies that included amphiphysin, Hu, Yo, CV2, Ri, Ma2/Ta, recoverin, Tr, GAD65, and others measured via line blot (EUROLINE, Euroimmun, Lubeck, Germany) were detected (details regarding autoantibody detection using in-house and commercially available assays are described in the Supplementary methods). The antibody titers of anti-NMDAR antibodies in the CSF decreased as the volume of brain tumor reduced after surgical and irradiative therapies (Figure 2, titers were 1:4).
Immunolabeling of the patient's cerebrospinal fluid (CSF) sample with in-house tissue-based assay (TBA) (A-C) and cell-based assay (CBA) (D-I). Control CSF did not react with in-house TBA (A), whereas the initial patient's CSF immunolabeled neuropil on the rat hippocampus in the TBA (B). The patient's CSF showed reactivity [green, (D)], with human embryonic kidney (HEK) cells expressing the N-methyl-d-aspartate receptor (NMDAR). The commercial antibody against NMDAR1 (clone EPR2480Y, ab68144, Abcam) [red, (E)] colocalizes with that of the patient's CSF [yellow, (F)], whereas a control CSF is negative (G). Anti-NMDAR antibody titers (1:32) were confirmed using the fluorescent reactivity of CBA on the initial patient's CSF (H). Following tumor removal and whole-brain irradiation therapy, immunosignals in TBA (C) and CBA decreased, as did antibody titers of anti-NMDAR antibody (titers 1:4) (I). Nuclei were counterstained with 4', 6-diamino-2-phenylindole. Bars = 1000 mum (A-C), 50 mum (D-G), and 100 mum (H, I).
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F2
Patient's CSF immunolabeled neuropil of the rat hippocampus (Figure 2) and granular cell layers in the cerebellum (Supplementary Figure 2), which was consistent with the immunolabeling pattern of anti-NMDAR antibodies. NR1/NR2-transfected HEK293 cells (BIOCHIP, Euroimmun, Labor Berlin) confirmed anti-NMDAR antibodies in the CSF alone (Figure 2, antibody titers were 1:32). No onconeural antibodies that included amphiphysin, Hu, Yo, CV2, Ri, Ma2/Ta, recoverin, Tr, GAD65, and others measured via line blot (EUROLINE, Euroimmun, Lubeck, Germany) were detected (details regarding autoantibody detection using in-house and commercially available assays are described in the Supplementary methods). The antibody titers of anti-NMDAR antibodies in the CSF decreased as the volume of brain tumor reduced after surgical and irradiative therapies (Figure 2, titers were 1:4).
Hematoxylin and eosin (H&E) staining (A, B) and immunolabeling with the antibodies against grail fibrillary acidic protein (GFAP) (clone G-A-5; Sigma, MO, US) (C) and NR1 (D) for the tumor cells. Note that H&E staining of the removed brain specimens showed foci of highly proliferative atypical cells interposed with basophilic matrix (A). The tumor cells with irregular nuclei and frequent mitoses [(B), inset in (A)] were not immunolabeled with commercial antibodies against GFAP (C) nor NR1 subunit of NMDAR (D), while the basophilic matrix on H&E staining was immunolabeled with commercial antibody against GFAP (C). Asterisks in red (B-D) indicate the same blood vessel, and all bars = 100 mum.
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F3
Immunological profiles of the tumor were consistent with primary central nervous system diffuse large B-cell lymphoma (PCNS-DLBCL) (Supplementary Figure 3). In addition, immunolabeling with biopsied brain sections using anti-NR1 antibodies (clone EPR23397-66; Abcam, Cambridge, UK) revealed that the tumor cells did not express the NR1 subunit of NMDAR (Figure 3).