Paraneoplastic Calmodulin Kinase-Like Vesicle-Associated Protein (CAMKV) Autoimmune Encephalitis

Abstract

Objectives: To report an autoimmune paraneoplastic encephalitis characterized by immunoglobulin G (IgG) antibody targeting synaptic protein calmodulin kinase-like vesicle-associated (CAMKV).

Methods: Serum and cerebrospinal fluid (CSF) samples harboring unclassified antibodies on murine brain-based indirect immunofluorescence assay (IFA) were screened by human protein microarray. In 5 patients with identical cerebral IFA staining, CAMKV was identified as top-ranking candidate antigen. Western blots, confocal microscopy, immune-absorption, and mass spectrometry were performed to substantiate CAMKV specificity. Recombinant CAMKV-specific assays (cell-based [fixed and live] and Western blot) provided additional confirmation.

Results: Of 5 CAMKV-IgG positive patients, 3 were women (median symptom-onset age was 59 years; range, 53-74). Encephalitis-onset was subacute (4) or acute (1) and manifested with: altered mental status (all), seizures (4), hyperkinetic movements (4), psychiatric features (3), memory loss (2), and insomnia (2). Paraclinical testing revealed CSF lymphocytic pleocytosis (all 4 tested), electrographic seizures (3 of 4 tested), and striking MRI abnormalities in all (mesial temporal lobe T2 hyperintensities [all patients], caudate head T2 hyperintensities [3], and cortical diffusion weighted hyperintensities [2]). None had post-gadolinium enhancement. Cancers were uterine adenocarcinoma (3 patients: poorly differentiated or neuroendocrine-differentiated in 2, both demonstrated CAMKV immunoreactivity), bladder urothelial carcinoma (1), and non-Hodgkin lymphoma (1). Two patients developed encephalitis following immune checkpoint inhibitor cancer therapy (atezolizumab [1], pembrolizumab [1]). All treated patients (4) demonstrated an initial response to immunotherapy (corticosteroids [4], IVIG [2]), though 3 died from cancer.

Interpretation: CAMKV-IgG is a biomarker of immunotherapy-responsive paraneoplastic encephalitis with temporal and extratemporal features and uterine cancer as a prominent oncologic association. ANN NEUROL 2024;96:21-33.

Figure 1.. Characterization of CAMKV-IgG antibody

(A-D) Indirect immunofluorescence staining pattern of patient CAMKV-IgG binding to crytosectioned mouse tissue composite. Patient serum produces synaptic staining of (A and B) hippocampus (Hi, with relative sparing of the stratum lucidum [SL] and dentate gyrus [DG]), thalamus (Thal), basal ganglia (BG) and cerebral cortex (C). The staining is cerebrum-predominant with sparing of the cerebellar molecular layer (ML) and granular layer (GL) (D). Scale bar = 100 μm. (E) CAMKV ranked top neural protein hit in each of 5 patient serums by protein microarray as measured by AF647 secondary antibody signal (F) Mouse whole brain lysate, denatured and separated by electrophoresis, when probed with rabbit polyclonal CAMKV antibody or candidate patient (pt) serums 1–5 (diluted 1:100) produced bands ~ 70 kDa molecular weight. None of the 4 healthy control serums were reactive (not shown). (G) CAMKV was the top-ranked hit following mass-spectrometry of immunoprecipitated product of patient serum exposed to mouse whole brain protein extract. Immunoprecipitation of normal control serum with mouse whole brain extract did not contain CAMKV protein. (H) Patient 4 serum demonstrated reactivity with the dendrites and synaptic boutons of live rat neurons. This reactivity was not seen with CSF (Scale bar = 20 μm).

Figure 2.. Confirmation of CAMKV as the target antigen.

Confirmation of CAMKV as target antigen. (A) Confocal microscopy demonstrates patient IgG (green) colocalizing with CAMKV rabbit polyclonal antibody staining (red) in mouse hippocampus and cerebral cortex (yellow). Scale bar = 100 μm. (B) Western blot using recombinant CAMKV protein produced a ~70-kDa band when probed with commercial CAMKV antibody, or with patient serum or CSF (lanes 1–7). Patient 5 (lane 8) and control serum were negative (lane 9). (C and D) Patient IgG binding to HEK-293 cells transfected with cDNAs encoding green fluorescent protein (GFP)-tagged CAMKV (CAMKV-GFP). Primary antibody was applied to cells fixed (C) and live (D). Patient IgG (red) colocalized (yellow) with CAMKV-GFP (green) in both the fixed and live states and healthy control serums were negative. Scale bar = 50 μm. (E) Immunostaining (IFA) of mouse hippocampus produced by CAMKV-IgG positive serum (patient 1) is abrogated post-incubation with recombinant (r) CAMKV protein, but not post-incubation with recombinant CRMP-5 protein. CRMP-5-IgG positive control serum is not abrogated post-incubation with CAMKV protein. Scale bar = 100μm.

Figure 3.. Examples of MRI signal change in 5 CAMKV-IgG positive patients.

T2 FLAIR signal change was observed in the right mesial temporal lobe (A1), caudate nuclei bilaterally (more than adjacent putamina [arrows] A2), and the brainstem (A3) in patient 1. T2 FLAIR signal abnormality was observed in the bilateral mesial temporal lobes in patient 2 (B1). A parietal lobe subcortical T2 FLAIR lesion (B2), which also demonstrates diffusion restriction on diffusion weighted imaging (DWI, B3) was also observed in patient 2. T2 FLAIR hyperintensities of mesial temporal lobes (C1), bilateral caudate nuclei (C2) and striatum (C3) were observed in patient 3. Subtle bilateral insular hyperintensity was observed in patient 4 (D1, arrows). T2 FLAIR hyperintense lesions were observed in the cingulate gyrus bilaterally (D2) with restricted diffusion on DWI (D3) in patient 4. T2 FLAIR hyperintensities of bilateral mesial temporal lobes (E1), bilateral caudate nuclei (E2) and left putamen (arrow, E3) were observed in patient 5.

Figure 4.. Timeline of clinical and radiologic evolution for patient 3.

MRI imaging from multiple time points is presented alongside corresponding clinical information. Signal change in the bilateral caudate nuclei and mesial temporal lobes are shown at clinical presentation in images A1 and A2. Evolution of these changes with development of edema is demonstrated in images B1 and B2. There is further progression evident in images C1 and C2, with near resolution of the T2 FLAIR signal change and the development of severe atrophy in images D1 and D2.

Figure 5.. Histopathology of neoplastic tissue

Patient 5 (A-D): (A) The H&E stain of the biopsied endometrium of a high-grade endometrial carcinoma patient reveals neoplastic cells characterized by enlarged nuclei, peripheral accumulated chromatin, multiple nucleoli (indicated with arrowhead in the inset), and atypical mitosis (arrow). (B) Immunohistochemical staining with synaptophysin antibody supports neuroendocrine differentiation. (C) Immunostaining shows CAMKV granular cytoplasmic immunoreactivity in the neoplastic cells. (D) Negative control (bovine serum without CAMKV antibody). Patient 1 (E-F): (E) Immunohistochemistry reveals cytoplasmic CAMKV immunoreactivity in the neoplastic cells of endometrial carcinoma tissue (a CAMKV positive giant neoplastic cell is indicated with the arrow and inset). (F) Negative control. Scale bars=50μm.