The expanded clinical spectrum of anti-GABABR encephalitis and added value of KCTD16 autoantibodies

Abstract

In this study we report the clinical features of 32 patients with gamma aminobutyric acid B receptor (GABABR) antibodies, identify additional autoantibodies in patients with anti-GABABR encephalitis that mark the presence of an underlying small cell lung carcinoma and optimize laboratory methods for the detection of GABABR antibodies. Patients (n = 3225) were tested for the presence of GABABR antibodies using cell-based assay, immunohistochemistry and live hippocampal neurons. Clinical data were obtained retrospectively. Potassium channel tetramerization domain-containing (KCTD)16 antibodies were identified by immunoprecipitation, mass spectrometry analysis and cell-based assays. KCTD16 antibodies were identified in 23/32 patients with anti-GABABR encephalitis, and in 1/26 patients with small cell lung carcinoma and Hu antibodies, but not in 329 healthy subjects and disease controls. Of the anti-GABABR encephalitis patients that were screened sufficiently, 18/19 (95%) patients with KCTD16 antibodies had a tumour versus 3/9 (33%) anti-GABABR encephalitis patients without KCTD16 antibodies (P = 0.001). In most cases this was a small cell lung carcinoma. Patients had cognitive or behavioural changes (97%) and prominent seizures (90%). Thirteen patients developed a refractory status epilepticus with intensive care unit admittance (42%). Strikingly, 4/32 patients had a rapidly progressive dementia. The addition of KCTD16 to the GABABR cell-based assay improved sensitivity of the in-house fixed cell-based assay, without loss of specificity. Twenty-two of 26 patients improved (partially) to immunotherapy or chemotherapy. Anti-GABABR encephalitis is a limbic encephalitis with prominent, severe seizures, but patients can also present with rapidly progressive dementia. The co-occurrence of KCTD16 antibodies points towards a paraneoplastic origin. The addition of KCTD16 improves the sensitivity of the cell-based assay.

Keywords: antineuronal autoantibodies; autoimmune encephalitis; neuronal surface antigens; paraneoplastic neurological disorders.

Figure 1

KCTD16 antibodies are associated with an underlying tumour. (A) Bar diagram depicting percentages of patients with or without an underlying tumour. Patients with KCTD16 antibodies more frequently have an underlying tumour. Fisher exact test, P = 0.001. (B) Scatterplot depicting serum and CSF anti-GABA_BR titres of patients with or without KCTD16 antibodies; lines indicate median values. GABA_BR antibody titres in serum do not differ between patients with or without KCTD16 antibodies, whereas antibody titres in CSF are significantly higher in patients with KCT16 antibodies. Mann-Whitney test, P = 0.24 (serum), P = 0.01 (CSF). (C) Bar diagram depicting percentages of patients with a status epilepticus. Status epilepticus tended to occur more frequently in patients with KCTD16 antibodies when compared to patients without KCTD16 antibodies. Fisher exact test, P = 0.045 (P-values between 0.01 and 0.05 should be considered with caution). (D) Scatterplot depicting mRS at disease maximum, lines indicate median values. Maximum disease severity does not differ between patients with or without KCTD16 antibodies. Mann-Whitney test, P = 0.59. (E) Scatterplot depicting minimal mRS after treatment, lines indicate median values. Response to treatment does not differ between patients with or without KCTD16 antibodies. Mann-Whitney test, P = 0.20. (F) Immunohistochemistry of SCLC tissue from Patient 5, stained with haematoxylin and eosin (HE), normal rabbit serum and KCTD16 antibody. The image shows specific KCTD16 expression in tumour cells, which is absent in healthy lung tissue. Staining was performed on sequential slides and images were taken in the same area of the sample. Scale bars = 25 µm.

Figure 2

Kaplan Meier curve of survival comparing tumour and non-tumour patients. Showing the data of patients with (n = 21) and without tumours (n = 7). Median survival was 17 months (95% CI 7.80–26.20), not different between patients with tumours (15 months, 95% CI 11.04–18.96), or without (no median number as >50% survived, P = 0.36).

Figure 3

Diagnostic tests for GABA_B receptor antibodies. (A) Immunohistochemistry of adult rat brain stained with patient CSF or control CSF. The patient CSF shows brain-wide neuropil staining, here exemplified by an image of the hippocampus. Scale bars = 500 µm. (B) Immunocytochemistry of living rat hippocampal neurons. Labelling with the patient serum (green) results in a dot-like pattern along the neurites. Scale bars = 10 µm. (C) Live in-house CBA of HEK cells transfected with GABA_B1-GFP and GABA_B2 (green) and stained with patient serum or control serum (red). The patient serum labels the surface of cells transfected with GABA_B receptor. Scale bars = 20 µm. (D) Bar diagram representing the percentages of positive and negative tests for the different laboratory techniques that are used for the detection of GABA_B receptor antibodies. For one patient, CSF was not available to perform CBA, but this sample did test positive for GABA_B receptor in live CBA (Dalmau lab, Barcelona).

Figure 4

Endpoint titrations with fixed cell-based assay. (A) Titration of serum of an anti-KCTD16-negative patient (red) using a fixed CBA of HEK cells transfected with GABA_B1-GFP and GABA_B2 (green) with or without co-transfection of KCTD16. Staining of cells co-transfected with KCTD16 can be detected up to a dilution of 1:3200, as opposed to without KCTD16 co-transfection, up to a dilution of 1:800. (B) Serum titres detected with a fixed CBA with or without co-transfection of KCTD16. Higher serum titres are detected with the addition of KCTD16 to the CBA. Median serum titre detected with the GABA_B1/2 assay was 200 (IQR 60–1600, range 0–25 600), and with addition of KCTD16 3200 (IQR 3200–9600, range 0–64 000; P < 0.0001). (C) CSF titres detected with or without co-transfection of KCTD16. Higher CSF titres are detected with the addition of KCTD16 to the CBA. Median CSF titre detected with the GABA_B1/2 assay was 64 (IQR 7–160, range 0–512), and with addition of KCTD16 128 (IQR 48–512, range 4–2048; P = 0.001). (D) Serum and CSF titres detected with a fixed CBA without KCTD16 co-transfection. Patients with an underlying tumour do not have higher titres in serum and CSF than patients without an underlying tumour. Mann-Whitney test, serum P = 0.23, CSF P = 0.41. Symbols in B–D refer to individual patients, which are explained in greater detail in Supplementary Tables 1 and 3–5.

Figure 5

GABA_B receptor with and without KCTD16 co-expression. Schematic representation of the possible effect of the addition of KCTD16 to the CBA. (A) GABA_B1 and GABA_B2 subunits expressed without co-expression of KCTD16. (B) GABA_B1 and GABA_B2 subunits expressed with co-expression of KCTD16 resulting in a conformational change in the GABA_B receptor that allows for more efficient antibody binding. (C) GABA_B1 and GABA_B2 subunits expressed with co-expression of KCTD16 resulting in clustering of GABA_B receptors via the unknown scaffold protein ‘X’ resulting in more dense antibody labelling.