Clinical significance of Kelch-like protein 11 antibodies

Abstract

Objective: To report the clinical and oncologic associations of antibodies against Kelch-like protein 11 (KLHL11-ab), recently suggested as biomarkers of a paraneoplastic brainstem cerebellar syndrome associated with testicular seminoma, and to determine the value of immunohistochemistry as a screening technique.

Methods: Studies included 432 sera or CSF from 329 patients with paraneoplastic (157) or autoimmune neurologic syndromes (172); 63 with neurologic symptoms and benign teratomas; 28 with small-cell lung cancer, and 12 healthy subjects. KLHL11-abs were examined using a cell-based assay (CBA) with HEK293 cells transfected with a human KLHL11 clone. The CBA specificity was confirmed by immunoprecipitation. All positive samples were examined by immunohistochemistry on rat brain sections.

Results: KLHL11-abs were detected in 32 patients by CBA, and patients' antibodies immunoprecipitated KLHL11. Using rat brain immunohistochemistry, only 7 samples (22%) were positive. Patients' median age was 28 years (range 9-76 years), and 16 (50%) were women. Tumors were identified in 23/32 (72%) patients, including 14 teratomas and 7 seminomas or mixed germ cell tumors. Thirteen (41%) patients had cerebellar ataxia (7) or encephalitis with brainstem cerebellar symptoms (6), 7 (22%) anti-NMDA receptor (NMDAR) encephalitis (5 with ovarian teratoma), 5 (16%) opsoclonus-myoclonus, 3 (9%) limbic encephalitis, and 4 (12%) diverse neurologic symptoms (3 with benign teratomas). Concurrent autoantibodies occurred in 14 (44%) patients (7 anti-NMDAR, 6 Ma2, and 1 Hu).

Conclusions: KLHL11-abs associate with a spectrum of syndromes and tumors wider than those previously reported; 44% of patients have concurrent neuronal antibodies, some of them (anti-NMDAR) pathogenically relevant. Brain immunostaining is not useful for routine screening of KLHL11-abs.

Figure 1. Human KLHL11-ab demonstrated in a cell-based assay and by immunoprecipitation

(A) Reactivity (green) of a representative patient's serum with HEK293 cells expressing KLHL11. No immunoreactivity is observed with a serum control (D). (B and E) Reactivity (red) of a commercial KLHL11-ab. (C and F) Merged reactivities of the indicated samples (patient and control samples) with the commercial KLHL11-ab, showing a perfect colocalization with patients' antibodies. Scale bar = 10 μm. (G) Western blot showing that the indicated patients' serum samples with KLHL11-ab (lanes 2–5) immunoprecipitated KLHL11. Lane 1 shows the immunoprecipitation using the commercial antibody, and lanes 6–9 demonstrate the lack of KLHL11 immunoprecipitation with the indicated control samples. Lane 10 is the lack of immunoprecipitation of KLHL11 using untransfected HEK293 cells and serum of a patient with KLHL11-ab.

Figure 2. Detection of KLHL11-ab by immunohistochemistry on rat tissue

Immunohistochemistry on rat brain sections showing cytoplasmic staining of neurons of deep cerebellar nuclei incubated by a representative patient's serum with KLHL11-ab (A) and a negative control (B). Scale bar = 200 μm. Panel C shows the reactivity (green) of a representative patient's serum with HEK293 cells expressing KLHL11. No immunoreactivity is observed with a serum control (F). Panels D and G show the reactivity (red) of a commercial KLHL11-ab. Panels E and H show the merged reactivities of the indicated samples (patient's and control samples) with the commercial KLHL11-ab, showing a perfect colocalization with patient's antibodies (E). Scale bar = 10 μm.