Standardized test for anti-Tr/DNER in patients with paraneoplastic cerebellar degeneration

Abstract

Objective: To determine sensitivity and specificity of a standardized recombinant cell-based indirect immunofluorescence assay (RC-IFA) for anti-Tr antibodies in comparison to a reference procedure.

Methods: Delta/Notch-like epidermal growth factor-related receptor (DNER) was expressed in HEK293 and used as a substrate for RC-IFA. HEK293 control cells expressing CDR2/Yo and CDR2L as well as mock-transfected HEK293 cells were used as controls. Serum samples from 38 patients with anti-Tr antibodies (33 with paraneoplastic cerebellar degeneration [PCD] and Hodgkin lymphoma), 66 patients with anti-Tr-negative PCD, 53 patients with Hodgkin lymphoma without neurologic symptoms, 40 patients with rheumatic diseases, and 42 healthy blood donors were tested for anti-DNER reactivity in the RC-IFA. In addition, RC-IFA results were compared to those from a commercial tissue-based IFA using monkey cerebellum.

Results: Using the RC-IFA, anti-DNER was detected in all anti-Tr-positive patients but in none of the controls (sensitivity 100%, 95% confidence interval [CI] 92.8%-100%; specificity 100%, 95% CI 98.7%-100%). In comparison, anti-Tr was not detected in 4 samples with low-titer autoantibodies using the commercial tissue-based assay. Preadsorption of sera with either recombinant full-length DNER or its extracellular domain selectively abolished anti-Tr reactivity.

Conclusion: Anti-Tr antibodies bind to the extracellular domain of DNER and can be detected by RC-IFA using HEK293 cells expressing the recombinant receptor. The new method performs better than a frequently used commercial tissue-based indirect immunofluorescence assay (IFA) in samples with low-titer antibodies.

Classification of evidence: This study provides Class II evidence that RC-IFA accurately detects anti-Tr as compared to conventional IFA.

Figure 1. Determination of anti-Tr/DNER by indirect immunofluorescence

(A) Slide with 10 reaction fields as used for the study. Each reaction field contained 9 biochips with HEK293 cells expressing Delta/Notch-like epidermal growth factor-related receptor (DNER), CDR2/Yo, or CDR2L, and mock-transfected control cells fixed with either acetone or formalin as well as cryosections of monkey cerebellum. (B) Binding of serum IgG of a patient with paraneoplastic cerebellar degeneration and Hodgkin lymphoma (a–c) to Purkinje cell somata and dendritic trees (anti-Tr) pattern on monkey cerebellum (a, d) and to DNER-expressing cells (b, e) but not control cells used as internal control (c, f). None of these reactivities were seen with serum from controls (d–f). (C) Preincubation with a lysate of HEK293 expressing DNER (a, b) completely abolished the anti-Tr/DNER fluorescence (a, c: monkey cerebellum; b, d: DNER-expressing cells), but it was not abolished following incubation with a similar lysate from wild-type HEK293 (c, d).

Figure 2. Comparative analysis of anti-Tr and anti-DNER titers as detected using mouse cerebellum and HEK293 cells transfected with human full-length DNER

Each dot represents the endpoint titer of a single serum (N = 38). All dots in individual squares represent identical results. Gray fields: line of equal endpoint titer between tissue and recombinant cells. DNER = Delta/Notch-like epidermal growth factor-related receptor.