Antibodies to Zic4 in paraneoplastic neurologic disorders and small-cell lung cancer

Abstract

Objective: To determine whether serum Zic4 antibodies associate with paraneoplastic neurologic disorders (PND) and small-cell lung cancer (SCLC), and the association of these antibodies with other onconeuronal immunities associated with SCLC.

Design/methods: The authors studied 498 patients (215 with PND and 283 without PND or without cancer). The presence of antibodies was tested with immunoblots of Zic4, HuD, and CRMP5 proteins. The tumor expression of these proteins was determined by immunohistochemistry.

Results: Zic4 antibodies were identified in 61 patients. Ninety-two percent of patients with Zic4 antibodies had SCLC; detection of these antibodies segregated with the presence of PND (p = 0.031). Intrathecal synthesis of Zic4 antibodies was demonstrated in 5/7 patients with PND. None of 175 control patients without PND or cancer had Zic4 antibodies. Because of the robust association between Zic autoimmunity and SCLC, all patients were tested for other SCLC-related antibodies; concurrent Zic4, Hu, or CRMP5 antibodies occurred in the serum or CSF of 27% of SCLC patients with PND. Patients with isolated Zic4 antibodies were more likely to develop predominant cerebellar dysfunction than patients with several immunities (p < 0.001). Tumors of patients with and without onconeuronal antibodies coexpressed Zic, Hu, and CRMP5 proteins, indicating that the tumor expression of these antigens is necessary, but not sufficient, for immunologic activation.

Conclusions: In patients with neurologic symptoms of unknown cause detection of Zic4 antibodies predicts a neoplasm, usually a SCLC, and suggests that the neurologic disorder is paraneoplastic. Detection of Zic4 antibodies often associates with anti-Hu or CRMP5 antibodies. Patients with isolated Zic4 antibodies are more likely to develop cerebellar dysfunction than those with concurrent immunities.

Figure 1

Immunoblot and immunohistochemistry of Zic4 antibodies. (A) Immunoblot of recombinant Zic4 protein with sera of four small-cell lung cancer (SCLC) patients with paraneoplastic neurologic disorders (PND) and Zic4 antibodies (lanes 3 to 6), serum of a normal individual (lane 1), and serum of a patient with SCLC without Zic4 antibodies (lane 2). (B) Immunolabeling of rat cerebellum with Zic4 antibodies. The predominant reactivity corresponds to the granule cell layer of the cerebellum; minor reactivity is barely perceived in other areas (brainstem at bottom left, and brain at top left). Section not counter-stained (×10).

Figure 2

Coexpression of immunoreactive Zic4, Hu, and CRMP5 proteins by tumors. Consecutive sections of small-cell lung cancer (SCLC) from a patient without paraneoplastic neurologic disorders (PND) and without antineuronal antibodies and a Merkel cell carcinoma of a patient with PND and anti-Zic4 and anti-Hu antibodies, incubated with biotinylated immunoglobulin G (IgG) from sera of patients harboring only anti-Zic4, anti-Hu, or anti-CRMP antibodies. In both tumors there are areas with intense coexpression of the three antigens (Zic, Hu, CRMP proteins) and other areas with unequal antigen expression (i.e., in the SCLC there is an extensive area with minimal expression of Hu proteins but intense expression of Zic and CRMP proteins). Row “normal” corresponds to biotinylated IgG from a normal individual and serves as a negative control. The specific reactivity of each biotinylated IgG (anti-Zic4, anti-Hu, and anti-CRMP) was confirmed with immunocompetition assays (sections counterstained with hematoxylin, ×200).