(D) IgG eluted from excised nitrocellulose region corresponding to the ~ 90 kDa immunoreactive band yielded the ANNA3 immunostaining pattern when applied to mouse tissue sections; IgG eluted from the nitrocellulose region corresponding to ~170 kDa did not. (anti-Hu) and ANNA2 (anti-Ri) are recognized biomarkers of paraneoplastic autoimmunity and incorporated into serological evaluations of patients with suspected neurological autoimmunity. They also serve as biomarkers of cancer even in the absence of neurological symptoms.[2-6] A third ANNA (ANNA3) was described in 11 patients with multifocal neurological presentations and cancer; its detection is based on the characteristic immunofluorescence staining on mouse tissue sections.[7] Although relatively rare, serological detection of this autoantibody is critical in delineating the underlying pathophysiology, and expediting diagnosis and treatment; ignorance of the antigens molecular identity has precluded its ready detection in clinical practice. Here we report the identity of the ANNA3 antigen and a broadened clinical phenotype of seropositive patients. == Methods == The Mayo Clinic Institutional Review Board approved this study. ANNA3-IgG positive patients were identified through the Mayo Clinic Neuroimmunology Laboratory database (1/1/1993-02/1/2021); 32 patients had residual specimens (serum, 21; CSF, 3; both, 8); non-uniform clinical information was available for 30. Control patients for serology studies included 21 healthy subjects, 10 anti-nuclear antibody-IgG patients, 30 small cell lung cancer (SCLC) patients, 60 patients with neural autoantibodies to intracellular antigens (ANNA1-IgG, 34; ANNA2-IgG, 7; collapsin-response-mediator-protein-5 [CRMP5]-IgG, 12; or amphiphysin-IgG, 7) and 24 to cell-surface antigens (aquaporin-4-IgG, 12; Leucine-rich-glioma-inactivated-1[LGI1]-IgG, 12). In addition, we reviewed 100 consecutive Mayo Clinic patients charts with paraneoplastic antibody evaluations ordered in the Neuroimmunology Laboratory to assess the frequency of malignancies and compare it with the ANNA3-IgG patient cohort. Seropositive patients were identified by the characteristic staining on murine tissue indirect immunofluorescence assay (IFA,Figure 1).[7] The SCC37 SCLC-cell line was reactive when tested by indirect immunofluorescence with patients IgG and a nuclear lysate preparation was used as an antigen source.[8,9] Purified patient IgG was used to immunoprecipitate SCC37 nuclear proteins that were eluted, separated by gel electrophoresis and characterized by mass spectometry, as previously described.[10] Dachshund-homolog 1 (DACH1) was confirmed as the antigen by using a commercial DACH1-specific IgG (Thermo Fisher) for immunohistochemical colocalization, antigen-specific Western blot (with DACH1 polypeptide [residues 282-758, produced in anE. colisystem using the BL21-Codon plus-[DE3]-RIPL strain, Agilent] and DACH1-overexpressing HEK293-cell lysate), cell-based immunofluorescence assay (CBA) using HEK293-cells transiently transfected with DACH1-C-EGFP plasmid (GenScript,NM_001366712.1), and immune absorption experiments.[10] CBAs (serum tested at 1:500 dilution, CSF at 1:2) were interpreted blinded by two experienced readers (AZ, EPF). == Figure 1. Discovery of ANNA3 antibodies in serum samples. == (A) ANNA3-IgG binds selectively to nuclei in cerebellar Purkinje neurons and glomerular podocytes by indirect immunofluorescence on murine tissues, and (B) to nuclei of the SCC37 small-cell carcinoma cell line. (C) Western blot demonstrated ~90 kDa immunoreactive band in SCC37 TSPAN2 nuclear extract with 19/19 tested ANNA3-positive sera; control sera were nonreactive. *indicates patients with multiple specimens. (D) IgG eluted from excised nitrocellulose region corresponding to the ~ 90 kDa immunoreactive band yielded the ANNA3 KY02111 immunostaining pattern when applied to mouse tissue sections; IgG eluted from the nitrocellulose region corresponding to ~170 kDa did not. GL = granular layer; ML = molecular layer; Pc = Purkinje cells. Po = glomerular podocytes. For the tumor pathology, heat-induced antigen retrieval was performed on deparaffinized PFA-fixed sections. Immunostaining used IgGs specific for DACH1, synaptophysin (Leica), chromogranin A (Invitrogen) and thyroid transcription factor (TTF-1, DAKO) and the EnVision FLEX immunohistochemistry system (Dako). Stained sections were counterstained with hematoxylin (Dako). == Results == == Antigen characterization (Figure 1) == Immunoreactivity in SCC37 cells was confirmed immunohistochemically KY02111 and by western blot. ANNA3-IgG-positive sera bound to a protein (<100 kDa) by western blot using SCC37 nuclear extracts and IgG eluted from the nitrocellulose-corresponding band yielded the ANNA3-IgG staining pattern on IFA. Mass spectrometric analysis revealed KY02111 DACH1 peptides as most abundant amongst proteins captured by ANNA3-positive patients IgG. == Confirmation of DACH1 as ANNA3 autoantigen (Figure 2) == == Figure 2. Confirmation of DACH1 as the antigenic target of ANNA3. == (A) By indirect immunofluorescence on murine tissues, IgG in ANNA3-positive patient serum (red) co-localizes with rabbit anti-DACH1-IgG (green; merged images: yellow); no colocalization with control serum. (B) Western blot shows binding of IgG in 25 ANNA3-positive sera to recombinant DACH1 (~ 120 KDa) in lysate of transected HEK293 cells, which is identified by positive control rabbit anti-DACH1 IgG; all control patient IgGs are negative. (C) Western blot shows binding of IgG in 25 ANNA3-positive sera to a recombinant DACH1 polypeptide (residues 282-758, ~ 55 KDa, identified by positive control.