Anti-Neuronal Nuclear Antibody 3 Autoimmunity targets Dachshund homolog 1

Abstract

The antigen specificity of Anti-Neuronal Nuclear Antibody-type 3 (ANNA3)-IgG is unknown. We identified Dachshund-homolog 1 (DACH1) as the ANNA3 autoantigen and confirmed it by antigen-specific assays, immunohistochemical colocalization and immune absorption experiments. Patients’ median age was 63.5 years (range, 49-88); 67% were female. Neurological manifestations (information available for 30 patients) included one or more of neuropathy, 12; cognitive difficulties, 11; ataxia, 8; dysautonomia, 7. Evidence of a neoplasm was present in 27/30 (90%), most of neuroendocrine lineage. DACH1-IgG is rare and represents a novel proposed biomarker of neurological autoimmunity and cancer.

Introduction

Novel neural autoantibodies are increasingly validated as biomarkers of paraneoplastic neurological autoimmunity and cancer.[1] Antineuronal nuclear antibody (ANNA) type 1 (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 antigen’s 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 an E. coli system 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 nuclear extract with 19/19 tested ANNA3-positive sera; control sera were non-reactive. *indicates patients with multiple specimens. (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. 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 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 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 rabbit anti-DACH1 IgG); all control patient IgGs are negative. (D) IgG in ANNA3-positive patient serum (red), but not in control serum, binds to permeabilized DACH1-GFP-transfected HEK293 cells (green); yellow indicates colocalization. (E) The immunostaining pattern typical of ANNA3-IgG was eliminated by pre-absorbing a positive patient’s serum with lysate of DACH1 overexpressing HEK293 cells; the staining pattern of a control ANNA1-IgG positive patient’s serum was not diminished by lysate absorption.

We confirmed DACH1 was the pertinent ANNA3 antigen: Firstly, IgG in ANNA3-positive sera colocalized with a commercial DACH1-specific-IgG by confocal microscopy. Secondly, IgG in patients’ sera bound to DACH1 by WB using HEK293 DACH1-overexpressing lysate or recombinant DACH1 polypeptide. By DACH1-CBA, IgG in all ANNA3-positive specimens, but in no controls, were positive. Finally, preabsorption of serum with HEK293 DACH1-overexpressing lysate eliminated tissue binding of ANNA3-IgG but not ANNA1-IgG.

Clinical Characteristics (Supplementary table)

Clinical information was available for 30 patients: median age, 63.5 years (range, 49-88); 20 were female. Clinical phenotypes varied and included neuropathy in 12, cognitive difficulties or encephalitis in 11, ataxia in 9, and autonomic involvement in 7. An additional two patients had diarrhea or esophageal spasms. Chorioretinopathy, optic neuropathy or Adie’s pupils were documented in 3 individual patients. One of 2 patients with sensory neuronopathy had co-existing ANNA1-IgG. Two patients had a neurodegenerative disease but a malignant neoplasm was found in both. CSF was inflammatory in 4/6 with interpretable results with either pleocytosis or CSF-unique oligoclonal bands.

Eight of 11 patients with available information showed improvement in their neurological syndrome (all had immunotherapy and/or cancer treatment). For two patients with SCLC, neurological symptoms worsened or appeared during immune checkpoint inhibitor cancer immunotherapy.

Oncological findings

Cancer was evident in 27 of 30 (90%) of which 22 were confirmed histopathologically, 4 had a lung mass with or without hilar adenopathy and 1 had an FDG-avid PET mass. In four patients, more than one malignancy was documented. Neuroendocrine tumors accounted for 14/22 (64%) of histopathologically-confirmed neoplasms. Others included: carcinomas of colon, breast, lung and ovary, a poorly differentiated carcinoma of unknown primary and an ovarian teratoma. Of 3 patients without imaging evidence of cancer, 2 had a neuroendocrine tumor-predicting neural autoantibody profile and the other was a smoker. In 12/23 (52%) patients with available information, the cancer was identified after neurological symptom onset. When compared with a group tested for paraneoplastic neural autoantibody evaluation, ANNA3-seropositive patients had higher frequency of cancer (27/30 vs 15/100, p=0.0001) and a higher proportion of neoplasms were neuroendocrine tumors (14/22 vs 2/15, p=0.0031).

Additional serological findings

In cases with paired serum and CSF, ANNA3-IgG was detected in both, except in one CSF with co-existing CRMP5-IgG that was negative by IFA but positive by CBA. Fourteen patients had co-existing neural autoantibodies, most commonly predicting a neuroendocrine tumor: neuronal intermediate filament (NIF), 3; CRMP5, 3; voltage-gated calcium channel (P/Q type), 3; ANNA1, 2; SRY-Box-transcription-factor-1 (SOX1), 2; Purkinje-cell-autoantibody-type-2/microtubule-associated-protein-1B (PCA2/MAP1B), 1.

Immunoreactivity of patients’ tumor tissues

Lymph-node SCLC metastatic tumors were available from two patients. In both, the neoplastic cells were compatible with a neuroendocrine tumor and expressed DACH1; DACH1 was not expressed by normal lymph-node tissue (Figure 3).

Figure 3. DACH1-immunoreactivity in metastatic tumor cells of two ANNA3-positive patients.

Case 1 (A-H): Resected mediastinal lymph node containing SCLC cells; H&E stain (A)and (E). The tumorous tissue is hypercellular with irregularly shaped tumor cells of increased nuclear: cytoplasmic ratio. The nuclei vary in size and exhibit coarse chromatin (E, inset). Synaptophysin (B, F) and chromogranin A (C, G) contour the tumor cells with a trabecular growth pattern typical of neuroendocrine differentiation. The nuclei of the tumor parenchymal cells are DACH1 immunoreactive (D, H).

Case 2 (I-P): Resected right lower paratracheal lymph node tissue shows diffuse metastatic neoplastic cells infiltration (I and M). Tumor cells show obvious atypia with increased nuclear: cytoplasmic ratio, increased mitosis, and apoptosis. Synaptophysin (J, N) and TTF1 (K, O) immunoreactivities are consistent with neuroendocrine differentiation. The nuclei of the tumor parenchymal cells are immunoreactive for DACH1 (L, P).

Normal lymph-node tissue (Q-S) shows no DACH1 immunoreactivity.

Scale bars: 50 μm (A-D, R); 20 μm (E-H, M-P, S); 100 μm (I-L, Q)

Discussion

For two decades ANNA3 has been recognized by immunostaining pattern, as a serological accompaniment of paraneoplastic neurological autoimmunity.[7] This study identified and confirmed the DACH1 protein as the antigenic specificity of ANNA3-IgG, enabling future studies into disease presentation and pathogenesis.

The neurological manifestations of ANNA3 seropositivity are diverse, and can involve multiple levels of the neuraxis but, despite this, in most patients the final clinical diagnosis was a paraneoplastic neurologic disorder.[1, 4, 11] High-risk paraneoplastic neurologic phenotypes, as defined in the recent criteria, are common in ANNA3-seropositive patients.[1] Occasionally alternative explanations were identified for the neurologic features but the antibody still had oncological significance. Future studies will determine if a more homogeneous neurologic phenotype emerges now that serological diagnosis will be enhanced due to the identification of the antigenic target.

DACH1-IgG seropositivity merits evaluation for occult cancer, regardless of neurological diagnosis.[2, 3] Ninety percent of the patients in this report had evidence of malignancy; the cancer was neuroendocrine in the majority of cases, mainly SCLC, a cancer recognized to associate strongly with neural autoantibodies and autoimmunity.[3] The link of ANNA3-IgG to SCLC was strengthened by our demonstration of DACH1 immunoreactivity in metastatic SCLC-cells of seropositive patients. The neurological and oncological correlations and the detection of DACH1 in tumors of ANNA3-seropositive patients, fulfills diagnostic criteria for establishing neural autoantibodies as portending high risk for paraneoplastic neurological autoimmunity.[1]

As immune checkpoint inhibitor cancer immunotherapy is becoming first line treatment for SCLC, neural autoantibodies hold promise for predicting patients prone to develop autoimmune complications.[12] As recently described in other cases, the neurological syndrome appeared or aggravated in two patients of this report after the administration of immune checkpoint inhibitors.[13]

In almost half of the patients, ANNA3-IgG was accompanied by co-existing neural antibodies, a majority of which predicted a neuroendocrine cancer. The occurrence of multiple neural autoantibodies in a patient with paraneoplastic autoimmunity is well documented and reflects the multiple immunogens driving the tumor immune response; the antibodies will more often predict the malignancy rather than the neurological phenotype.[2, 3, 14, 15].

ANNA3-IgG is rare. In a 5-year period (2016-2020), we identified 9 individual patients while we identified 473 ANNA1-positive patients, 236 PCA1-positive, 49 PCA2-positive, 45 ANNA2-positive and 21 PCA-Tr-positive. In terms of autoantibody detection specificity, none of 145 controls yielded a positive result by DACH1-CBA. Increasingly, recombinant antigen assays are introduced to optimize initial screening sensitivity. Further investigation is needed to define if screening with a DACH1-antigen specific assays will increase sensitivity without affecting specificity.

DACH1 is a transcription factor first described in the drosophila, involved in regulation of organogenesis. In addition to our finding DACH1 immunoreactivity in SCLC tumors, other cancers encountered in the ANNA3-IgG positive patients have been reported to express DACH1.[16-19] In breast cancer, DACH1 has been assigned tumor suppressor function.[17, 18]

DACH1-IgG production presumably reflects T lymphocyte-dependent activation of B lymphocytes during a patient’s protective anti-tumor immune response. Future studies are needed to determine the pathogenic role of DACH1-IgG and DACH1-specific cytotoxic T-cells in paraneoplastic neurologic disease.

Defining the antigenic target of ANNA3-IgG enables more sensitive and certain serological diagnosis, leading to earlier diagnosis of a patient’s neurological presentation as paraneoplastic and earlier appropriate treatment, thus improving the neurological and oncological outcome.

What is the current knowledge on the topic?

Anti-Neuronal Nuclear Antibody-type 3 (ANNA3) was the third anti-neuronal nuclear antibody proposed as a biomarker of paraneoplastic neurological autoimmunity; its detection has relied on identifying its characteristic immunostaining pattern on rodent tissue sections, but its antigen is not known.

What question did this study address?

What is the autoantigen defined by antineuronal nuclear antibody-type 3 (ANNA3) and the clinical phenotype of seropositive patients?

What does this study add to our knowledge?

In this study we identified dachshund-homolog 1 (DACH1), a tumor suppressor protein, as the antigenic target of ANNA3-IgG. Neurologic manifestations were diverse and while a homogeneous neurologic phenotype was lacking, 90% had evidence of a malignancy, the majority being of neuroendocrine lineage.

How might this potentially impact on the practice of neurology?

The rare detection DACH1-IgG should lead to consideration of a diagnosis of a paraneoplastic neurologic disorder and prompt a search for an underlying cancer. Identification of DACH1 as the autoantigen of ANNA3-IgG will enable future studies into its potential neurologic accompaniments and its pathogenesis.